Figure 3 ( A ) Lysates from spleen - derived T cells of wild - type ( Bim + / + ) and Bim - knockout ( Bim − / − ) mice were immunoblotted and probed with indicated antibodies . Statistics were from three knockout and control mice . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 . ( B ) T cells from Bim + / + and Bim − / − mice were cultured with / without Baf for 4 hr preceding harvest , immunoblotted , and probed with the indicated antibodies . ( C ) Spleen - derived T cells from Bim + / + or Bim − / − mice were stained with rabbit anti - LC3 antibody . Representative confocal images and quantification from triplicates ( n = 100 - 110 ) are shown . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 0001 . ( D ) Bim + / + and Bim − / − mouse liver lysates were immunoblotted and probed with the indicated antibodies ( n = 3 ) . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 . ( E ) Bim + / + and Bim − / − mouse liver lysates were immunoblotted and probed with the indicated antibodies ( n = 3 ) . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 . ( F ) RNA from Bim wild - type or Bim - knockout mouse livers was analyzed by qRT - PCR for p62 / beta - actin mRNA . The mean ± SD from three mice per group is shown . NS , not significant .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Lysates from spleen-derived T cells of wild-type (Bim+/+) and Bim-knockout (Bim−/−) mice were immunoblotted and probed with indicated antibodies. Statistics were from three knockout and control mice. Data are shown as mean ± SD. ∗∗∗p < 0.001.(B) T cells from Bim+/+ and Bim−/− mice were cultured with/without Baf for 4 hr preceding harvest, immunoblotted, and probed with the indicated antibodies.(C) Spleen-derived T cells from Bim+/+ or Bim−/− mice were stained with rabbit anti-LC3 antibody. Representative confocal images and quantification from triplicates (n = 100-110) are shown. Data are shown as mean ± SD. ∗∗∗p < 0.0001.(D) Bim+/+ and Bim−/− mouse liver lysates were immunoblotted and probed with the indicated antibodies (n = 3). Data are shown as mean ± SD. ∗∗p < 0.01.(E) Bim+/+ and Bim−/− mouse liver lysates were immunoblotted and probed with the indicated antibodies (n = 3). Data are shown as mean ± SD. ∗∗∗p < 0.001.(F) RNA from Bim wild-type or Bim-knockout mouse livers was analyzed by qRT-PCR for p62/beta-actin mRNA. The mean ± SD from three mice per group is shown. NS, not significant.


Figure 4 ( A ) HeLa cells were treated with control siRNA , Bim siRNA + control siRNA , control siRNA + Beclin 1 siRNA , or Bim siRNA + Beclin 1 siRNA . Blots were probed as indicated . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 . ( B ) HeLa cells were treated with control siRNA , Bim siRNA + control siRNA , control siRNA + Beclin 1 siRNA , or Bim siRNA + Beclin 1 siRNA . After 24 hr , cells were transfected with GFP - PX for 24 hr . GFP - PX vesicles were assessed with a confocal microscope . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 . Arrows label cells with increased number / size of GFP - PX vesicles . ( C ) GFP - mRFP - LC3 HeLa cells were treated with control , Bim siRNA , or Bcl - 2 siRNA and then analyzed with Cellomics microscope . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 . ( D ) GFP - mRFP - LC3 stable HeLa cells were treated with control siRNA , Bim siRNA + control siRNA , control siRNA + Beclin 1 siRNA , or Bim siRNA + Beclin 1 siRNA and then analyzed with Cellomics microscope and matching confocal images are shown . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) HeLa cells were treated with control siRNA, Bim siRNA + control siRNA, control siRNA + Beclin 1 siRNA, or Bim siRNA + Beclin 1 siRNA. Blots were probed as indicated. Data are shown as mean ± SD. ∗∗p < 0.01.(B) HeLa cells were treated with control siRNA, Bim siRNA + control siRNA, control siRNA + Beclin 1 siRNA, or Bim siRNA + Beclin 1 siRNA. After 24 hr, cells were transfected with GFP-PX for 24 hr. GFP-PX vesicles were assessed with a confocal microscope. Data are shown as mean ± SD. ∗∗p < 0.01. Arrows label cells with increased number/size of GFP-PX vesicles.(C) GFP-mRFP-LC3 HeLa cells were treated with control, Bim siRNA, or Bcl-2 siRNA and then analyzed with Cellomics microscope. Data are shown as mean ± SD. ∗∗∗p < 0.001.(D) GFP-mRFP-LC3 stable HeLa cells were treated with control siRNA, Bim siRNA + control siRNA, control siRNA + Beclin 1 siRNA, or Bim siRNA + Beclin 1 siRNA and then analyzed with Cellomics microscope and matching confocal images are shown. Data are shown as mean ± SD. ∗∗∗p < 0.001.


Figure 5 ( A ) Bim ( EL ) EE / empty vector ( IP negative control ) or Bim ( EL ) EE / Beclin 1 - Flag ( in duplicate ) were transfected into HeLa cells . After 20 hr , one set of cells with Bim ( EL ) EE / Beclin 1 - Flag were starved in HBSS for 2 hr . Anti - Flag antibody ( M2 ) was used for immunoprecipitation , and blots were probed as indicated . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 . ( B ) Panel i : HeLa cells were treated with control siRNA or Bim siRNA . After 48 hr , one set of transfections were starved in HBSS for 2 hr . Blots were probed as indicated . LC3 - II / tubulin ratio of siRNA - transfected cells is set as 1 ( n = 3 ) . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 ; NS , not significant . Panel ii : HeLa cells were treated with control siRNA or Bim siRNA . After 48 hr , one set of transfections were treated with Baf for 2 hr ; one set of transfections were starved and treated with Baf for 2 hr . Blots were probed as indicated and analyzed as in ( Bi ) . ∗ ∗ p < 0 . 01 ; NS , not significant . ( C ) HeLa cells were transfected with control plasmid / GFP - LC3 ( 3 : 1 ) , or Bim ( EL ) EE / GFP - LC3 ( 3 : 1 ) . After 20 hr , one set of transfected cells were starved in HBSS for 2 hr and then fixed . The percentages of cells with GFP - LC3 vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 . NS , not significant . ( D ) BimS - Beclin 1 binding is not sensitive to HBSS starvation . BimEL - EE ( EL ) / vector ( IP negative control ) , BimEL - EE ( EL ) / Beclin 1 - Flag ( two replicates ) , BimL - EE ( L ) / Beclin 1 - Flag ( two replicates ) or BimS - EE ( S ) / Beclin 1 - Flag ( two replicates ) were transfected into HeLa cells . After 20 hr , one of EL - Beclin 1 - Flag transfections , one of L - Beclin - Flag transfections and one of S - Beclin 1 - Flag transfections were subjected to 2 hr HBSS starvation . Anti - Flag ( M2 ) was used for immunoprecipitation . ( E ) HBSS starvation increases Bim T116 phosphorylation . Bim ( EL ) EE or BimEE ( EL ) - T116A were transfected into HeLa cells . After 20 hr , one set of Bim ( EL ) EE transfections and one of Bim ( EL ) EE - T116A were starved for 2 hr in HBSS . Blots were probed with the indicated antibodies . ( F ) Bim T116 phosphorylation disables Bim - Beclin 1 interaction . Bim ( EL ) EE / vector ( IP negative control ) , Bim ( EL ) EE / Beclin 1 - Flag ( in duplicate ) , or Bim ( EL ) EE - T116E ( phospho - mimic , designated as T116E here ) / Beclin - Flag ( in duplicate ) were transfected into HeLa cells . After 20 hr , one set of Bim ( EL ) EE / Beclin 1 - Flag transfections and one of Bim ( EL ) EE - T116E / Beclin 1 - Flag transfections were starved for 2 hr in HBSS ( Starv ) . Anti - Flag ( M2 ) was used for immunoprecipitation . ( G ) HeLa cells were transfected with control plasmid / GFP - LC3 , Bim ( EL ) - EE / GFP - LC3 ( 3 : 1 ) , or Bim ( EL ) - EE T116E / GFP - LC3 ( 3 : 1 ) . After 20 hr , one set of transfected cells were starved in HBSS for 2 hr and the cells were then fixed . The percentages of cells with GFP - LC3 vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 ; NS , not significant .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Bim(EL)EE/empty vector (IP negative control) or Bim(EL)EE/Beclin 1-Flag (in duplicate) were transfected into HeLa cells. After 20 hr, one set of cells with Bim(EL)EE/Beclin 1-Flag were starved in HBSS for 2 hr. Anti-Flag antibody (M2) was used for immunoprecipitation, and blots were probed as indicated. Data are shown as mean ± SD. ∗∗∗p < 0.001.(B) Panel i: HeLa cells were treated with control siRNA or Bim siRNA. After 48 hr, one set of transfections were starved in HBSS for 2 hr. Blots were probed as indicated. LC3-II/tubulin ratio of siRNA-transfected cells is set as 1 (n = 3). Data are shown as mean ± SD. ∗∗∗p < 0.001; NS, not significant. Panel ii: HeLa cells were treated with control siRNA or Bim siRNA. After 48 hr, one set of transfections were treated with Baf for 2 hr; one set of transfections were starved and treated with Baf for 2 hr. Blots were probed as indicated and analyzed as in (Bi). ∗∗p < 0.01; NS, not significant.(C) HeLa cells were transfected with control plasmid /GFP-LC3 (3:1), or Bim(EL)EE/GFP-LC3 (3:1). After 20 hr, one set of transfected cells were starved in HBSS for 2 hr and then fixed. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01. NS, not significant.(D) BimS-Beclin 1 binding is not sensitive to HBSS starvation. BimEL-EE (EL)/vector (IP negative control), BimEL-EE (EL)/Beclin 1-Flag (two replicates), BimL-EE (L)/Beclin 1-Flag (two replicates) or BimS-EE (S)/Beclin 1-Flag (two replicates) were transfected into HeLa cells. After 20 hr, one of EL-Beclin 1-Flag transfections, one of L-Beclin-Flag transfections and one of S-Beclin 1-Flag transfections were subjected to 2 hr HBSS starvation. Anti-Flag (M2) was used for immunoprecipitation.(E) HBSS starvation increases Bim T116 phosphorylation. Bim(EL)EE or BimEE(EL)-T116A were transfected into HeLa cells. After 20 hr, one set of Bim(EL)EE transfections and one of Bim(EL)EE-T116A were starved for 2 hr in HBSS. Blots were probed with the indicated antibodies.(F) Bim T116 phosphorylation disables Bim-Beclin 1 interaction. Bim(EL)EE /vector (IP negative control), Bim(EL)EE/Beclin 1-Flag (in duplicate), or Bim(EL)EE-T116E (phospho-mimic, designated as T116E here)/Beclin-Flag (in duplicate) were transfected into HeLa cells. After 20 hr, one set of Bim(EL)EE/Beclin 1-Flag transfections and one of Bim(EL)EE-T116E/Beclin 1-Flag transfections were starved for 2 hr in HBSS (Starv). Anti-Flag (M2) was used for immunoprecipitation.(G) HeLa cells were transfected with control plasmid /GFP-LC3, Bim(EL)-EE/GFP-LC3 (3:1), or Bim(EL)-EE T116E/GFP-LC3 (3:1). After 20 hr, one set of transfected cells were starved in HBSS for 2 hr and the cells were then fixed. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01; NS, not significant.


Figure 6 ( A ) Bim - 147 does not bind to Beclin 1 . Beclin 1 - Flag / vector ( IP negative control ) , Beclin 1 - Flag / Myc - BimEL - 1 - 147aa ( Myc - 147 ) , or Beclin 1 - Flag / Myc - BimEL - ΔBH3 ( Myc - ΔBH3 ) were transfected into HeLa cells . Anti - Myc ( Rabbit ) was used for immunoprecipitation . ( B ) Bim - 147 is competent for LC8 binding . LC8 / vector ( IP negative control ) , LC8 / Myc - BimEL - 1 - 147aa ( Myc - 147 ) , or LC8 / Myc - BimEL - ΔBH3 ( Myc - ΔBH3 ) were transfected into HeLa cells . Anti - Myc ( Rabbit ) was used for immunoprecipitation . ( C ) Mutating LC8 binding consensus sites within Bim disrupts the Bim - LC8 interaction . Bim ( EL ) EE / vector ( IP negative control ) , Bim ( EL ) EE / Myc - LC8 , Bim ( EL ) EE - S109A , S113A , T114A ( AAA ) / Myc - LC8 , or Bim ( EL ) EE - T116E / Myc - LC8 were transfected into HeLa cells . Anti - Myc ( Rabbit ) was used for immunoprecipitation . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 . ( D ) Mutating LC8 binding consensus sites within Bim disrupts the Bim - Beclin 1 interaction . Bim ( EL ) EE / vector ( IP negative control ) , Bim ( EL ) EE / Beclin 1 - Flag , Bim ( EL ) EE - S109A , S113A , T114A ( AAA ) / Beclin 1 - Flag , or Bim ( EL ) EE - T116E / Beclin 1 - Flag were transfected into HeLa cells . Anti - Flag ( M2 ) was used for immunoprecipitation . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 . ( E ) Bim - S does not bind to LC8 . BimEL - EE ( EL ) / vector ( IP negative control ) , BimEL - EE ( EL ) / Myc - LC8 , BimL - EE ( L ) / Myc - LC8 , or BimS - EE ( S ) / Myc - LC8 were transfected into HeLa cells . Anti - Myc ( Rabbit ) was used for immunoprecipitation . ( G ) LC8 enhances Bim - Beclin 1 interaction in vitro . His - Bim , His - LC8 and GST - Beclin 1 were expressed in BL21 ( DE3 ) E . coli and purified . Two micrograms of His - Bim was combined with 2 μg GST without or with 1 μg His - LC8 ( as controls ) ; 2 μg His - Bim was combined with 2 μg GST - Beclin 1 without or with 1 μg His - LC8 . The mixtures were incubated in buffer A for 3 hr . Glutathione beads were then used to pull down GST or GST - Beclin 1 . The pulldown products were detected with anti - Bim and anti - Beclin 1 . ( H ) LC8 siRNA knockdown reduces the Bim - Beclin 1 interaction . Control siRNA or LC8 siRNA were transfected into HeLa cells . After 48 hr , HA - Bim ( EL ) EE / vector ( IP negative control ) or HA - Bim ( EL ) EE / Beclin 1 - Flag was transfected into control siRNA transfected HeLa cells ; HA - Bim ( EL ) EE / Beclin 1 - Flag was transfected into LC8 siRNA - transfected HeLa cells . Anti - Flag ( M2 ) was used for immunoprecipitation . Note that in the presence of BimEE , endogenous LC8 was pulled down by Beclin 1 when cells were treated with control siRNA . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Bim-147 does not bind to Beclin 1. Beclin 1-Flag/vector (IP negative control), Beclin 1-Flag/Myc-BimEL-1-147aa (Myc-147), or Beclin 1-Flag/Myc-BimEL-ΔBH3 (Myc-ΔBH3) were transfected into HeLa cells. Anti-Myc (Rabbit) was used for immunoprecipitation.(B) Bim-147 is competent for LC8 binding. LC8/vector (IP negative control), LC8/Myc-BimEL-1-147aa (Myc-147), or LC8/Myc-BimEL-ΔBH3 (Myc-ΔBH3) were transfected into HeLa cells. Anti-Myc (Rabbit) was used for immunoprecipitation.(C) Mutating LC8 binding consensus sites within Bim disrupts the Bim-LC8 interaction. Bim(EL)EE/vector (IP negative control), Bim(EL)EE/Myc-LC8, Bim(EL)EE-S109A, S113A, T114A (AAA)/ Myc-LC8, or Bim(EL)EE-T116E/ Myc-LC8 were transfected into HeLa cells. Anti-Myc (Rabbit) was used for immunoprecipitation. Data are shown as mean ± SD. ∗∗∗p < 0.001.(D) Mutating LC8 binding consensus sites within Bim disrupts the Bim-Beclin 1 interaction. Bim(EL)EE/vector (IP negative control), Bim(EL)EE/Beclin 1-Flag, Bim(EL)EE-S109A, S113A, T114A (AAA)/Beclin 1-Flag, or Bim(EL)EE-T116E/Beclin 1-Flag were transfected into HeLa cells. Anti-Flag (M2) was used for immunoprecipitation. Data are shown as mean ± SD. ∗∗∗p < 0.001.(E) Bim-S does not bind to LC8. BimEL-EE (EL)/vector (IP negative control), BimEL-EE (EL)/Myc-LC8, BimL-EE (L)/Myc-LC8, or BimS-EE (S)/Myc-LC8 were transfected into HeLa cells. Anti-Myc (Rabbit) was used for immunoprecipitation.(G) LC8 enhances Bim-Beclin 1 interaction in vitro. His-Bim, His-LC8 and GST-Beclin 1 were expressed in BL21(DE3) E. coli and purified. Two micrograms of His-Bim was combined with 2 μg GST without or with 1 μg His-LC8 (as controls); 2 μg His-Bim was combined with 2 μg GST-Beclin 1 without or with 1 μg His-LC8. The mixtures were incubated in buffer A for 3 hr. Glutathione beads were then used to pull down GST or GST-Beclin 1. The pulldown products were detected with anti-Bim and anti-Beclin 1.(H) LC8 siRNA knockdown reduces the Bim-Beclin 1 interaction. Control siRNA or LC8 siRNA were transfected into HeLa cells. After 48 hr, HA-Bim(EL)EE/vector (IP negative control) or HA-Bim(EL)EE/Beclin 1-Flag was transfected into control siRNA transfected HeLa cells; HA-Bim(EL)EE/Beclin 1-Flag was transfected into LC8 siRNA-transfected HeLa cells. Anti-Flag (M2) was used for immunoprecipitation. Note that in the presence of BimEE, endogenous LC8 was pulled down by Beclin 1 when cells were treated with control siRNA. Data are shown as mean ± SD. ∗∗∗p < 0.001.


Figure 7 ( A ) HeLa cells were transfected with vector ( vec ) / GFP - LC3 , BimEL - EE ( EL ) / GFP - LC3 , BimL - EE ( L ) / GFP - LC3 , BimS - EE ( S ) / GFP - LC3 , or BimEL - EE - AAA ( AAA ) / GFP - LC3 ( 3 : 1 ) . The percentages of cells with GFP - LC3 vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ p < 0 . 01 ; NS , not significant . Note that BimEL - EE - S109A , S113A , T114A is designated as BimEL - EE - AAA . ( B ) Bim bridges the Beclin 1 - LC8 interaction . Dynein light chain1 ( Myc - LC8 ) / empty vectors ( IP negative control ) , Myc - LC8 / Beclin 1 - Flag / empty vector , Myc - LC8 / Beclin 1 - Flag / BimEL - EE ( EL ) , Myc - LC8 / Beclin 1 - Flag / BimL - EE ( L ) , Myc - LC8 / Beclin 1 - Flag / BimS - EE ( S ) , and Myc - LC8 / Beclin 1 - Flag / BimEL - EE - S109A , S113A , T114A ( AAA ) were transfected into HeLa cells . Anti - Flag ( M2 ) was used for immunoprecipitation . ( C ) Starvation reduces the ability of Bim to bridge the Beclin 1 - LC8 interaction . Myc - LC8 / empty vectors ( IP negative control ) , Myc - LC8 / Beclin 1 - Myc / empty vector , or Myc - LC8 / Beclin 1 - Flag / HA - BimEL - EE ( HA - Bim ) ( two replicates ) were transfected into HeLa cells . After 20 hr , one set of cells with Myc - LC8 / Beclin 1 - Flag / HA - HA - Bim was starved in HBSS for 2 hr . Anti - Flag ( M2 ) antibody was used for immunoprecipitation . ( D ) HeLa cells were cultured with DMEM with 10 % serum ( Control ) or starved ( Starve ) in HBSS for 2 hr . The fixed cells were stained with Beclin 1 and tubulin antibodies and analyzed by confocal microscopy . White boxes show the areas where Beclin 1 is enriched . Yellow boxes show enlarged areas . ( E ) HeLa cells were treated with control siRNA or LC8 siRNA . After 24 hr , cells were split . Vector / GFP - LC3 or Bim ( EL ) EE / GFP - LC3 ( 3 : 1 ) were transfected into the control siRNA - transfected or LC8 siRNA - transfected cells . The percentages of cells with GFP - LC3 vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 ; NS , not significant . ( F ) HeLa cells were treated with control siRNA , Bim siRNA , or LC8 siRNA for 48 hr . Cells were then fixed in 37 ° C , 4 % PFA for 10 min . Cells were stained with Beclin 1 and tubulin antibodies and analyzed by confocal microscopy . White boxes / triangle show areas where Beclin 1 is enriched . Yellow boxes show enlarged areas . Colocalizations were quantified from images in 12 - 15 cells with Volocity program ( Colocalization coefficient Mx ) . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) HeLa cells were transfected with vector (vec) /GFP-LC3, BimEL-EE (EL)/GFP-LC3, BimL-EE (L)/GFP-LC3, BimS-EE (S)/GFP-LC3, or BimEL-EE-AAA (AAA)/GFP-LC3 (3:1). The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗∗p < 0.001; ∗∗p < 0.01; NS, not significant. Note that BimEL-EE-S109A, S113A, T114A is designated as BimEL-EE-AAA.(B) Bim bridges the Beclin 1-LC8 interaction. Dynein light chain1 (Myc-LC8)/empty vectors (IP negative control), Myc-LC8/Beclin 1-Flag/empty vector, Myc-LC8/Beclin 1-Flag/BimEL-EE (EL), Myc-LC8/Beclin 1-Flag/BimL-EE (L), Myc-LC8/Beclin 1-Flag/BimS-EE (S), and Myc-LC8/Beclin 1-Flag/BimEL-EE-S109A, S113A, T114A (AAA) were transfected into HeLa cells. Anti-Flag (M2) was used for immunoprecipitation.(C) Starvation reduces the ability of Bim to bridge the Beclin 1-LC8 interaction. Myc-LC8/empty vectors (IP negative control), Myc-LC8/Beclin 1-Myc/empty vector, or Myc-LC8/Beclin 1-Flag/HA-BimEL-EE (HA-Bim) (two replicates) were transfected into HeLa cells. After 20 hr, one set of cells with Myc-LC8/Beclin 1-Flag/HA-HA-Bim was starved in HBSS for 2 hr. Anti-Flag (M2) antibody was used for immunoprecipitation.(D) HeLa cells were cultured with DMEM with 10% serum (Control) or starved (Starve) in HBSS for 2 hr. The fixed cells were stained with Beclin 1 and tubulin antibodies and analyzed by confocal microscopy. White boxes show the areas where Beclin 1 is enriched. Yellow boxes show enlarged areas.(E) HeLa cells were treated with control siRNA or LC8 siRNA. After 24 hr, cells were split. Vector/GFP-LC3 or Bim(EL)EE/GFP-LC3 (3:1) were transfected into the control siRNA-transfected or LC8 siRNA-transfected cells. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01; NS, not significant.(F) HeLa cells were treated with control siRNA, Bim siRNA, or LC8 siRNA for 48 hr. Cells were then fixed in 37°C, 4% PFA for 10 min. Cells were stained with Beclin 1 and tubulin antibodies and analyzed by confocal microscopy. White boxes/triangle show areas where Beclin 1 is enriched. Yellow boxes show enlarged areas. Colocalizations were quantified from images in 12-15 cells with Volocity program (Colocalization coefficient Mx). Data are shown as mean ± SD. ∗∗∗p < 0.001.


Figure 1 Figure 1 . Breast cancer cells derived from tumor - draining lymph nodes gain aggressive malignancy ( A and B ) Inguinal LNs were harvested from 4T1 - injected mice at the indicated week after initial injection . LN tissues were fixed and paraffin - embedded . Immunoflurorescence was performed with anti - GFP antibody to visualize GFP + 4T1 cells . ( A ) Confocal micrographs of the representative field of the LN section . Scale bar : 10 μm . Data information : In ( A - G ) , all experimental data were verified in at least two independent experiments . All values are presented as mean or mean ± SD . Figure 1 . Breast cancer cells derived from tumor - draining lymph nodes gain aggressive malignancy ( A and B ) Inguinal LNs were harvested from 4T1 - injected mice at the indicated week after initial injection . LN tissues were fixed and paraffin - embedded . Immunoflurorescence was performed with anti - GFP antibody to visualize GFP + 4T1 cells . ( B ) Histograms illustrated that the dynamic changes of GFP + 4T1 cells metastasized to the inguinal LN . Cells were counted in whole sections ( 2 non - consecutive sections per LN per mouse ) . Each group was composed of at least two mice . Data information : In ( A - G ) , all experimental data were verified in at least two independent experiments . All values are presented as mean or mean ± SD . ( C - I ) 4T1 - injected or EMT6 - injected mice were sacrificed at the indicated week after initial injection . Collagenase - digested specimens from the primary tumors or inguinal LN tissues were cultured in culture dish for cancer cell enrichment . The detailed process is described in the methods section . After several days selection / culture , viable 4T1PT , 4T1LN , EMT6PT , and EMT6LN cells were used for the following assays : ( C ) Soft - agar colony forming activity was examined in 4T1PT and 4T1LN cells ( 5x102 cells / well , n = 4 wells per group ) . Data information : In ( A - G ) , all experimental data were verified in at least two independent experiments . All values are presented as mean or mean ± SD . In ( C ) , * * P = 0 . 000088 and * * P = 0 . 000004 for the 4T1LN cells at week 2 and week 3 , respectively . ( C - I ) 4T1 - injected or EMT6 - injected mice were sacrificed at the indicated week after initial injection . Collagenase - digested specimens from the primary tumors or inguinal LN tissues were cultured in culture dish for cancer cell enrichment . The detailed process is described in the methods section . After several days selection / culture , viable 4T1PT , 4T1LN , EMT6PT , and EMT6LN cells were used for the following assays : ( D and E ) Tumorigenesis assays were determined in BALB / c mice ( n = 3 mice per group ) orthotopically injected with 5x102 4T1PT or 4T1LN cells . Tumor mass ( D ) and tumor weight ( E ) were measured on day 28 . Scale bar : 1 cm . Data information : In ( A - G ) , all experimental data were verified in at least two independent experiments . All values are presented as mean or mean ± SD . ( D and E ) Tumorigenesis assays were determined in BALB / c mice ( n = 3 mice per group ) orthotopically injected with 5x102 4T1PT or 4T1LN cells . Tumor mass ( D ) and tumor weight ( E ) were measured on day 28 . Scale bar : 1 cm . Data information : In ( A - G ) , all experimental data were verified in at least two independent experiments . All values are presented as mean or mean ± SD . In ( E ) , * * P = 0 . 000006 . In ( G ) * * P = 0 . 000734 . Level of significance was determined using two - tailed unpaired t - test . ( F and G ) BALB / c mice ( n = 3 mice per group ) were injected with 2x105 4T1PT or 4T1LN cells via tail - vein . Lung colonization was examined by lung morphology ( F ) and the numbers of tumor nodule ( G ) on day 21 . Scale bar : 1 cm . Data information : In ( A - G ) , all experimental data were verified in at least two independent experiments . All values are presented as mean or mean ± SD . ( F and G ) BALB / c mice ( n = 3 mice per group ) were injected with 2x105 4T1PT or 4T1LN cells via tail - vein . Lung colonization was examined by lung morphology ( F ) and the numbers of tumor nodule ( G ) on day 21 . Scale bar : 1 cm . Data information : In ( A - G ) , all experimental data were verified in at least two independent experiments . All values are presented as mean or mean ± SD . ( H and I ) NSG mice ( n = 5 mice per group ) were injected with 4T1PT ( 5x102 ) , 4T1LN ( 5x102 ) , EMT6PT ( 1x102 ) , or EMT6LN ( 1x102 ) cells . Distant organ metastasis was examined by bioluminscent images on day 28 . The bioluminescent signal ( pseudocolor ) was recorded as photons per second per centimeter squared per steradian ( p / s / cm2 / sr ) and the luminescent image was overlaid on the photographic image . ( H and I ) NSG mice ( n = 5 mice per group ) were injected with 4T1PT ( 5x102 ) , 4T1LN ( 5x102 ) , EMT6PT ( 1x102 ) , or EMT6LN ( 1x102 ) cells . Distant organ metastasis was examined by bioluminscent images on day 28 . The bioluminescent signal ( pseudocolor ) was recorded as photons per second per centimeter squared per steradian ( p / s / cm2 / sr ) and the luminescent image was overlaid on the photographic image .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1 Figure 1. Breast cancer cells derived from tumor-draining lymph nodes gain aggressive malignancy (A and B) Inguinal LNs were harvested from 4T1-injected mice at the indicated week after initial injection. LN tissues were fixed and paraffin-embedded. Immunoflurorescence was performed with anti-GFP antibody to visualize GFP+ 4T1 cells. (A) Confocal micrographs of the representative field of the LN section. Scale bar: 10 μm. Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD. Figure 1. Breast cancer cells derived from tumor-draining lymph nodes gain aggressive malignancy (A and B) Inguinal LNs were harvested from 4T1-injected mice at the indicated week after initial injection. LN tissues were fixed and paraffin-embedded. Immunoflurorescence was performed with anti-GFP antibody to visualize GFP+ 4T1 cells. (B) Histograms illustrated that the dynamic changes of GFP+ 4T1 cells metastasized to the inguinal LN. Cells were counted in whole sections (2 non-consecutive sections per LN per mouse). Each group was composed of at least two mice.Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD. (C-I) 4T1-injected or EMT6-injected mice were sacrificed at the indicated week after initial injection. Collagenase-digested specimens from the primary tumors or inguinal LN tissues were cultured in culture dish for cancer cell enrichment. The detailed process is described in the methods section. After several days selection/culture, viable 4T1PT, 4T1LN, EMT6PT, and EMT6LN cells were used for the following assays: (C) Soft-agar colony forming activity was examined in 4T1PT and 4T1LN cells (5x102 cells/well, n = 4 wells per group). Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD. In (C), **P = 0.000088 and **P = 0.000004 for the 4T1LN cells at week 2 and week 3, respectively.(C-I) 4T1-injected or EMT6-injected mice were sacrificed at the indicated week after initial injection. Collagenase-digested specimens from the primary tumors or inguinal LN tissues were cultured in culture dish for cancer cell enrichment. The detailed process is described in the methods section. After several days selection/culture, viable 4T1PT, 4T1LN, EMT6PT, and EMT6LN cells were used for the following assays:(D and E) Tumorigenesis assays were determined in BALB/c mice (n = 3 mice per group) orthotopically injected with 5x102 4T1PT or 4T1LN cells. Tumor mass (D) and tumor weight (E) were measured on day 28. Scale bar: 1 cm.Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD.(D and E) Tumorigenesis assays were determined in BALB/c mice (n = 3 mice per group) orthotopically injected with 5x102 4T1PT or 4T1LN cells. Tumor mass (D) and tumor weight (E) were measured on day 28. Scale bar: 1 cm.Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD. In (E), **P = 0.000006. In (G) **P = 0.000734. Level of significance was determined using two-tailed unpaired t-test.(F and G) BALB/c mice (n = 3 mice per group) were injected with 2x105 4T1PT or 4T1LN cells via tail-vein. Lung colonization was examined by lung morphology (F) and the numbers of tumor nodule (G) on day 21. Scale bar: 1 cm.Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD.(F and G) BALB/c mice (n = 3 mice per group) were injected with 2x105 4T1PT or 4T1LN cells via tail-vein. Lung colonization was examined by lung morphology (F) and the numbers of tumor nodule (G) on day 21. Scale bar: 1 cm.Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD.(H and I) NSG mice (n = 5 mice per group) were injected with 4T1PT (5x102), 4T1LN (5x102), EMT6PT (1x102), or EMT6LN (1x102) cells. Distant organ metastasis was examined by bioluminscent images on day 28. The bioluminescent signal (pseudocolor) was recorded as photons per second per centimeter squared per steradian (p/s/cm2/sr) and the luminescent image was overlaid on the photographic image.(H and I) NSG mice (n = 5 mice per group) were injected with 4T1PT (5x102), 4T1LN (5x102), EMT6PT (1x102), or EMT6LN (1x102) cells. Distant organ metastasis was examined by bioluminscent images on day 28. The bioluminescent signal (pseudocolor) was recorded as photons per second per centimeter squared per steradian (p/s/cm2/sr) and the luminescent image was overlaid on the photographic image.


Figure 2 ( C and D ) Distant organ metastasis was examined by bioluminscent images of 4T1 tumor - bearing mice after tumor resection with ( n = 13 mice ) or without ( n = 10 mice ) tumor - draining lymph node dissection on week 5 . The bioluminescent signal ( pseudocolor ) was recorded as photons per second per centimeter squared per steradian ( p / s / cm2 / sr ) and the luminescent image was overlaid on the photographic image . For the additional details , see the methods section . ( E and I ) Summary table of distant organ metastasis derived from each surgery group . ( E and I ) Summary table of distant organ metastasis derived from each surgery group . ( G and H ) Distant organ metastasis was examined by bioluminscent images of inguinal lymph node pre - removed tumor - bearing mice ( n = 9 mice ) or sham surgery tumor - bearing mice ( n = 5 mice ) after tumor resection on week 5 . The bioluminescent signal ( pseudocolor ) was recorded as photons per second per centimeter squared per steradian ( p / s / cm2 / sr ) and the luminescent image was overlaid on the photographic image . For the additional details , see the methods section .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 (C and D) Distant organ metastasis was examined by bioluminscent images of 4T1 tumor-bearing mice after tumor resection with (n=13 mice) or without (n=10 mice) tumor-draining lymph node dissection on week 5. The bioluminescent signal (pseudocolor) was recorded as photons per second per centimeter squared per steradian (p/s/cm2/sr) and the luminescent image was overlaid on the photographic image. For the additional details, see the methods section. (E and I) Summary table of distant organ metastasis derived from each surgery group. (E and I) Summary table of distant organ metastasis derived from each surgery group.(G and H) Distant organ metastasis was examined by bioluminscent images of inguinal lymph node pre-removed tumor-bearing mice (n=9 mice) or sham surgery tumor-bearing mice (n=5 mice) after tumor resection on week 5. The bioluminescent signal (pseudocolor) was recorded as photons per second per centimeter squared per steradian (p/s/cm2/sr) and the luminescent image was overlaid on the photographic image. For the additional details, see the methods section.


Figure 3 Figure 3 . Up - regulation of Il - 17rb contributes to the aggressive malignancy phenotypes of breast cancer cell derived from tumor - draining lymph node ( A ) Gene expression profiles were shown at 4T1PT to 4T1LN cells . Five genes encoding cell surface proteins were identified among up - regulated genes . ( B ) mRNA expression of each candidate gene in 4T1PT and 4T1LN cells was determined by RT - qPCR . Gapdh was used as an internal control . ( C and D ) Il - 17rb , Gpr56 , and Scara5 expression in 4T1PT and 4T1LN cells were examined by Western blotting analysis . ( C and D ) Il - 17rb , Gpr56 , and Scara5 expression in 4T1PT and 4T1LN cells were examined by Western blotting analysis . ( E ) Western blotting and RT - qPCR analysis of Il - 17rb , Gpr56 , and Scara5 expression in 4T1 cells transduced with Il - 17rb , Gpr56 , Scara5 , or control LacZ shRNA lentivirus respectively . ( F ) Soft - agar colony forming activity was examined in lentivirus transduced shIl - 17rb , shGpr56 , shScara5 , or shLacZ 4T1 cells ( 5x102 cells / well , n = 6 wells per group ) . ( G ) Western blotting analysis was used to detect ectopic expression of Il - 17rb in 4T1 cells infected with either lentiviruses carrying Il - 17rb cDNA or empty vector . ( H ) Soft - agar colony forming activity was examined using control or Il - 17rb over - expressing 4T1 cells ( 5x102 cells / well , n = 6 wells per group ) . ( I ) Western blotting analysis was used to examine Il - 17rb in a representative Il - 17rbDel 4T1 clone derived from Il - 17rb knocked - out 4T1 cells using a CRISPR / Cas9 system . ( J and K ) Tumorigenesis assays were determined in BALB / c mice ( n = 3 mice per group ) orthotopically injected with 5x102 4T1 cells from WT or Il - 17rbDel clone . Tumor mass ( J ) and weight ( K ) were measured on day 28 . Scale bar : 1 cm . ( L and M ) BALB / c mice ( n = 3 mice per group ) were injected with 5x105 4T1 cells from WT or Il - 17rbDel clone via tail - vein . Lung colonization was examined by lung morphology ( L ) and the numbers of tumor nodule ( M ) on day 21 . Scale bar : 1 cm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 Figure 3. Up-regulation of Il-17rb contributes to the aggressive malignancy phenotypes of breast cancer cell derived from tumor-draining lymph node (A) Gene expression profiles were shown at 4T1PT to 4T1LN cells. Five genes encoding cell surface proteins were identified among up-regulated genes. (B) mRNA expression of each candidate gene in 4T1PT and 4T1LN cells was determined by RT-qPCR. Gapdh was used as an internal control.(C and D) Il-17rb, Gpr56, and Scara5 expression in 4T1PT and 4T1LN cells were examined by Western blotting analysis.(C and D) Il-17rb, Gpr56, and Scara5 expression in 4T1PT and 4T1LN cells were examined by Western blotting analysis.(E) Western blotting and RT-qPCR analysis of Il-17rb, Gpr56, and Scara5 expression in 4T1 cells transduced with Il-17rb, Gpr56, Scara5, or control LacZ shRNA lentivirus respectively.(F) Soft-agar colony forming activity was examined in lentivirus transduced shIl-17rb, shGpr56, shScara5, or shLacZ 4T1 cells (5x102 cells/well, n = 6 wells per group).(G) Western blotting analysis was used to detect ectopic expression of Il-17rb in 4T1 cells infected with either lentiviruses carrying Il-17rb cDNA or empty vector.(H) Soft-agar colony forming activity was examined using control or Il-17rb over-expressing 4T1 cells (5x102 cells/well, n = 6 wells per group).(I) Western blotting analysis was used to examine Il-17rb in a representative Il-17rbDel 4T1 clone derived from Il-17rb knocked-out 4T1 cells using a CRISPR/Cas9 system.(J and K) Tumorigenesis assays were determined in BALB/c mice (n = 3 mice per group) orthotopically injected with 5x102 4T1 cells from WT or Il-17rbDel clone. Tumor mass (J) and weight (K) were measured on day 28. Scale bar: 1 cm.(L and M) BALB/c mice (n = 3 mice per group) were injected with 5x105 4T1 cells from WT or Il-17rbDel clone via tail-vein. Lung colonization was examined by lung morphology (L) and the numbers of tumor nodule (M) on day 21. Scale bar: 1 cm.


Figure 4 ( B and C ) Inguinal lymph node - injected BALB / c mice were sacrificed at the indicated week after initial injection . Total cells isolated from inguinal lymph node tissues were transwell co - cultured with 4T1 cells . Inguinal lymph node tissues came from un - injection BALB / c mice as control . After 5 - day co - culture , 4T1 cells at lower well were examined in the RT - qPCR ( B ) analyses of Il - 17rb expression . Gapdh was used as an internal control or as a loading control . ( B and C ) 4T1 - injected BALB / c mice were sacrificed at the indicated week after initial injection . Total cells isolated from inguinal lymph node tissues were transwell co - cultured with 4T1 cells . Inguinal lymph node tissues came from un - injection BALB / c mice as control . After 5 - day co - culture , 4T1 cells at lower well were examined in the Western blotting ( C ) analyses of Il - 17rb expression . Gapdh was used as an internal control or as a loading control . ( D ) Soft - agar colony forming activity was examined using co - cultured 4T1 cells at lower well ( 5x102 cells / well , n = 4 wells per group ) . ( E and F ) 4T1 - injected BALB / c mice were sacrificed at three weeks post initial injection . Each lymphocyte subsets isolated from inguinal lymph node tissues by FACS sorting were transwell co - cultured with 4T1 cells . After 5 - day co - culture , 4T1 cells at lower well were used for the RT - qPCR ( E ) analyses of Il - 17rb expression . Gapdh was used as internal control or as a loading control . ( E and F ) 4T1 - injected BALB / c mice were sacrificed at three weeks post initial injection . Each lymphocyte subsets isolated from inguinal lymph node tissues by FACS sorting were transwell co - cultured with 4T1 cells . After 5 - day co - culture , 4T1 cells at lower well were used for the Western blotting ( F ) analyses of Il - 17rb expression . Gapdh was used as internal control or as a loading control . ( G ) Percentage of CD4 + Foxp3 + Tregs of total CD4 + cells in inguinal LN , spleen and peripheral blood of BALB / c mice ( n = 3 mice per group ) injected with 4T1 ( Blue ) or γ - irradiated 4T1 ( Red ) cancer cells was analyzed by FACS . ( H ) 4T1 cells co - cultured with the indicated lymphocyte populations isolated from inguinal LN of 4T1 tumor - bearing mice by FACS sorting . After 5 - day co - culture , 4T1 cells at lower well were analyzed for Il - 17rb expression by RT - qPCR analysis . Gapdh was used as internal control .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B and C) Inguinal lymph node-injected BALB/c mice were sacrificed at the indicated week after initial injection. Total cells isolated from inguinal lymph node tissues were transwell co-cultured with 4T1 cells. Inguinal lymph node tissues came from un-injection BALB/c mice as control. After 5-day co-culture, 4T1 cells at lower well were examined in the RT-qPCR (B)analyses of Il-17rb expression. Gapdh was used as an internal control or as a loading control.(B and C) 4T1-injected BALB/c mice were sacrificed at the indicated week after initial injection. Total cells isolated from inguinal lymph node tissues were transwell co-cultured with 4T1 cells. Inguinal lymph node tissues came from un-injection BALB/c mice as control. After 5-day co-culture, 4T1 cells at lower well were examined in the Western blotting (C) analyses of Il-17rb expression. Gapdh was used as an internal control or as a loading control.(D) Soft-agar colony forming activity was examined using co-cultured 4T1 cells at lower well (5x102 cells/well, n = 4 wells per group).(E and F) 4T1-injected BALB/c mice were sacrificed at three weeks post initial injection. Each lymphocyte subsets isolated from inguinal lymph node tissues by FACS sorting were transwell co-cultured with 4T1 cells. After 5-day co-culture, 4T1 cells at lower well were used for the RT-qPCR (E)analyses of Il-17rb expression. Gapdh was used as internal control or as a loading control.(E and F) 4T1-injected BALB/c mice were sacrificed at three weeks post initial injection. Each lymphocyte subsets isolated from inguinal lymph node tissues by FACS sorting were transwell co-cultured with 4T1 cells. After 5-day co-culture, 4T1 cells at lower well were used for the Western blotting (F) analyses of Il-17rb expression. Gapdh was used as internal control or as a loading control.(G) Percentage of CD4+Foxp3+ Tregs of total CD4+ cells in inguinal LN, spleen and peripheral blood of BALB/c mice (n = 3 mice per group) injected with 4T1 (Blue) or γ-irradiated 4T1 (Red) cancer cells was analyzed by FACS.(H) 4T1 cells co-cultured with the indicated lymphocyte populations isolated from inguinal LN of 4T1 tumor-bearing mice by FACS sorting. After 5-day co-culture, 4T1 cells at lower well were analyzed for Il-17rb expression by RT-qPCR analysis. Gapdh was used as internal control.


Figure 5 Figure 5 . Depletion of Tregs in the tumor - draining lymph node microenvironment abolishes Il - 17rb induction and enhanced malignancy in breast cancer cells . ( A ) 4T1 - injected BALB / c mice were treated with control IgG , anti - CD4 or anti - CD25 neutralizing antibody at the day 15 and day 18 after initial injection . Mice were sacrificed at three weeks after initial injection . Depletion of CD4 + Foxp3 + Tregs in inguinal LNs was confirmed by FACS analysis . ( B ) Western blotting analysis of Il - 17rb expression was performed in 4T1LN or 4T1PT cells collected from control IgG - , anti - CD4 , or anti - CD25 antibody - treated 4T1 tumor - bearing mice . ( C and D ) BALB / c mice were orthotopically injected with 5x102 4T1LN or 4T1PT cells collected from control IgG - , anti - CD4 , or anti - CD25 antibody - treated 4T1 tumor - bearing mice . ( n = 3 mice per group ) . Tumor mass ( D ) and tumor weight ( E ) were measured on day 28 . Scale bar : 1 cm . ( C and D ) BALB / c mice were orthotopically injected with 5x102 4T1LN or 4T1PT cells collected from control IgG - , anti - CD4 , or anti - CD25 antibody - treated 4T1 tumor - bearing mice . ( n = 3 mice per group ) . Tumor mass ( D ) and tumor weight ( E ) were measured on day 28 . Scale bar : 1 cm . ( E ) NSG mice ( n = 5 mice per group ) were injected with 5x102 4T1LN or 4T1PT cells collected from control IgG - , anti - CD4 , or anti - CD25 antibody - treated 4T1 tumor - bearing mice . Distant organ metastasis was examined by bioluminscent images on day 21 . The bioluminescent signal ( pseudocolor ) was recorded as photons per second per centimeter squared per steradian ( p / s / cm2 / sr ) and the luminescent image was overlaid on the photographic image .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5 Figure 5. Depletion of Tregs in the tumor-draining lymph node microenvironment abolishes Il-17rb induction and enhanced malignancy in breast cancer cells. (A) 4T1-injected BALB/c mice were treated with control IgG, anti-CD4 or anti-CD25 neutralizing antibody at the day 15 and day 18 after initial injection. Mice were sacrificed at three weeks after initial injection. Depletion of CD4+Foxp3+ Tregs in inguinal LNs was confirmed by FACS analysis. (B) Western blotting analysis of Il-17rb expression was performed in 4T1LN or 4T1PT cells collected from control IgG-, anti-CD4, or anti-CD25 antibody-treated 4T1 tumor-bearing mice.(C and D) BALB/c mice were orthotopically injected with 5x102 4T1LN or 4T1PT cells collected from control IgG-, anti-CD4, or anti-CD25 antibody-treated 4T1 tumor-bearing mice. (n = 3 mice per group). Tumor mass (D) and tumor weight (E) were measured on day 28. Scale bar: 1 cm.(C and D) BALB/c mice were orthotopically injected with 5x102 4T1LN or 4T1PT cells collected from control IgG-, anti-CD4, or anti-CD25 antibody-treated 4T1 tumor-bearing mice. (n = 3 mice per group). Tumor mass (D) and tumor weight (E) were measured on day 28. Scale bar: 1 cm.(E) NSG mice (n = 5 mice per group) were injected with 5x102 4T1LN or 4T1PT cells collected from control IgG-, anti-CD4, or anti-CD25 antibody-treated 4T1 tumor-bearing mice. Distant organ metastasis was examined by bioluminscent images on day 21. The bioluminescent signal (pseudocolor) was recorded as photons per second per centimeter squared per steradian (p/s/cm2/sr) and the luminescent image was overlaid on the photographic image.


Figure 6 Figure 6 . TGF - β1 secreted from Tregs induces Il - 17rb expression of breast cancer cells via Smad2 / 3 / 4 signaling . ( A ) CD4 + CD25 + Tregs were isolated from inguinal LN of BALB / c mice injected with 4T1 cells for three weeks , and were transwell cocultured with 4T1 cells in the presence of control IgG or neutralizing antibodies against IL - 10 or TGF - β1 . After 5 - day coculture , 4T1 cells at lower well were analyzed for Il - 17rb expression by RT - qPCR analysis . Gapdh was used as internal control . ( B and C ) Western blotting analyzed Il - 17rb in 4T1 cells treated with recombinant TGF - β1 ( B ) proteins for 5 days . ( B and C ) Western blotting analyzed Il - 17rb in 4T1 cells treated with recombinant IL - 10 ( C ) proteins for 5 days . ( D ) Western blotting analyzed Il - 17rb in shLacZ or shTgfbr1 4T1 cells treated with recombinant TGF - β1 for 5 days . ( E ) 4T1 - injected BALB / c mice were sacrificed at three weeks post initial injection . Total cells isolated from inguinal lymph node tissues were transwell co - cultured with 4T1 cells . After 5 - day coculture , soft - agar colony forming activity was determined in co - cultured shLacZ or shTgfbr1 4T1 cells at lower well . ( 5x102 cells / well , n = 6 wells per group ) . ( F - H ) Western blotting analyzed Il - 17rb in shLacZ , shSmad2 ( F ) 4T1 cells treated with recombinant TGF - β1 for 5 days . ( F - H ) Western blotting analyzed Il - 17rb in shLacZ , shSmad3 ( G ) 4T1 cells treated with recombinant TGF - β1 for 5 days . ( F - H ) Western blotting analyzed Il - 17rb in shLacZ , shSmad4 ( H ) 4T1 cells treated with recombinant TGF - β1 for 5 days . ( I and J ) WT 4T1 ( I ) or Il - 17rbDel 4T1 ( J ) cells were treated with or without recombinant TGF - β1 for 5 days . After treatment , nuclear translocation of NF - κB p65 was assayed by Western blotting in cells treated with recombinant Il - 17b for 2 hours . Nuclear Matrix Protein p84 was used as a loading control of nuclear extracts . f triplicate measurement . In ( B , C , D , F , G , H , I , and J ) , the intensity of each band was quantified using the Image J software . Gapdh , α - tubulin , or p84 was used as a loading control . Relative expression ( RE ) of protein levels in each sample to control 4T1 cells or Il - 17rbDel 4T1 clone is indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6 Figure 6. TGF-β1 secreted from Tregs induces Il-17rb expression of breast cancer cells via Smad2/3/4 signaling. (A) CD4+CD25+ Tregs were isolated from inguinal LN of BALB/c mice injected with 4T1 cells for three weeks, and were transwell cocultured with 4T1 cells in the presence of control IgG or neutralizing antibodies against IL-10 or TGF-β1. After 5-day coculture, 4T1 cells at lower well were analyzed for Il-17rb expression by RT-qPCR analysis. Gapdh was used as internal control. (B and C) Western blotting analyzed Il-17rb in 4T1 cells treated with recombinant TGF-β1 (B)proteins for 5 days.(B and C) Western blotting analyzed Il-17rb in 4T1 cells treated with recombinant IL-10 (C) proteins for 5 days.(D) Western blotting analyzed Il-17rb in shLacZ or shTgfbr1 4T1 cells treated with recombinant TGF-β1 for 5 days.(E) 4T1-injected BALB/c mice were sacrificed at three weeks post initial injection. Total cells isolated from inguinal lymph node tissues were transwell co-cultured with 4T1 cells. After 5-day coculture, soft-agar colony forming activity was determined in co-cultured shLacZ or shTgfbr1 4T1 cells at lower well. (5x102 cells/well, n = 6 wells per group).(F-H) Western blotting analyzed Il-17rb in shLacZ, shSmad2 (F)4T1 cells treated with recombinant TGF-β1 for 5 days.(F-H) Western blotting analyzed Il-17rb in shLacZ,shSmad3 (G)4T1 cells treated with recombinant TGF-β1 for 5 days.(F-H) Western blotting analyzed Il-17rb in shLacZ, shSmad4 (H) 4T1 cells treated with recombinant TGF-β1 for 5 days. (I and J) WT 4T1 (I) or Il-17rbDel 4T1 (J) cells were treated with or without recombinant TGF-β1 for 5 days. After treatment, nuclear translocation of NF-κB p65 was assayed by Western blotting in cells treated with recombinant Il-17b for 2 hours. Nuclear Matrix Protein p84 was used as a loading control of nuclear extracts.f triplicate measurement. In (B, C, D, F, G, H, I, and J), the intensity of each band was quantified using the Image J software. Gapdh, α-tubulin, or p84 was used as a loading control. Relative expression (RE) of protein levels in each sample to control 4T1 cells or Il-17rbDel 4T1 clone is indicated.


Figure 7Figure 7 . IL - 17RB expression is correlated with the elevated prevalence of regulatory T cells in the tumor - draining lymph nodes of breast cancer patients . ( A and C ) Representative IHC images of IL - 17RB ( A ) and Foxp3 ( C ) were taken in breast specimens from primary tumors and their paired LN metastasis specimens . Scale bar : 10 μmFigure 7 . IL - 17RB expression is correlated with the elevated prevalence of regulatory T cells in the tumor - draining lymph nodes of breast cancer patients . ( A and C ) Representative IHC images of IL - 17RB ( A ) and Foxp3 ( C ) were taken in breast specimens from primary tumors and their paired LN metastasis specimens . Scale bar : 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Figure 7. IL-17RB expression is correlated with the elevated prevalence of regulatory T cells in the tumor-draining lymph nodes of breast cancer patients. (A and C) Representative IHC images of IL-17RB (A) and Foxp3 (C) were taken in breast specimens from primary tumors and their paired LN metastasis specimens. Scale bar: 10 μmFigure 7. IL-17RB expression is correlated with the elevated prevalence of regulatory T cells in the tumor-draining lymph nodes of breast cancer patients. (A and C) Representative IHC images of IL-17RB (A) and Foxp3 (C) were taken in breast specimens from primary tumors and their paired LN metastasis specimens. Scale bar: 10 μm.


Figure 8 Figure 8 . Elevated IL - 17RB expression in cancer cells derived from tumor - draining lymph nodes in breast cancer patients correlates with aggressive growth nature . ( A ) Freshly collected humanbreast cancer ( hBC ) specimens ( PT , primary tumors ; LN , paired LN metastasis from the same breast cancer patient ) were digested with collagenase - containing buffer overnight . Depletion of hematopoietic cells by CD45 Dyna beads ( Invitrogen ) enriched the EpCAM + cells for following xenografts . Percentages of EpCAM + cells in human primary breast cancer cells were confirmed by FACS analysis ( Left panel : before enrichment ; Right panel : after enrichment ) . All of the histograms were shown that they were EpCAM expressing ( black line ) , compared to isotype control ( gray filled ) . ( B ) Tumorigenesis assay were determined in NOD / SCID / IL2Rγnull mice orthotopically injected with hBCPT and hBCLN cells . Representative data on day 180 were shown . ( D and E ) H & E stainings and IHC stainings of pan - keratin were shown in the representative cases of fat - pad sections from ( B ) . Scale bar : 1 mm ( left panel ) , 100 μm ( right panel ) . ( D and E ) H & E stainings and IHC stainings of pan - keratin were shown in the representative cases of fat - pad sections from ( B ) . Scale bar : 1 mm ( left panel ) , 100 μm ( right panel ) . ( F ) IHC stainings of IL - 17RB were shown in the representative cases of fat - pad sections from ( B ) . Scale bar : 10 μm . ( G ) The correlation of tumor volumes and IL - 17RB expression ( composite IHC score ) in hBCLN cells - injected NOD / SCID / IL2Rγnull mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8 Figure 8. Elevated IL-17RB expression in cancer cells derived from tumor-draining lymph nodes in breast cancer patients correlates with aggressive growth nature. (A) Freshly collected humanbreast cancer (hBC) specimens (PT, primary tumors; LN, paired LN metastasis from the same breast cancer patient) were digested with collagenase-containing buffer overnight. Depletion of hematopoietic cells by CD45 Dyna beads (Invitrogen) enriched the EpCAM+ cells for following xenografts. Percentages of EpCAM+ cells in human primary breast cancer cells were confirmed by FACS analysis (Left panel: before enrichment; Right panel: after enrichment). All of the histograms were shown that they were EpCAM expressing (black line), compared to isotype control (gray filled). (B) Tumorigenesis assay were determined in NOD/SCID/IL2Rγnull mice orthotopically injected with hBCPT and hBCLN cells. Representative data on day 180 were shown.(D and E) H&E stainings and IHC stainings of pan-keratin were shown in the representative cases of fat-pad sections from (B). Scale bar: 1 mm (left panel), 100 μm (right panel).(D and E) H&E stainings and IHC stainings of pan-keratin were shown in the representative cases of fat-pad sections from (B). Scale bar: 1 mm (left panel), 100 μm (right panel).(F) IHC stainings of IL-17RB were shown in the representative cases of fat-pad sections from (B). Scale bar: 10 μm.(G) The correlation of tumor volumes and IL-17RB expression (composite IHC score) in hBCLN cells-injected NOD/SCID/IL2Rγnull mice.


Figure 2 ( B ) Bar diagram representation of the relative SUMO conjugation activity of Nse2 , full - length Smc5 - Nse2 , ΔHinge / Smc5 - Nse2 , ΔHead / Smc5 - Nse2 and Arm / Smc5 - Nse2 truncation constructs ( schematic representation above ) . Orange bars indicate the presence of ssDNA ( virion ϕx174 ) and red bars absence of ssDNA . Reaction rates were performed at least in three different independent experiments ( see Fig EV2 ) . Data values are mean ± s . e . m . ; and n = 3 technical replicates . Significance was measured by a two - tailed unpaired t - test relative to wild - type . * * P < 0 . 01 ( C ) SYPRO - stained ( left ) and Western blot ( right ) time course SUMO conjugation reaction using and ΔHead / Smc5 - Nse2 truncation construct in the presence of ssDNA . T7 - tagged Smc5 and E1 were immunodetected by an anti - T7 antibody . Reactions were run at 30 ° C and stopped at indicated times by adding SDS - loading buffer ( D ) SYPRO - stained ( left ) and Western blot ( right ) time course SUMO conjugation reaction using and ΔHinge / Smc5 - Nse2 truncation construct in the presence of ssDNA . T7 - tagged Smc5 and E1 were immunodetected by an anti - T7 antibody . Reactions were run at 30 ° C and stopped at indicated times by adding SDS - loading buffer ( E ) Western blot of the time - course SUMO conjugation reaction in the presence of ssDNA ( virion ϕx174 ) using either Nse2 ( left ) or Arm / Smc5 - Nse2 complex ( right ) . The reactions were run in the presence or absence of cNse4 external substrate . Reaction was run at 30 ° C and stopped at indicated minutes by adding SDS - loading buffer ( N - S2 , cNse4 - SUMO2 ; N - 2S2 , cNse4 - 2SUMO2 ; N - 3S2 , cNse4 - 3SUMO2 and pS2 , poly - SUMO2 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 (B) Bar diagram representation of the relative SUMO conjugation activity of Nse2, full-length Smc5-Nse2, ΔHinge/Smc5-Nse2, ΔHead/Smc5-Nse2 and Arm/Smc5-Nse2 truncation constructs (schematic representation above). Orange bars indicate the presence of ssDNA (virion ϕx174) and red bars absence of ssDNA. Reaction rates were performed at least in three different independent experiments (see Fig EV2). Data values are mean ± s.e.m.; and n=3 technical replicates. Significance was measured by a two-tailed unpaired t-test relative to wild-type. **P<0.01  (C) SYPRO-stained (left) and Western blot (right) time course SUMO conjugation reaction using and ΔHead/Smc5-Nse2 truncation construct in the presence of ssDNA. T7-tagged Smc5 and E1 were immunodetected by an anti-T7 antibody. Reactions were run at 30 °C and stopped at indicated times by adding SDS-loading buffer  (D) SYPRO-stained (left) and Western blot (right) time course SUMO conjugation reaction using and ΔHinge/Smc5-Nse2 truncation construct in the presence of ssDNA. T7-tagged Smc5 and E1 were immunodetected by an anti-T7 antibody. Reactions were run at 30 °C and stopped at indicated times by adding SDS-loading buffer  (E) Western blot of the time-course SUMO conjugation reaction in the presence of ssDNA (virion ϕx174) using either Nse2 (left) or Arm/Smc5-Nse2 complex (right). The reactions were run in the presence or absence of cNse4 external substrate. Reaction was run at 30°C and stopped at indicated minutes by adding SDS-loading buffer (N-S2, cNse4-SUMO2; N-2S2, cNse4-2SUMO2; N-3S2, cNse4-3SUMO2 and pS2, poly-SUMO2)


Figure 3 ( D ) Bar diagram representation of the SUMO conjugation rates of activity assays of Arm / Smc5 - Nse2 KE mutants in the presence ( orange bars ) or absence ( red bars ) of ssDNA ( virion ϕx174 ) , relative to wild - type ( set to 1 ) . Reactions rates were performed at least in three different independent experiments . Data values are mean ± s . e . m . and n = 3 technical replicates . Significance was measured by a two - tailed unpaired t - test relative to wild - type . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 (D) Bar diagram representation of the SUMO conjugation rates of activity assays of Arm/Smc5-Nse2 KE mutants in the presence (orange bars) or absence (red bars) of ssDNA (virion ϕx174), relative to wild-type (set to 1). Reactions rates were performed at least in three different independent experiments. Data values are mean ± s.e.m. and n=3 technical replicates. Significance was measured by a two-tailed unpaired t-test relative to wild-type. *P<0.05, **P<0.01, ***P<0.001


Figure 4 ( C ) Degree of conformational changes in CM induced by 60b linear ssDNA . Blue bar , Arm / Smc5 - Nse2 ( wild - type ) ; grey bar , Arm / Smc5 - Nse2 ( K333E / K344E ) ; green bar , Arm / Smc5 - Nse2 ( K764E ) ; pink bar , Arm / Smc5 - Nse2 ( K333E / K344E / K764E ) ; red bar , Arm / Smc5 - Nse2 ( K743E / K745E ) . Reactions were performed at least in three different independent experiments . Data values are mean ± s . d . and n = 3 technical replicates . Significance was measured by a two - tailed unpaired t - test relative to wild - type . * * * * P < 0 . 0001

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 (C) Degree of conformational changes in CM induced by 60b linear ssDNA. Blue bar, Arm/Smc5-Nse2 (wild-type); grey bar, Arm/Smc5-Nse2 (K333E/K344E); green bar, Arm/Smc5-Nse2 (K764E); pink bar, Arm/Smc5-Nse2 (K333E/K344E/K764E); red bar, Arm/Smc5-Nse2 (K743E/K745E). Reactions were performed at least in three different independent experiments. Data values are mean ± s.d. and n=3 technical replicates. Significance was measured by a two-tailed unpaired t-test relative to wild-type. ****P < 0.0001


Figure 5 ( B ) Growth test analysis of wild - type , nse2 - CH and the indicated smc5 - KE mutants ; 10 - fold serial dilutions of the liquid cultures were spotted in YPD and pictures taken after 48 hours ( C ) Same as in ( B ) but using the nse2 - CH , smc5 - 3KE , smc5 - K743 , 745E , smc5 - K743 , 745R , smc5 - 7KE and smc5 - 7KR mutants

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5 (B) Growth test analysis of wild-type, nse2-CH and the indicated smc5-KE mutants; 10-fold serial dilutions of the liquid cultures were spotted in YPD and pictures taken after 48 hours  (C) Same as in (B) but using the nse2-CH, smc5-3KE, smc5-K743,745E, smc5-K743,745R, smc5-7KE and smc5-7KR mutants


Figure 6 ( A ) Protein extracts from exponentially growing wild - type , smc5 - 3KE , smc5 - K743 , 745E , smc5 - 4KE and smc5 - K743 , 745R cells were prepared under denaturing conditions ; 6xHis - Flag tagged SUMO ( HF - SUMO ) was pulled down from protein extracts to purify sumoylated species and analyzed by western blot . A strain with no HF - tag was used as control ; arrow points to unmodified form of the protein , vertical bar to sumoylated forms ( B ) Left , same as in ( A ) , but using wild - type and two independent clones of smc5 - 7KE ; right , same as in ( A ) , but using wild - type , smc5 - 7KE and smc5 - 7KR mutant cells ( C ) Quantification of Smc5 sumoylated species in pull downs from the indicated smc5 - KE mutants , relative to wild - type controls from at least three independent experiments . Boxes , 25 - 75 % data range ; whiskers , total data range ; black bar , median ; grey cross , mean . * * * * P < 0 . 0001 ; * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 . 1 - way ANOVA , n = number of samples analyzed ( D ) Co - immunoprecipitation analysis of the Smc5 - Nse2 interactions . NSE2 - 6HA cells were transformed with centromeric plasmids expressing the indicated SMC5 alleles , and protein extracts from exponentially growing cells subjected to anti - HA immunoprecipitation . A strain with no HA tag was used as control ( E ) Same as in ( D ) , but using SMC6 - 6HA cells ( F ) Quantification of Smc5 - 6HA signal from immunofluorescence on chromosome spreads prepared from exponentially growing cultures of the indicated genotypes . The mean value on wild - type spreads was arbitrarily set to 1 ; each dot represents one nucleus ; red line , median . 1 - way ANOVA , n = number of nuclei analyzed . * * * * P < 0 . 0001

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6 (A) Protein extracts from exponentially growing wild-type, smc5-3KE, smc5-K743,745E, smc5-4KE and smc5-K743,745R cells were prepared under denaturing conditions; 6xHis-Flag tagged SUMO (HF-SUMO) was pulled down from protein extracts to purify sumoylated species and analyzed by western blot. A strain with no HF-tag was used as control; arrow points to unmodified form of the protein, vertical bar to sumoylated forms  (B) Left, same as in (A), but using wild-type and two independent clones of smc5-7KE; right, same as in (A), but using wild-type, smc5-7KE and smc5-7KR mutant cells  (C) Quantification of Smc5 sumoylated species in pull downs from the indicated smc5-KE mutants, relative to wild-type controls from at least three independent experiments. Boxes, 25-75% data range; whiskers, total data range; black bar, median; grey cross, mean. ****P < 0.0001; ***P<0.001; **P < 0.01; *P<0.05. 1-way ANOVA, n = number of samples analyzed  (D) Co-immunoprecipitation analysis of the Smc5-Nse2 interactions. NSE2-6HA cells were transformed with centromeric plasmids expressing the indicated SMC5 alleles, and protein extracts from exponentially growing cells subjected to anti-HA immunoprecipitation. A strain with no HA tag was used as control  (E) Same as in (D), but using SMC6-6HA cells  (F) Quantification of Smc5-6HA signal from immunofluorescence on chromosome spreads prepared from exponentially growing cultures of the indicated genotypes. The mean value on wild-type spreads was arbitrarily set to 1; each dot represents one nucleus; red line, median. 1-way ANOVA, n=number of nuclei analyzed. ****P < 0.0001


Figure 1 ( b ) RNAscope analysis of Lgr5 mRNA in DSS colitis demonstrating loss of Lgr5 transcript ( N = 3 per condition ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . Scale bars = 50µm ( c ) Hematoxylin and Eosin staining of the colonic epithelium following DSS induced damaged . Crypts are dramatically injured by d5 . At d8 through d12 , regenerating crypts are observed . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . Scale bars = 50µm ( d - e ) Compared to untreated controls , DSS treatment ablates Lgr5 - GFP + stem cells and results in functional loss of tdTomato + lineage tracing as early as d5 . Lgr5 - GFP + stem cells reappear by d12 ( N = 3 - 4 per condition ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . Scale bars = 50µm Data are represented as mean ± SD ( e ) analyzed using Student ' s t - test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . ( f ) Effects of DSS colitis injury on RNA expression levels of stem ( Lgr5 and Krt19 ) and progenitor ( Atoh1 and Notch1 ) cell markers . Lgr5 mRNA expression is significantly decreased after exposure to DSS ( N = 6 - 7 per condition ) . Data are represented as mean ± SEM analyzed using Student ' s t - test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Mice body weight ( h ) are comparable between three treatments : DSS + DT ( N = 12 ) , only DSS ( N = 8 ) or only DT ( N = 3 ) . Data are represented as mean ± SEM analyzed using Student ' s t - test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Mice survival ( i ) are comparable between three treatments : DSS + DT ( N = 12 ) , only DSS ( N = 8 ) or only DT ( N = 3 ) . Data are represented as mean ± SEM analyzed using Student ' s t - test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . ( j ) Hematoxylin and Eosin staining of the colonic epithelium shows no morphological difference between DSS alone and DSS + DT treated mice . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . scale bar ( j ) = 500µm . ( k ) The extent of epithelial damage is similar in mice treated with DSS alone versus DSS + DT ( to ablate Lgr5 + stem cells ) ( N = 3 DSS ; N = 6 DSS + DT ) . Data are represented as mean ± SEM analyzed using Student ' s t - test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . ( l ) RNAscope for Lgr5 in Lgr5DTR - GFP mice showing loss of Lgr5 transcripts after DT treatment ( N = 3 - 4 per condition ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . Scale bars =

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(b) RNAscope analysis of Lgr5 mRNA in DSS colitis demonstrating loss of Lgr5 transcript (N=3 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50µm(c) Hematoxylin and Eosin staining of the colonic epithelium following DSS induced damaged. Crypts are dramatically injured by d5. At d8 through d12, regenerating crypts are observed. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50µm(d-e) Compared to untreated controls, DSS treatment ablates Lgr5-GFP+ stem cells and results in functional loss of tdTomato+ lineage tracing as early as d5. Lgr5-GFP+ stem cells reappear by d12 (N=3-4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50µm Data are represented as mean ± SD (e) analyzed using Student's t-test. *P≤0.05, ** P≤0.01.(f) Effects of DSS colitis injury on RNA expression levels of stem (Lgr5 and Krt19) and progenitor (Atoh1 and Notch1) cell markers. Lgr5 mRNA expression is significantly decreased after exposure to DSS (N=6-7 per condition). Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05, ** P≤0.01.Mice body weight (h) are comparable between three treatments: DSS+DT (N=12), only DSS (N=8) or only DT (N=3). Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05, ** P≤0.01.Mice survival (i) are comparable between three treatments: DSS+DT (N=12), only DSS (N=8) or only DT (N=3). Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05, ** P≤0.01.(j) Hematoxylin and Eosin staining of the colonic epithelium shows no morphological difference between DSS alone and DSS+DT treated mice. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. scale bar (j) =500µm.(k) The extent of epithelial damage is similar in mice treated with DSS alone versus DSS+DT (to ablate Lgr5+ stem cells) (N=3 DSS; N=6 DSS+DT). Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05, ** P≤0.01.(l) RNAscope for Lgr5 in Lgr5DTR-GFP mice showing loss of Lgr5 transcripts after DT treatment (N=3-4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50µm


Figure 2 ( b - c ) Krt19 + cells lineage trace entire colonic crypts during both normal homeostasis and injury ( N = 3 - 4 per condition ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines outline the basement membrane . Scale bars ( b ) = 45µm Data are represented as mean ± SEM * P ≤ 0 . 05 , * * P ≤ 0 . 01 . DT ablation of Krt19 + cells ( N ≥ 3 per condition ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines outline the basement membrane . scale bar ( e ) = 30µmDT ablation of Krt19 + cells significantly reduced the number of lineage labeled crypts ( N ≥ 3 per condition ) . Data are represented as mean ± SEM using Student ' s t - test * P ≤ 0 . 05 , * * P ≤ 0 . 01 . ( g ) Mice body weight and survival remain unchanged between DSS and DSS + DT treated groups ( N = 6 control ; N = 7 DT ) . Data are represented as mean ± SEM using Student ' s t - test P ≤ 0 . 05 , * * P ≤ 0 . 01 . Hematoxylin and Eosin staining of the colonic epithelium after DSS versus DSS + DT showing more damage is seen in mice treated with both DSS + DT treatment ( N = 6 control ; N = 7 DT ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines outline the basement membrane . scale bar ( h ) = 1mm . quantification of damaged epithelial area ( i ) , showing more damage is seen in mice treated with both DSS + DT treatment ( N = 6 control ; N = 7 DT ) . Data are represented as mean ± SEM using Student ' s t - test * P ≤ 0 . 05 , * * P ≤ 0 . 01 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(b-c) Krt19+ cells lineage trace entire colonic crypts during both normal homeostasis and injury (N=3-4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines outline the basement membrane. Scale bars (b) =45µm Data are represented as mean ± SEM *P≤0.05, **P≤0.01.DT ablation of Krt19+ cells (N≥3 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines outline the basement membrane. scale bar (e) =30µmDT ablation of Krt19+ cells significantly reduced the number of lineage labeled crypts (N≥3 per condition). Data are represented as mean ± SEM using Student's t-test *P≤0.05, **P≤0.01.(g) Mice body weight and survival remain unchanged between DSS and DSS+DT treated groups (N=6 control; N=7 DT). Data are represented as mean ± SEM using Student's t-test P≤0.05, **P≤0.01.Hematoxylin and Eosin staining of the colonic epithelium after DSS versus DSS+DT showing more damage is seen in mice treated with both DSS+DT treatment (N=6 control; N=7 DT). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines outline the basement membrane. scale bar (h) =1mm.quantification of damaged epithelial area (i), showing more damage is seen in mice treated with both DSS+DT treatment (N=6 control; N=7 DT). Data are represented as mean ± SEM using Student's t-test *P≤0.05, **P≤0.01.


Figure 3 ( b - c ) Notch1 + progenitors are able to generate Lgr5 + stem cells and contribute to epithelial renewal in homeostasis ; however , do not contribute to regeneration after colitis ( N = 4 per condition ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . Scale bars = 50μm . Data are represented as mean ± SD analyzed using two - way ANOVA with Sidak ' s multiple comparisons test . * * * * P ≤ 0 . 0001 . Images of whole mount showing Notch1 lineage labeled crypts . No partially labeled crypts were found after Lgr5 + stem cell ablation . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . Scale bars = 50μm . Images of tissue sections Notch1CreERT2 ; ROSA26tdTomato ; Lgr5DTR - GFP mice ( f ) showing Notch1 lineage labeled crypts . No partially labeled crypts were found after Lgr5 + stem cell ablation . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . Scale bars = 50μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(b-c) Notch1+ progenitors are able to generate Lgr5+ stem cells and contribute to epithelial renewal in homeostasis; however, do not contribute to regeneration after colitis (N=4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars=50μm. Data are represented as mean ± SD analyzed using two-way ANOVA with Sidak's multiple comparisons test. ****P≤0.0001.Images of whole mount showing Notch1 lineage labeled crypts. No partially labeled crypts were found after Lgr5+ stem cell ablation. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars=50μm.Images of tissue sections Notch1CreERT2; ROSA26tdTomato;Lgr5DTR-GFP mice (f) showing Notch1 lineage labeled crypts. No partially labeled crypts were found after Lgr5+ stem cell ablation. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars=50μm.


Figure 4 ( a - b ) Atoh1 + progenitors show stem cell capacity during normal homeostasis and regeneration . At early stages of recovery , d10 - 12 , Atoh1 + cells lineage trace the colonic crypt ( N = 4 per condition ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . Scale bars = 50μm Data are represented as mean ± SD analyzed using two - way ANOVA with Sidak ' s multiple comparisons test * P ≤ 0 . 05 , * * * P ≤ 0 . 001 . ( c - d ) By d30 after DSS , Atoh1 derived stem cells continue to lineage trace complete colonic crypts ( N = 4 per condition ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . Scale bars first two panels of c = 50μm ; scale bars ( last panel of c = 100μm . Data are represented as mean ± SD analyzed using two - way ANOVA with Sidak ' s multiple comparisons test * P ≤ 0 . 05 , * * * P ≤ 0 . 001 . Whole mount ( f ) images of Atoh1 lineage labeled crypts . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . Scale bars = 50μmsection ( g ) images of Atoh1 lineage labeled crypts . Majority of crypts are labeled after Lgr5 + stem cell ablation and remain label with the appearance of Lgr5 + stem cells ( inset , arrowheads ) . Atoh1 lineage labeled crypts remain by d30 . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . scale bars = 100μm . ( h ) Significantly increased labeling of crypt base was observed at d8 in DT treated mice ( N = 4 per condition ) . Data are represented as mean ± SD analyzed using Student ' s t - test with Welch ' s correction * P ≤ 0 . 05 , * * * P ≤ 0 . 001 . ( j - k ) Scattered labeling of Atoh1 + cells in control conditions , while DSS injury lead to Atoh1 lineage labeled crypts lacking Lgr5 + stem cells . Data information : Nuclei are counterstained with Dapi ( blue ) ; white dashed lines indicate basement membrane . Scale bars = 50μm Data are represented as mean ± SD analyzed using Student ' s t - test with Welch ' s correction * P ≤ 0 . 05 , * * * P ≤ 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(a-b) Atoh1+ progenitors show stem cell capacity during normal homeostasis and regeneration. At early stages of recovery, d10-12, Atoh1+ cells lineage trace the colonic crypt (N=4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50μm Data are represented as mean ± SD analyzed using two-way ANOVA with Sidak's multiple comparisons test *P≤0.05, ***P≤0.001.(c-d) By d30 after DSS, Atoh1 derived stem cells continue to lineage trace complete colonic crypts (N=4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars first two panels of c =50μm; scale bars (last panel of c =100μm. Data are represented as mean ± SD analyzed using two-way ANOVA with Sidak's multiple comparisons test *P≤0.05, ***P≤0.001.Whole mount (f) images of Atoh1 lineage labeled crypts. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50μmsection (g) images of Atoh1 lineage labeled crypts. Majority of crypts are labeled after Lgr5+ stem cell ablation and remain label with the appearance of Lgr5+ stem cells (inset, arrowheads). Atoh1 lineage labeled crypts remain by d30. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. scale bars =100μm.(h) Significantly increased labeling of crypt base was observed at d8 in DT treated mice (N=4 per condition). Data are represented as mean ± SD analyzed using Student's t-test with Welch's correction *P≤0.05, ***P≤0.001.(j-k) Scattered labeling of Atoh1+ cells in control conditions, while DSS injury lead to Atoh1 lineage labeled crypts lacking Lgr5+ stem cells. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50μm Data are represented as mean ± SD analyzed using Student's t-test with Welch's correction *P≤0.05, ***P≤0.001.