figf4 ( a ) The proportions of EGFP - positive SK - N - SH cells with EGFP - HDQ74 aggregates or cell death as in Figure 1c , treated with or without 10 μM calpastatin , 50 μM N - acetyl - Leu - Leu - methional ( ALLM ) or 50 μM calpeptin for 48 h post - transfection . DMSO was control for ALLM and calpeptin . Error bars show 95 % confidence interval . ( b ) HeLa cells were transfected with EGFP - HDQ74 construct along with siGLO ( control siRNA ) in presence or absence of calpain 1 or calpain 2 siRNA ( 1 : 3 ratio ) for 96 h . EGFP - and siGLO - positive cells were assessed for aggregation and cell death as in Figure 1c . Error bars show 95 % confidence interval . ( c ) HeLa cells , transfected with siGLO or siRNAs for calpain 1 or calpain 2 for 96 h , were analyzed with anti - calpain 1 or anti - calpain 2 domain III antibodies . ( d ) Clearance of soluble EGFP - HDQ74 in stable PC12 cells as in Figure 1b , treated with drugs ( concentrations in a ) for 96 h . Densitometry is relative to actin . Error bars show s . e . m . ( e ) A53T α - synuclein clearance in undifferentiated or nonmitotic neuronally differentiated ( with 100 ng ml - 1 nerve growth factor for 5 d ) stable PC12 cells as in Figure 1a , treated with or without 10 μM calpastatin for 24 h . Densitometry is relative to actin . Error bars show s . e . m . ( f ) COS - 7 cells , transfected with EGFP - HDQ74 along with empty vector ( pcDNA3 . 1 ) or constitutive active ( CA ) m - calpain ( 1 : 3 ratio ) for 48 h , were assessed for aggregation and cell death as in Figure 1c . Error bars show 95 % confidence interval . ( g ) SK - N - SH cells , transfected with EGFP - HDQ74 along with empty vector ( pcDNA3 . 1 ) or constitutive active m - calpain ( 1 : 3 ratio ) for 4 h , were subsequently treated with DMSO ( control ) , 1 μM verapamil or 1 μM clonidine for 48 h . The proportions of EGFP - positive cells with aggregates were assessed as in Figure 1c . Error bars show 95 % confidence interval . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 .

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

figf4(a) The proportions of EGFP-positive SK-N-SH cells with EGFP-HDQ74 aggregates or cell death as in Figure 1c, treated with or without 10 μM calpastatin, 50 μM N-acetyl-Leu-Leu-methional (ALLM) or 50 μM calpeptin for 48 h post-transfection. DMSO was control for ALLM and calpeptin. Error bars show 95% confidence interval.(b) HeLa cells were transfected with EGFP-HDQ74 construct along with siGLO (control siRNA) in presence or absence of calpain 1 or calpain 2 siRNA (1:3 ratio) for 96 h. EGFP- and siGLO-positive cells were assessed for aggregation and cell death as in Figure 1c. Error bars show 95% confidence interval.(c) HeLa cells, transfected with siGLO or siRNAs for calpain 1 or calpain 2 for 96 h, were analyzed with anti-calpain 1 or anti-calpain 2 domain III antibodies.(d) Clearance of soluble EGFP-HDQ74 in stable PC12 cells as in Figure 1b, treated with drugs (concentrations in a) for 96 h. Densitometry is relative to actin. Error bars show s.e.m.(e) A53T α-synuclein clearance in undifferentiated or nonmitotic neuronally differentiated (with 100 ng ml-1 nerve growth factor for 5 d) stable PC12 cells as in Figure 1a, treated with or without 10 μM calpastatin for 24 h. Densitometry is relative to actin. Error bars show s.e.m.(f) COS-7 cells, transfected with EGFP-HDQ74 along with empty vector (pcDNA3.1) or constitutive active (CA) m-calpain (1:3 ratio) for 48 h, were assessed for aggregation and cell death as in Figure 1c. Error bars show 95% confidence interval.(g) SK-N-SH cells, transfected with EGFP-HDQ74 along with empty vector (pcDNA3.1) or constitutive active m-calpain (1:3 ratio) for 4 h, were subsequently treated with DMSO (control), 1 μM verapamil or 1 μM clonidine for 48 h. The proportions of EGFP-positive cells with aggregates were assessed as in Figure 1c. Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05.


figf5 ( a ) The proportions of EGFP - positive MEFs with aggregates , transfected with EGFP - HDQ74 and pcDNA3 . 1 ( empty vector ) in Atg5 + / + cells , or transfected with EGFP - HDQ74 and pcDNA3 . 1 , WT Atg5 or K130R Atg5 constructs in Atg5 - / - cells for 4 h ( 1 : 3 ratios ) , were treated for next 48 h with or without 10 μM calpastatin . All data are from the same experiment , but odds ratios for reference conditions in specific experiments have been set to 1 , to facilitate comparisons . Atg5 - / - cells had increased EGFP - HDQ74 aggregates compared to Atg5 + / + cells . Error bars show 95 % confidence interval . ( b ) HeLa cells were transfected with siGLO ( control ) with or without calpain 1 siRNA or calpain 2 siRNA ( 1 : 3 ratio ) for 96 h , followed by transfection with EGFP - LC3 construct for 4 h . Cells were fixed after a further 2 h . The proportions of transfected cells with > 5 EGFP - LC3 vesicles were assessed . Error bars show 95 % confidence interval . ( c ) Endogenous LC3 - II levels in SK - N - SH cells treated with or without 10 μM or 30 μM calpastatin for 24 h . ( d ) HeLa cells stably expressing EGFP - LC3 were treated for 4 h with 400 nM bafilomycin A1 in presence or absence of 10 μM calpastatin , 50 μM ALLM or 50 μM calpeptin . Calpain inhibitors were treated for 24 h before bafilomycin A1 addition . EGFP - LC3 - II was detected with anti - EGFP antibody . Data from different calpain inhibitors are from different blots . ( e ) COS - 7 cells , transfected with myc - LC3 and EGFP - C1 with pcDNA3 . 1 ( empty vector ) or constitutively active ( CA ) m - calpain ( 1 : 1 : 3 ratio ) for 4 h , were analyzed after 24 h post - transfection for LC3 - II levels by immunoblotting with anti - myc antibody . Error bars show s . e . m . ( f ) COS - 7 cells , transfected with EGFP - LC3 and pcDNA3 . 1 ( empty vector ) or CA m - calpain ( 1 : 3 ratio ) for 4 h and fixed 24 h post - transfection , were assessed for proportions of EGFP - positive cells with > 5 EGFP - LC3 vesicles . Error bars show 95 % confidence interval . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

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

figf5(a) The proportions of EGFP-positive MEFs with aggregates, transfected with EGFP-HDQ74 and pcDNA3.1 (empty vector) in Atg5+/+ cells, or transfected with EGFP-HDQ74 and pcDNA3.1, WT Atg5 or K130R Atg5 constructs in Atg5-/- cells for 4 h (1:3 ratios), were treated for next 48 h with or without 10 μM calpastatin. All data are from the same experiment, but odds ratios for reference conditions in specific experiments have been set to 1, to facilitate comparisons. Atg5-/- cells had increased EGFP-HDQ74 aggregates compared to Atg5+/+ cells. Error bars show 95% confidence interval.(b) HeLa cells were transfected with siGLO (control) with or without calpain 1 siRNA or calpain 2 siRNA (1:3 ratio) for 96 h, followed by transfection with EGFP-LC3 construct for 4 h. Cells were fixed after a further 2 h. The proportions of transfected cells with >5 EGFP-LC3 vesicles were assessed. Error bars show 95% confidence interval.(c) Endogenous LC3-II levels in SK-N-SH cells treated with or without 10 μM or 30 μM calpastatin for 24 h.(d) HeLa cells stably expressing EGFP-LC3 were treated for 4 h with 400 nM bafilomycin A1 in presence or absence of 10 μM calpastatin, 50 μM ALLM or 50 μM calpeptin. Calpain inhibitors were treated for 24 h before bafilomycin A1 addition. EGFP-LC3-II was detected with anti-EGFP antibody. Data from different calpain inhibitors are from different blots.(e) COS-7 cells, transfected with myc-LC3 and EGFP-C1 with pcDNA3.1 (empty vector) or constitutively active (CA) m-calpain (1:1:3 ratio) for 4 h, were analyzed after 24 h post-transfection for LC3-II levels by immunoblotting with anti-myc antibody. Error bars show s.e.m.(f) COS-7 cells, transfected with EGFP-LC3 and pcDNA3.1 (empty vector) or CA m-calpain (1:3 ratio) for 4 h and fixed 24 h post-transfection, were assessed for proportions of EGFP-positive cells with >5 EGFP-LC3 vesicles. Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


figf6 ( a ) A53T α - synuclein clearance in stable PC12 cells as in Figure 1a , treated with or without 100 nM PACAP , with or without 10 μM calpastatin or 500 μM 2 ′ 5 ′ ddA for 24 h . ( b ) The proportion of EGFP - positive cells with aggregates in SK - N - SH cells as in Figure 1c , treated for 48 h with 1 μM PACAP or 200 μM NF449 . Error bars show 95 % confidence interval . ( c ) Aggregation in COS - 7 cells transfected with either pcDNA3 : 1 ( empty vector ) or constitutively active ( CA ) m - calpain and EGFP - HDQ74 ( 3 : 1 ratio ) for 4 h and then treated with or without 500 μM 2 ′ 5 ′ ddA for 48 h . Error bars show 95 % confidence interval . ( d ) HeLa cells transfected with control or Gsα siRNA for 72 h were analyzed for Gsα levels by immunoblotting with anti - Gsα antibody . Aggregation was assessed in HeLa cells transfected with control or Gsα siRNA and EGFP - HDQ74 for 72 h . Error bars show 95 % confidence interval . ( e ) Endogenous LC3 - II levels in HeLa cells transfected with control or Gsα siRNA for 72 h . ( f ) Endogenous LC3 - II levels in PC12 cells treated with or without 200 μM NF449 for 24 h . ( g ) Endogenous LC3 - II levels in HeLa cells transfected with control or Gsα siRNA for 72 h and treated with or without 400 nM bafilomycin A1 for the last 4 h . Densitometric analysis is relative to actin . Error bars show s . e . m . ( h ) The proportions of EGFP - positive HeLa cells with aggregates , transfected with control or Gsα siRNA for 48 h , and then retransfected together with pcDNA 3 . 1 ( empty vector ) or CA m - calpain and EGFP - HDQ74 ( 3 : 1 ratio ) for a further 48 h . Error bars show 95 % confidence interval . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

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

figf6(a) A53T α-synuclein clearance in stable PC12 cells as in Figure 1a, treated with or without 100 nM PACAP, with or without 10 μM calpastatin or 500 μM 2′5′ddA for 24 h.(b) The proportion of EGFP-positive cells with aggregates in SK-N-SH cells as in Figure 1c, treated for 48 h with 1 μM PACAP or 200 μM NF449. Error bars show 95% confidence interval.(c) Aggregation in COS-7 cells transfected with either pcDNA3:1 (empty vector) or constitutively active (CA) m-calpain and EGFP-HDQ74 (3:1 ratio) for 4 h and then treated with or without 500 μM 2′5′ddA for 48 h. Error bars show 95% confidence interval.(d) HeLa cells transfected with control or Gsα siRNA for 72 h were analyzed for Gsα levels by immunoblotting with anti-Gsα antibody. Aggregation was assessed in HeLa cells transfected with control or Gsα siRNA and EGFP-HDQ74 for 72 h. Error bars show 95% confidence interval.(e) Endogenous LC3-II levels in HeLa cells transfected with control or Gsα siRNA for 72 h.(f) Endogenous LC3-II levels in PC12 cells treated with or without 200 μM NF449 for 24 h.(g) Endogenous LC3-II levels in HeLa cells transfected with control or Gsα siRNA for 72 h and treated with or without 400 nM bafilomycin A1 for the last 4 h. Densitometric analysis is relative to actin. Error bars show s.e.m.(h) The proportions of EGFP-positive HeLa cells with aggregates, transfected with control or Gsα siRNA for 48 h, and then retransfected together with pcDNA 3.1 (empty vector) or CA m-calpain and EGFP-HDQ74 (3:1 ratio) for a further 48 h. Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


figf7 ( a ) The proportions of EGFP - positive COS - 7 cells with aggregates or cell death , after transfection with EGFP - HDQ74 and rheb or pcDNA3 . 1 ( empty vector ) ( 1 : 3 ratio ) for 4 h and treatment with or without 10 μM calpastatin or 50 μM calpeptin for 48 h post - transfection . The control condition for assessing the effect of calpain inhibitors in rheb - transfected cells was taken as 1 . Error bars show 95 % confidence interval . ( b ) The proportions of EGFP - positive COS - 7 cells with > 5 EGFP - LC3 vesicles , transfected with EGFP - LC3 and pcDNA3 . 1 ( empty vector ) or rheb ( 1 : 3 ratio ) for 4 h and treated with or without 10 μM calpastatin or 50 μM calpeptin for 24 h post - transfection . Error bars show 95 % confidence interval . ( c ) The proportions of EGFP - positive COS - 7 cells with aggregates , transfected with EGFP - HDQ74 and constitutively active ( CA ) m - calpain or pcDNA3 . 1 ( empty vector ) ( 1 : 3 ratio ) for 4 h and treated with or without 0 . 2 μM rapamycin for 48 h post - transfection . Error bars show 95 % confidence interval . ( d ) The proportions of EGFP - positive COS - 7 cells with > 5 EGFP - LC3 vesicles , transfected with EGFP - LC3 and CA m - calpain or pcDNA3 . 1 ( empty vector ) ( 1 : 3 ratio ) for 4 h and treated with or without 0 . 2 μM rapamycin for 24 h post - transfection . Error bars show 95 % confidence interval . ( e , f ) The proportions of EGFP - positive COS - 7 cells with aggregates ( e ) and cell death ( f ) as in Figure 1c , treated with or without 0 . 2 μM rapamycin , 10 μM calpastatin or both for 48 h . Error bars show 95 % confidence interval . ( g ) Clearance of soluble EGFP - HDQ74 in stable PC12 cells as in Figure 1b , treated with or without 0 . 2 μM rapamycin , 10 μM calpastatin or both for the 48 h switch - off period . Error bars show s . e . m . ( h ) Clearance of A53T α - synuclein in stable PC12 cells as in Figure 1a , treated with or without 0 . 2 μM rapamycin , 10 μM calpastatin or both for the 8 h switch - off period . Error bars show s . e . m . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

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

figf7(a) The proportions of EGFP-positive COS-7 cells with aggregates or cell death, after transfection with EGFP-HDQ74 and rheb or pcDNA3.1 (empty vector) (1:3 ratio) for 4 h and treatment with or without 10 μM calpastatin or 50 μM calpeptin for 48 h post-transfection. The control condition for assessing the effect of calpain inhibitors in rheb-transfected cells was taken as 1. Error bars show 95% confidence interval.(b) The proportions of EGFP-positive COS-7 cells with >5 EGFP-LC3 vesicles, transfected with EGFP-LC3 and pcDNA3.1 (empty vector) or rheb (1:3 ratio) for 4 h and treated with or without 10 μM calpastatin or 50 μM calpeptin for 24 h post-transfection. Error bars show 95% confidence interval.(c) The proportions of EGFP-positive COS-7 cells with aggregates, transfected with EGFP-HDQ74 and constitutively active (CA) m-calpain or pcDNA3.1 (empty vector) (1:3 ratio) for 4 h and treated with or without 0.2 μM rapamycin for 48 h post-transfection. Error bars show 95% confidence interval.(d) The proportions of EGFP-positive COS-7 cells with >5 EGFP-LC3 vesicles, transfected with EGFP-LC3 and CA m-calpain or pcDNA3.1 (empty vector) (1:3 ratio) for 4 h and treated with or without 0.2 μM rapamycin for 24 h post-transfection. Error bars show 95% confidence interval.(e,f) The proportions of EGFP-positive COS-7 cells with aggregates (e) and cell death (f) as in Figure 1c, treated with or without 0.2 μM rapamycin, 10 μM calpastatin or both for 48 h. Error bars show 95% confidence interval.(g) Clearance of soluble EGFP-HDQ74 in stable PC12 cells as in Figure 1b, treated with or without 0.2 μM rapamycin, 10 μM calpastatin or both for the 48 h switch-off period. Error bars show s.e.m.(h) Clearance of A53T α-synuclein in stable PC12 cells as in Figure 1a, treated with or without 0.2 μM rapamycin, 10 μM calpastatin or both for the 8 h switch-off period. Error bars show s.e.m. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


figf8 ( a ) Western blot showing EGFP - HDQ71 - induced degeneration of rod photoreceptors at 9 d . p . f . as indicated by the decrease in the abundance of of monomeric ( ∼ 36 kDa ) and dimeric ( ∼ 72 kDa ) rhodopsin . Rhodopsin expression is further reduced by 3 μM ( ± ) - Bay K8644 , and recovered upon treatment with 3 μM verapamil , 100 μM 2 ′ 5 ′ ddA , 3 μM clonidine and 1 μM calpastatin . No general toxicity to the zebrafish at these concentrations was observed . ( b ) Expression of EGFP - HDQ71 significantly reduces the levels of rhodopsin expression when compared to wild - type ( WT ) nontransgenic controls . This effect is further abrogated by 3 μM ( ± ) - Bay K8644 , whereas 3 μM verapamil , 100 μM 2 ′ 5 ′ ddA , 3 μM clonidine and 1 μM calpastatin all protected against rod photoreceptor degeneration . Error bars show s . e . m . ( c ) Magnified bright field and EGFP images of zebrafish eye sections , taken at the ventral marginal zone for each of the indicated treatments ( with concentrations as in b ) , are shown . Scale bar , 10 μm . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 . ( d ) The total number of aggregates across 8 × 12 μm sections was counted and compared to the EGFP - HDQ71 DMSO - treated control . 3 μM ( ± ) - Bay K8644 was found to significantly increase the aggregate burden , whereas 3 μM verapamil , 1 μM calpastatin , 3 μM clonidine and 100 μM 2 ′ 5 ′ ddA decreased the aggregate load . As in our cell models , these aggregates were insoluble in 4 % triton / SDS . Error bars show s . e . m . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 .

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

figf8(a) Western blot showing EGFP-HDQ71-induced degeneration of rod photoreceptors at 9 d.p.f. as indicated by the decrease in the abundance of of monomeric (∼36 kDa) and dimeric (∼72 kDa) rhodopsin. Rhodopsin expression is further reduced by 3 μM (±)-Bay K8644, and recovered upon treatment with 3 μM verapamil, 100 μM 2′5′ddA, 3 μM clonidine and 1 μM calpastatin. No general toxicity to the zebrafish at these concentrations was observed. (b) Expression of EGFP-HDQ71 significantly reduces the levels of rhodopsin expression when compared to wild-type (WT) nontransgenic controls. This effect is further abrogated by 3 μM (±)-Bay K8644, whereas 3 μM verapamil, 100 μM 2′5′ddA, 3 μM clonidine and 1 μM calpastatin all protected against rod photoreceptor degeneration. Error bars show s.e.m.(c) Magnified bright field and EGFP images of zebrafish eye sections, taken at the ventral marginal zone for each of the indicated treatments (with concentrations as in b), are shown. Scale bar, 10 μm. ***P 0.001; **P 0.01; *P 0.05. (d) The total number of aggregates across 8 × 12 μm sections was counted and compared to the EGFP-HDQ71 DMSO-treated control. 3 μM (±)-Bay K8644 was found to significantly increase the aggregate burden, whereas 3 μM verapamil, 1 μM calpastatin, 3 μM clonidine and 100 μM 2′5′ddA decreased the aggregate load. As in our cell models, these aggregates were insoluble in 4% triton/SDS. Error bars show s.e.m. ***P 0.001; **P 0.01; *P 0.05.


Figure 1B : CL formation was measured in the presence of 60 nmol MLCL ( 18 : 2 ) 3 , 40 nmol PC ( 18 : 2 ) 2 , 4 µg TAZ , and 0 . 2 mg MBP or 0 . 2 mg Alb . C : CL formation was measured as in B with different amounts of MBP or Alb . G : CL formation was measured in the presence of 40 nmol PC ( 18 : 2 ) 2 , 10 nmol MLCL ( 18 : 2 ) 3 , 8 µg TAZ , 1250 nmol Triton X - 100 ( TX ) , and 5 . 6 nmol bacteriorhodopsin ( BR ) . H : PC formation was measured in the presence of 180 nmol CL ( 18 : 2 ) 4 , 20 nmol LPC16 : 0 , 8 µg TAZ , 20 mM Ca2 + , and 5 . 6 nmol BR . J : Transacylations were measured as in I in the presence of 0 . 9 g BR / g lipid . The ratio of total linoleoyl transfer over total oleoyl transfer was calculated .

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 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

Figure 1B: CL formation was measured in the presence of 60 nmol MLCL(18:2)3, 40 nmol PC(18:2)2, 4 µg TAZ, and 0.2 mg MBP or 0.2 mg Alb. C: CL formation was measured as in B with different amounts of MBP or Alb. G: CL formation was measured in the presence of 40 nmol PC(18:2)2, 10 nmol MLCL(18:2)3, 8 µg TAZ, 1250 nmol Triton X-100 (TX), and 5.6 nmol bacteriorhodopsin (BR). H: PC formation was measured in the presence of 180 nmol CL(18:2)4, 20 nmol LPC16:0, 8 µg TAZ, 20 mM Ca2+, and 5.6 nmol BR. J: Transacylations were measured as in I in the presence of 0.9 g BR/ g lipid. The ratio of total linoleoyl transfer over total oleoyl transfer was calculated.


Figure 2A : Inner ( IM ) and outer ( OM ) mitochondrial membranes were traced in randomly collected electron micrographs in order to calculate the IM / OM surface area ratio . Multiple micrographs were analyzed per biological replica .

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

Figure 2A: Inner (IM) and outer (OM) mitochondrial membranes were traced in randomly collected electron micrographs in order to calculate the IM/OM surface area ratio. Multiple micrographs were analyzed per biological replica.


Figure 3A : Mitochondrial membranes were traced in electron micrographs to determine the IM / OM surface area ratio . Tracings of the IM are shown in yellow boxes . C : Protein abundances were measured in adult hearts by quantitative Western blotting with fluorescence - labeled secondary antibodies . The abundances of IM proteins ( NDUFB6 , UQCR2 , ATP5A1 ) are expressed relative to the abundance of the OM protein TOM70 .

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

Figure 3A: Mitochondrial membranes were traced in electron micrographs to determine the IM/OM surface area ratio. Tracings of the IM are shown in yellow boxes.C: Protein abundances were measured in adult hearts by quantitative Western blotting with fluorescence-labeled secondary antibodies. The abundances of IM proteins (NDUFB6, UQCR2, ATP5A1) are expressed relative to the abundance of the OM protein TOM70.


Figure 4Tissues were analyzed by quantitative electron microscopy . Multiple micrographs were analyzed per biological replica F , G : Tissues were analyzed by label - free quantitative proteomics . C I - V , complex I - V ; CAC , citric acid cycle enzymes ; MICOS , contact site complex ; mt TA , mitochondrial transaminases ; NS , not significant ; PDH pyruvate dehydrogenase ; VDAC , voltage - dependent anion channel

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

Figure 4Tissues were analyzed by quantitative electron microscopy. Multiple micrographs were analyzed per biological replica F, G: Tissues were analyzed by label-free quantitative proteomics. C I-V, complex I-V; CAC, citric acid cycle enzymes; MICOS, contact site complex; mt TA, mitochondrial transaminases; NS, not significant; PDH pyruvate dehydrogenase; VDAC, voltage-dependent anion channel


Figure 1A . Immunofluorescence of SATB2 ( green ) and the GSC marker SOX2 or OLIG2 ( red ) on frozen tissue sections of human GBM surgical specimens . SATB2 is preferentially expressed by GSCs in human GBMs . Scale bar , 25 μm . B . Quantification of the fraction of SATB2 + cells in SOX2 + or OLIG2 + cells in human GBMs . More than 90 % SOX2 + or OLIG2 + cells showed SATB2 staining . n = 3 GBMs . C . qPCR analysis of SATB2 mRNA expression in GSCs and matched non - stem tumor cells ( NSTCs ) ( n = 5 ) . D . Immunoblot analysis of SATB2 , SOX2 and OLIG2 expression in cell lysates of GSCs and matched NSTCs . E . Immunofluorescenc of SATB2 ( green ) and SOX2 ( red ) in T3359 GSCs and matched NSTCs . Scale bar , 50 μm . F . qPCR analysis of SATB2 mRNA expression in GSCs and neural progenitor cells ( NPCs ) ( n = 3 ) . G . Immunoblot analysis of SATB2 and SOX2 expression in cell lysates of GSCs and NPCs . H . Immunoblot analysis of SATB2 , GSC marker SOX2 and differentiation marker GFAP expression during serum - induced GSC differentiation .

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 B-PANEL_START 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 B-PANEL_START 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

Figure 1A. Immunofluorescence of SATB2 (green) and the GSC marker SOX2 or OLIG2 (red) on frozen tissue sections of human GBM surgical specimens. SATB2 is preferentially expressed by GSCs in human GBMs. Scale bar, 25 μm. B. Quantification of the fraction of SATB2+ cells in SOX2+ or OLIG2+ cells in human GBMs. More than 90% SOX2+ or OLIG2+ cells showed SATB2 staining. n=3 GBMs. C. qPCR analysis of SATB2 mRNA expression in GSCs and matched non-stem tumor cells (NSTCs) (n=5).D. Immunoblot analysis of SATB2, SOX2 and OLIG2 expression in cell lysates of GSCs and matched NSTCs.E. Immunofluorescenc of SATB2 (green) and SOX2 (red) in T3359 GSCs and matched NSTCs. Scale bar, 50 μm.F. qPCR analysis of SATB2 mRNA expression in GSCs and neural progenitor cells (NPCs) (n=3).G. Immunoblot analysis of SATB2 and SOX2 expression in cell lysates of GSCs and NPCs.H. Immunoblot analysis of SATB2, GSC marker SOX2 and differentiation marker GFAP expression during serum-induced GSC differentiation.


Figure 2A . Immunoblot analysis of SATB2 expression in GSCs transduced with lentiviral - mediated non - targeting shRNA ( shNT ) or SATB2 shRNA ( shSATB2 ) . B . Cell viability of GSCs transduced with shNT or shSATB2 ( n = 5 ) . C . EdU incorporation assay of GSCs transduced with shNT or shSATB2 . Scale bar : 50 μm . D . Quantification of ( C ) showing the percentage of EdU + cells ( n = 5 ) . E . Tumorsphere images of GSCs transduced with shNTor shSATB2 . Scale bar : 100 μm . Quantification of the diameter of tumorspheres formed by GSCs expressing shNT or shSATB2 ( F : n = 9 ; G : n = 5 ) . G . Quantification of the number of tumorspheres formed by GSCs expressing shNT or shSATB2 ( F : n = 9 ; G : n = 5 ) . H . In vitro limiting dilution analysis of the tumorsphere formations of GSCs expressing shNT or shSATB2 . Silencing SATB2 attenuated the self - renewal capacity of GSCs .

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 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 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 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 O O O

Figure 2A. Immunoblot analysis of SATB2 expression in GSCs transduced with lentiviral-mediated non-targeting shRNA (shNT) or SATB2 shRNA (shSATB2).B. Cell viability of GSCs transduced with shNT or shSATB2 (n=5).C. EdU incorporation assay of GSCs transduced with shNT or shSATB2. Scale bar: 50 μm. D. Quantification of (C) showing the percentage of EdU+ cells (n=5). E. Tumorsphere images of GSCs transduced with shNTor shSATB2. Scale bar: 100 μm. Quantification of the diameter of tumorspheres formed by GSCs expressing shNT or shSATB2 (F: n=9; G: n=5).G. Quantification of the number of tumorspheres formed by GSCs expressing shNT or shSATB2 (F: n=9; G: n=5).H. In vitro limiting dilution analysis of the tumorsphere formations of GSCs expressing shNT or shSATB2. Silencing SATB2 attenuated the self-renewal capacity of GSCs.


Figure 3 A . Bioluminescent images of the GBM xenografts derived from the luciferase - labeled T3359 GSCs expressing NT or SATB2 shRNA . Representative images on day 21 posttransplantation are shown ( n = 5 mice per group ) . Silencing SATB2 significantly delayed GBM growth . C . Kaplan - Meier survival curves of mice intracranially implanted with T3359 GSCs expressing shNT or shSATB2 ( shNT : n = 7 mice ; shSATB2 - 1 or shSATB2 - 2 : n = 5 mice ) . Median survival : shNT , 28 days ; shSATB2 - 1 , 48 days ; shSATB2 - 2 , 45 Days . Animals bearing GSC - derived xenografts expressing SATB2 shRNA survived longer than the control animals . D . Immunofluorescence of SATB2 ( Green ) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 5 tumors per group ) . Scale bar : 40 μm . E . Quantification of SATB2 intensity in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 5 tumors per group ) . F . Immunofluorescence of Ki67 ( Green ) in tumor xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 5 tumors per group ) . Scale bar : 40 μm . G . Quantification of Ki67 positive cells in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 5 tumors per group ) . H . Immunofluorescence of SOX2 ( Red ) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 4 tumors per group ) . Scale bar : 40 μm . I . Quantification of SOX2 positive cells in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 4 tumors per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Bioluminescent images of the GBM xenografts derived from the luciferase-labeled T3359 GSCs expressing NT or SATB2 shRNA. Representative images on day 21 posttransplantation are shown (n=5 mice per group). Silencing SATB2 significantly delayed GBM growth . C. Kaplan-Meier survival curves of mice intracranially implanted with T3359 GSCs expressing shNT or shSATB2 (shNT: n=7 mice; shSATB2-1 or shSATB2-2: n=5 mice). Median survival: shNT, 28 days; shSATB2-1, 48 days; shSATB2-2, 45 Days. Animals bearing GSC-derived xenografts expressing SATB2 shRNA survived longer than the control animals.  D. Immunofluorescence of SATB2 (Green) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group). Scale bar: 40 μm. E. Quantification of SATB2 intensity in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group).  F. Immunofluorescence of Ki67 (Green) in tumor xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group). Scale bar: 40 μm. G. Quantification of Ki67 positive cells in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group).  H. Immunofluorescence of SOX2 (Red) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=4 tumors per group). Scale bar: 40 μm. I. Quantification of SOX2 positive cells in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=4 tumors per group).


Figure 4 A . Heatmap analysis of differentially expressed genes between SATB2 silencing H2S GSCs ( shSATB2 ) and control H2S GSCs ( shNT ) . Differentially expressed genes had a 1 . 8 - fold or greater expression difference . Among differentially expressed genes , 160 are upregulated and 185 are downregulated . C . qPCR analysis of FOXM1 and SATB2 mRNA expression in GSCs transduced with shNT or shSATB2 ( n = 3 ) . D . Immunoblot analysis of FOXM1 and SATB2 expression in GSCs transduced with shNT or shSATB2 . E . Immunoblot analysis of FOXM1 and SATB2 expression in tumor xenografts - derived from T3359 GSCs expressing shNT or shSATB2 . F . Heatmap analysis of FOXM1 downstream targets between SATB2 silencing H2S GSCs ( shSATB2 ) and control H2S GSCs ( shNT ) from microarray analysis . Differentially expressed genes had a 1 . 5 - fold or greater expression difference . G . qPCR analysis of FOXM1 downstream targets in H2S GSCs transduced with shNT or shSATB2 ( n = 3 ) .

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

Figure 4 A. Heatmap analysis of differentially expressed genes between SATB2 silencing H2S GSCs (shSATB2) and control H2S GSCs (shNT). Differentially expressed genes had a 1.8-fold or greater expression difference. Among differentially expressed genes, 160 are upregulated and 185 are downregulated. C. qPCR analysis of FOXM1 and SATB2 mRNA expression in GSCs transduced with shNT or shSATB2 (n=3).D. Immunoblot analysis of FOXM1 and SATB2 expression in GSCs transduced with shNT or shSATB2.E. Immunoblot analysis of FOXM1 and SATB2 expression in tumor xenografts-derived from T3359 GSCs expressing shNT or shSATB2. F. Heatmap analysis of FOXM1 downstream targets between SATB2 silencing H2S GSCs (shSATB2) and control H2S GSCs (shNT) from microarray analysis. Differentially expressed genes had a 1.5-fold or greater expression difference.  G. qPCR analysis of FOXM1 downstream targets in H2S GSCs transduced with shNT or shSATB2 (n=3).


Figure 5 A . Immunoblot analysis of FOXM1 and SATB2 expression in T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 . B . Cell viability assay of T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( n = 5 ) . Ectopic expression of FOXM1 restored the cell proliferation impaired by SATB2 silencing . C . Tumorsphere number of T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( n = 5 ) . Ectopic expression of FOXM1 restored the tumorsphere formation of GSCs impaired by SATB2 silencing . In vivo bioluminescent images of the tumor xenografts derived from luciferase - labeled T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 5 mice ; shNT + FOXM1 : n = 4 mice ; shSATB2 : n = 5 mice ; shSATB2 + FOXM1 : n = 5 mice ) . Representative images on day 21 posttransplantation are shown . Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly restored GBM tumor growth . E . In vivo bioluminescent images or quantification of the tumor xenografts derived from luciferase - labeled T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 5 mice ; shNT + FOXM1 : n = 4 mice ; shSATB2 : n = 5 mice ; shSATB2 + FOXM1 : n = 5 mice ) . Representative images on day 21 posttransplantation are shown . Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly restored GBM tumor growth . F . Kaplan - Meier survival curves of mice intracranially implanted with T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 8 mice ; shNT + FOXM1 : n = 7 mice ; shSATB2 : n = 6 mice ; shSATB2 + FOXM1 : n = 8 mice ) . Median survival : shNT , 30 days ; shNT + FOXM1 , 28 days ; shSATB2 , 50 . 5 Days ; shSATB2 + FOXM1 , 34 days . Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly attenuated the increased survival of mice bearing the GSC - derived GBMs . G . Immunofluorescence of Ki67 ( Green ) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 6 tumors ; shNT + FOXM1 : n = 5 tumors ; shSATB2 : n = 6 tumors ; shSATB2 + FOXM1 : n = 6 tumors ) . Scale bar : 40 μm . H . Quantification of Ki67 positive cells in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 6 tumors ; shNT + FOXM1 : n = 5 tumors ; shSATB2 : n = 6 tumors ; shSATB2 + FOXM1 : n = 6 tumors ) . I . Immunofluorescence of SOX2 ( Red ) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 6 tumors ; shNT + FOXM1 : n = 5 tumors ; shSATB2 : n = 6 tumors ; shSATB2 + FOXM1 : n = 6 tumors ) . Scale bar : 40 μm . J . Quantification of SOX2 positive cells in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 6 tumors ; shNT + FOXM1 : n = 5 tumors ; shSATB2 : n = 6 tumors ; shSATB2 + FOXM1 : n = 6 tumors ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunoblot analysis of FOXM1 and SATB2 expression in T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2.  B. Cell viability assay of T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (n=5). Ectopic expression of FOXM1 restored the cell proliferation impaired by SATB2 silencing.  C. Tumorsphere number of T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (n=5). Ectopic expression of FOXM1 restored the tumorsphere formation of GSCs impaired by SATB2 silencing.  In vivo bioluminescent images of the tumor xenografts derived from luciferase-labeled T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=5 mice; shNT+FOXM1: n=4 mice; shSATB2: n=5 mice; shSATB2+ FOXM1: n=5 mice). Representative images on day 21 posttransplantation are shown. Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly restored GBM tumor growth.  E. In vivo bioluminescent images or quantification of the tumor xenografts derived from luciferase-labeled T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=5 mice; shNT+FOXM1: n=4 mice; shSATB2: n=5 mice; shSATB2+ FOXM1: n=5 mice). Representative images on day 21 posttransplantation are shown. Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly restored GBM tumor growth.  F. Kaplan-Meier survival curves of mice intracranially implanted with T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=8 mice; shNT+FOXM1: n=7 mice; shSATB2: n=6 mice; shSATB2+ FOXM1: n=8 mice). Median survival: shNT, 30 days; shNT+FOXM1, 28 days; shSATB2, 50.5 Days; shSATB2+FOXM1, 34 days. Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly attenuated the increased survival of mice bearing the GSC-derived GBMs.  G. Immunofluorescence of Ki67 (Green) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors). Scale bar: 40 μm. H. Quantification of Ki67 positive cells in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors).  I. Immunofluorescence of SOX2 (Red) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors). Scale bar: 40 μm. J. Quantification of SOX2 positive cells in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors).


Figure 6 B . ChIP assays with the SATB2 antibody or IgG using GSCs , NSTCs and NPCs . PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus . Note that abundant SATB2 binds to the MAR of FOXM1 gene locus in GSCs . C . qPCR analysis of ChIP assays with the SATB2 antibody or IgG using GSCs transduced with shNT or shSATB2 ( n = 3 ) . PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus . Silencing SATB2 decreased its binding amount to the MAR of FOXM1 gene locus . D . CoIP assays of endogenous protein interaction in GSCs . Immunoblots of precipitated proteins or total lysates were performed using indicated antibodies . Note that SATB2 associates with endogenous CBP while not P300 . E . qPCR analysis of ChIP assays with the CBP antibody or IgG using GSCs transduced with shNT or shSATB2 ( n = 3 ) . PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus . Silencing SATB2 reduced the binding of CBP to the MAR of FOXM1 gene locus . F . qPCR analysis of ChIP assays with the indicated antibody ( AcH3K18 , AcH3K27 , AcH4 ) using T3359 GSCs transduced with shNT or shSATB2 ( n = 3 ) . PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus . Silencing SATB2 reduced acetylation of H3K18 , H3K27 and H4 levels on the MAR of FOXM1 locus . G . qPCR analysis of FOXM1 mRNA expression in T3359 GSCs transduced with shSATB2 or shCBP or both ( n = 3 ) . H . Immunoblot analysis of FOXM1 expression in T3359 GSCs transduced with shSATB2 or shCBP or both .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O 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 B. ChIP assays with the SATB2 antibody or IgG using GSCs, NSTCs and NPCs. PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus. Note that abundant SATB2 binds to the MAR of FOXM1 gene locus in GSCs.  C. qPCR analysis of ChIP assays with the SATB2 antibody or IgG using GSCs transduced with shNT or shSATB2 (n=3). PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus. Silencing SATB2 decreased its binding amount to the MAR of FOXM1 gene locus. D. CoIP assays of endogenous protein interaction in GSCs. Immunoblots of precipitated proteins or total lysates were performed using indicated antibodies. Note that SATB2 associates with endogenous CBP while not P300. E. qPCR analysis of ChIP assays with the CBP antibody or IgG using GSCs transduced with shNT or shSATB2 (n=3). PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus. Silencing SATB2 reduced the binding of CBP to the MAR of FOXM1 gene locus.  F. qPCR analysis of ChIP assays with the indicated antibody (AcH3K18, AcH3K27, AcH4) using T3359 GSCs transduced with shNT or shSATB2 (n=3). PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus. Silencing SATB2 reduced acetylation of H3K18, H3K27 and H4 levels on the MAR of FOXM1 locus.  G. qPCR analysis of FOXM1 mRNA expression in T3359 GSCs transduced with shSATB2 or shCBP or both (n=3).  H. Immunoblot analysis of FOXM1 expression in T3359 GSCs transduced with shSATB2 or shCBP or both.


Figure 7 A . Cell viability of T3359 GSCs treated with indicated doses of C646 or the vehicle control ( n = 5 ) . B . Tumorsphere images of T3359 GSCs treated with indicated doses of C646 or the vehicle control for 6 days . Scale bar : 100 μm . C . qPCR analysis of SATB2 , FOXM1 and FOXM1 downstream targets in T3359 GSCs treated with indicated doses of C646 or the vehicle control for 24 hours ( n = 3 ) . E . Bioluminescent imaging of tumor growth in mice bearing xenografts derived from the luciferase - labeled T3359 GSCs treated with C646 or the vehicle control at indicated days after GSC transplantation ( n = 5 mice per group ) . F . Quantification of tumor growth from ( E ) ( n = 5 mice per group ) . G . Kaplan - Meier survival curves of mice bearing T3359 GSC - derived xenografts treated with C646 or the vehicle control ( Vehicle control : n = 9 mice ; C646 : n = 8 mice ) . Median survival : Vehicle control , 27 days ; C646 , 33 . 5 days . H . Immunofluorescence of Ki67 ( Green ) in T3359 GSC - derived xenografts from mice treated with with C646 or the vehicle control ( n = 6 tumors per group ) . Scale bar : 40 μm . I . Quantification of Ki67 positive cells in T3359 GSC - derived xenografts from mice treated with with C646 or the vehicle control ( n = 6 tumors per group ) .

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

Figure 7 A. Cell viability of T3359 GSCs treated with indicated doses of C646 or the vehicle control (n=5).  B. Tumorsphere images of T3359 GSCs treated with indicated doses of C646 or the vehicle control for 6 days. Scale bar: 100 μm.  C. qPCR analysis of SATB2, FOXM1 and FOXM1 downstream targets in T3359 GSCs treated with indicated doses of C646 or the vehicle control for 24 hours (n=3). E. Bioluminescent imaging of tumor growth in mice bearing xenografts derived from the luciferase-labeled T3359 GSCs treated with C646 or the vehicle control at indicated days after GSC transplantation (n=5 mice per group). F. Quantification of tumor growth from (E) (n=5 mice per group). G. Kaplan-Meier survival curves of mice bearing T3359 GSC-derived xenografts treated with C646 or the vehicle control (Vehicle control: n=9 mice; C646: n= 8 mice). Median survival: Vehicle control, 27 days; C646, 33.5 days.  H. Immunofluorescence of Ki67 (Green) in T3359 GSC-derived xenografts from mice treated with with C646 or the vehicle control (n=6 tumors per group). Scale bar: 40 μm. I. Quantification of Ki67 positive cells in T3359 GSC-derived xenografts from mice treated with with C646 or the vehicle control (n=6 tumors per group).


Figure 1A Left , time lapse confocal images of PHcrac ( red ) and LimE ( green ) in waves at the basal surface of a giant cell . Right , intensity plot across the blue dotted arrow in image " 30 sec " . Various biosensors in excitable waves . snapshots of biosensors ( top ) and intensity plots ( bottom ) across the blue dotted arrows , with black plots showing the corresponding biosensors in images above , red ( B ) showing PHcrac in the same cell , Scatter plots of biosensor peak locations relative to the peak of PHcrac ( C ) across propagating waves ( blue solid lines indicate median ) . Positive values indicate that the peak precedes that of PHcrac along the direction of wave propagation . n = 26 , 26 , 24 , 21 , 21 , 32 cells for RalGDS , RBD , PKBA , , respectively . Scale bars in images above represent 20 μm . Grey arrows in all plots represent the direction of wave propagation . Various biosensors in excitable waves . D , snapshots of biosensors ( top ) and intensity plots ( bottom ) across the blue dotted arrows , with black plots showing the corresponding biosensors in images above , green ( D ) showing LimE . E , Scatter plots of biosensor peak locations relative to the peak of or the first peak of LimE ( E ) across propagating waves ( blue solid lines indicate median ) . Positive values indicate that the peak precedes that of LimE ) along the direction of wave propagation . n = 26 , 26 , 24 , 21 , 21 , 32 cells for , RacGEF1 , PAK1 - GBD , and Coronin , respectively . Scale bars in images above represent 20 μm . Grey arrows in all plots represent the direction of wave propagation . F Left , confocal images of PHcrac ( red ) and PAK1 - GBD ( green ) in the same cells treated with LatA . Scale bars represent 10 μm . Right , temporal profiles of normalized mean cytosolic intensities of PHcrac ( red ) and PAK1 - GBD ( green ) ( mean ± s . e . m . , n = 18 cells ) in response to cAMP .

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 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 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 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

Figure 1A Left, time lapse confocal images of PHcrac (red) and LimE (green) in waves at the basal surface of a giant cell. Right, intensity plot across the blue dotted arrow in image "30 sec".Various biosensors in excitable waves. snapshots of biosensors (top) and intensity plots (bottom) across the blue dotted arrows, with black plots showing the corresponding biosensors in images above, red (B) showing PHcrac in the same cell, Scatter plots of biosensor peak locations relative to the peak of PHcrac (C) across propagating waves (blue solid lines indicate median). Positive values indicate that the peak precedes that of PHcrac along the direction of wave propagation. n=26, 26, 24, 21, 21, 32 cells for RalGDS, RBD, PKBA, , respectively. Scale bars in images above represent 20 μm. Grey arrows in all plots represent the direction of wave propagation.Various biosensors in excitable waves. D, snapshots of biosensors (top) and intensity plots (bottom) across the blue dotted arrows, with black plots showing the corresponding biosensors in images above, green (D) showing LimE. E, Scatter plots of biosensor peak locations relative to the peak of or the first peak of LimE (E) across propagating waves (blue solid lines indicate median). Positive values indicate that the peak precedes that of LimE) along the direction of wave propagation. n=26, 26, 24, 21, 21, 32 cells for , RacGEF1, PAK1-GBD, and Coronin, respectively. Scale bars in images above represent 20 μm. Grey arrows in all plots represent the direction of wave propagation.F Left, confocal images of PHcrac (red) and PAK1-GBD (green) in the same cells treated with LatA. Scale bars represent 10 μm. Right, temporal profiles of normalized mean cytosolic intensities of PHcrac (red) and PAK1-GBD (green) (mean±s.e.m., n=18 cells) in response to cAMP.


Figure 2A Confocal image of LimE in waves ( scale bar , 20 μm ) . B Top , kymograph of LimE in a fixed region across which a piece of wave propagates , with white arrows pointing to the same puncta . Bottom , box plot of puncta ' s lifetime ( n = 100 puncta ) . H A two - dimensional snapshot of a propagating wave ( direction of propagation indicated with the dashed arrow ) . A line scan corresponding to the STEN - CEN profile is shown on the right .

O B-PANEL_START 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 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

Figure 2A Confocal image of LimE in waves (scale bar, 20 μm).B Top, kymograph of LimE in a fixed region across which a piece of wave propagates, with white arrows pointing to the same puncta. Bottom, box plot of puncta's lifetime (n=100 puncta).H A two-dimensional snapshot of a propagating wave (direction of propagation indicated with the dashed arrow). A line scan corresponding to the STEN-CEN profile is shown on the right.


Figure 3A Time lapse confocal images of PHcrac ( top ) and LimE ( bottom ) in the same giant cell ( scale bar , 20 μm ) . White arrows point to wave breaking events .

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

Figure 3A Time lapse confocal images of PHcrac (top) and LimE (bottom) in the same giant cell (scale bar, 20 μm). White arrows point to wave breaking events.


Figure 4E Time lapse confocal images of LimE at the basal surface of a giant cell , which recruits Inp54p to membrane induced by rapamycin at time 0 . The white arrow ( - 45 s ) points to a nascent wave before Inp54p recruitment , while the red ( 60 s ) arrow after . F Color coded overlays of LimE before and after Inp54p recruitment . White arrows point to nascent waves at time 0 . G Kymographs of LimE in regions outlined by dotted line boxes in f . J Time lapse confocal images of PHcrac at the bottom of giant cells under LatA treatment , where rapamycin is added at time 0 to induce Inp54p recruitment . White arrows point to wave patches right before rapamycin addition . K Confocal image of PI5K ( left ) and LimE ( right ) at the bottom of the same cell . All scale bars in images represent 20 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 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 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 4E Time lapse confocal images of LimE at the basal surface of a giant cell, which recruits Inp54p to membrane induced by rapamycin at time 0. The white arrow (-45 s) points to a nascent wave before Inp54p recruitment, while the red (60 s) arrow after.F Color coded overlays of LimE before and after Inp54p recruitment. White arrows point to nascent waves at time 0. G Kymographs of LimE in regions outlined by dotted line boxes in f.J Time lapse confocal images of PHcrac at the bottom of giant cells under LatA treatment, where rapamycin is added at time 0 to induce Inp54p recruitment. White arrows point to wave patches right before rapamycin addition.K Confocal image of PI5K (left) and LimE (right) at the bottom of the same cell. All scale bars in images represent 20 μm.


Figure 5A Time lapse confocal images of LimE at the basal surface of a giant cell , which recruits PKBA to membrane induced by rapamycin at time 0 . B T - stack of LimE in the region outlined by the colored line box in A . C Box plots of mean wave speed ( left ) , mean wave patch area ( middle ) , and average number of wave patches per frame ( right ) before and after PKBA recruitment ( red bars indicate median , n = 24 cells ) . Error bars are from the student ' s t - test . D Time lapse confocal images of PHcrac at the basal surface of a giant cell , which recruits PKBA to membrane induced by rapamycin at time 0 . E Confocal images of PHcrac ( left ) and PHcrac merged with LimE ( middle , PHcrac as red , LimE as green ) in a giant cell after steady PKBA recruitment . Right , intensity plots of PHcrac ( red ) and LimE ( green ) across the blue box . F Kymograph of PHcrac ( red ) merged with LimE ( green ) along the white dashed line from E . G Time lapse confocal images of PHcrac at the bottom of a giant cell treated with LatA , where rapamycin is added at time 0 to induce PKBA recruitment . All scale bars above represent 20 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 B-PANEL_START 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 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 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 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

Figure 5A Time lapse confocal images of LimE at the basal surface of a giant cell, which recruits PKBA to membrane induced by rapamycin at time 0.B T-stack of LimE in the region outlined by the colored line box in A.C Box plots of mean wave speed (left), mean wave patch area (middle), and average number of wave patches per frame (right) before and after PKBA recruitment (red bars indicate median, n=24 cells). Error bars are from the student's t-test.D Time lapse confocal images of PHcrac at the basal surface of a giant cell, which recruits PKBA to membrane induced by rapamycin at time 0.E Confocal images of PHcrac (left) and PHcrac merged with LimE (middle, PHcrac as red, LimE as green) in a giant cell after steady PKBA recruitment. Right, intensity plots of PHcrac (red) and LimE (green) across the blue box.F Kymograph of PHcrac (red) merged with LimE (green) along the white dashed line from E.G Time lapse confocal images of PHcrac at the bottom of a giant cell treated with LatA, where rapamycin is added at time 0 to induce PKBA recruitment. All scale bars above represent 20 μm.


Figure 6A Left , time lapse confocal images of LimE in single cells following RacGEF1ΔN recruitment , induced by rapamycin addition at time 0 . Right , temporal profile of normalized mean LimE intensity at cell cortex ( mean ± s . e . m . , n = 20 cells ) . B Time lapse confocal images of LimE at the basal surface of a giant cell , which recruits RacGEF1ΔN to membrane induced by rapamycin at time 0 . C T - stack of LimE in the region outlined by the colored line box in B . D Right , kymographs of LimE along a line across giant cells after RacGEF1ΔN recruitment . Top left , confocal image with a dotted line as an example used to make kymographs . White arrows in image and kymographs point to examples of actin puncta . Bottom left , box plot of lifetime of puncta ( red bar indicates median , n = 100 puncta from 10 cells ) . All scale bars above represent 20 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 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 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

Figure 6A Left, time lapse confocal images of LimE in single cells following RacGEF1ΔN recruitment, induced by rapamycin addition at time 0. Right, temporal profile of normalized mean LimE intensity at cell cortex (mean±s.e.m., n=20 cells).B Time lapse confocal images of LimE at the basal surface of a giant cell, which recruits RacGEF1ΔN to membrane induced by rapamycin at time 0.C T-stack of LimE in the region outlined by the colored line box in B.D Right, kymographs of LimE along a line across giant cells after RacGEF1ΔN recruitment. Top left, confocal image with a dotted line as an example used to make kymographs. White arrows in image and kymographs point to examples of actin puncta. Bottom left, box plot of lifetime of puncta (red bar indicates median, n=100 puncta from 10 cells). All scale bars above represent 20 μm.


Figure 7A Time lapse confocal images of PHcrac at the basal surface of a giant cell , which recruits RacGEF1ΔN to membrane at time 0 induced by rapamycin . B Confocal images of PHcrac ( left ) and PHcrac merged with LimE ( right , PHcrac as red , LimE as green ) in a giant cell after RacGEF1ΔN recruitment . C Kymograph of LimE ( top , green ) and PHcrac ( bottom , red ) across the dotted line in B . D Confocal image of RalGDS ( left ) in a giant cell after RacGEF1ΔN recruitment and the corresponding kymograph ( right ) . E Time lapse confocal images of PHcrac in single cells , which recruit RacGEF1ΔN to membrane induced by rapamycin at time 0 . White arrow points to PHcrac patches at cell cortex . F Kymographs of PHcrac at cell cortex before and after RacGEF1ΔN recruitment . Red dashed lines in F indicate rapamycin addition at time 0 . G Temporal profiles of normalized PHcrac intensities at cell cortex before and after RacGEF1ΔN recruitment ( mean ± s . e . m . , n = 20 cells ) . Red dashed lines in F and G indicate rapamycin addition at time 0 . H Distribution of different sizes of PHcrac patches at cell cortex under LatA treatment , with ( solid ) or without ( hollow ) RacGEF1ΔN recruitment ( ~ 6000 cells from 3 independent experiments ) . I Temporal profiles of normalized cytosolic intensities of PHcrac in response to 1 nM cAMP stimulation at time 0 , with ( red ) or without ( blue ) RacGEF1ΔN recruitment ( mean ± s . e . m . , n = 25 cells each ) . J Confocal images of GflB ( top ) and RapGAP1 ( bottom ) in waves at the basal surface of a giant cell . K Intensity plots of GflB ( blue ) and RapGAP1 ( orange ) across the green dotted arrow in J . L Scatter plots of first peak distances , of GflB relative to LimE , RapGAP1 relative to LimE , and RapGAP1 relative to GflB in the same giant cells ( red lines indicate median , n = 19 cells each ) . M Confocal images of GflB ( top ) and RapGAP1 ( bottom ) at the basal surface of the same giant cell after RacGEF1ΔN recruitment . N kymographs of GflB ( top ) and RapGAP1 ( bottom ) across the green dotted lines in m . All scale bars represent 20 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 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 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 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 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 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 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 B-PANEL_START 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 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

Figure 7A Time lapse confocal images of PHcrac at the basal surface of a giant cell, which recruits RacGEF1ΔN to membrane at time 0 induced by rapamycin.B Confocal images of PHcrac (left) and PHcrac merged with LimE (right, PHcrac as red, LimE as green) in a giant cell after RacGEF1ΔN recruitment.C Kymograph of LimE (top, green) and PHcrac (bottom, red) across the dotted line in B.D Confocal image of RalGDS (left) in a giant cell after RacGEF1ΔN recruitment and the corresponding kymograph (right).E Time lapse confocal images of PHcrac in single cells, which recruit RacGEF1ΔN to membrane induced by rapamycin at time 0. White arrow points to PHcrac patches at cell cortex.F Kymographs of PHcrac at cell cortex before and after RacGEF1ΔN recruitment. Red dashed lines in F indicate rapamycin addition at time 0.G Temporal profiles of normalized PHcrac intensities at cell cortex before and after RacGEF1ΔN recruitment (mean±s.e.m., n=20 cells). Red dashed lines in F and G indicate rapamycin addition at time 0.H Distribution of different sizes of PHcrac patches at cell cortex under LatA treatment, with (solid) or without (hollow) RacGEF1ΔN recruitment (~6000 cells from 3 independent experiments).I Temporal profiles of normalized cytosolic intensities of PHcrac in response to 1 nM cAMP stimulation at time 0, with (red) or without (blue) RacGEF1ΔN recruitment (mean±s.e.m., n=25 cells each).J Confocal images of GflB (top) and RapGAP1 (bottom) in waves at the basal surface of a giant cell.K Intensity plots of GflB (blue) and RapGAP1 (orange) across the green dotted arrow in J.L Scatter plots of first peak distances, of GflB relative to LimE, RapGAP1 relative to LimE, and RapGAP1 relative to GflB in the same giant cells (red lines indicate median, n=19 cells each).M Confocal images of GflB (top) and RapGAP1 (bottom) at the basal surface of the same giant cell after RacGEF1ΔN recruitment.N kymographs of GflB (top) and RapGAP1 (bottom) across the green dotted lines in m. All scale bars represent 20 μm.


Figure 8A , B Left , confocal images of LimE ( top ) and kymographs of cortical LimE ( bottom ) in single cells with PKBA ( A ) or RacGEF1ΔN ( B ) steadily recruited ( scale bars , 10 μm ) . Right , color coded overlays of LimE in single cells . C Box plots of roundness ( left ) and cell area ( middle ) comparing wt , cells with RacGEF1ΔN recruited , and spreading phases of oscillators after Inp54p recruitment ( n = 100 cells each ) . Right , box plots of coefficient of variance of cell area with the above conditions in a 10 - minute time window ( n = 30 cells each ) . Red bars indicate median . D Phalloidin staining of cells after perturbations . Maximal intensity projections are shown .

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

Figure 8A, B Left, confocal images of LimE (top) and kymographs of cortical LimE (bottom) in single cells with PKBA (A) or RacGEF1ΔN (B) steadily recruited (scale bars, 10 μm). Right, color coded overlays of LimE in single cells.C Box plots of roundness (left) and cell area (middle) comparing wt, cells with RacGEF1ΔN recruited, and spreading phases of oscillators after Inp54p recruitment (n=100 cells each). Right, box plots of coefficient of variance of cell area with the above conditions in a 10-minute time window (n=30 cells each). Red bars indicate median.D Phalloidin staining of cells after perturbations. Maximal intensity projections are shown.


Figure 1B The expression of mature miR - 200b and miR - 200a in the MOE of the NC and miR - 200b / a KD mice , as revealed by qPCR analysis ( n = 4 mice each group ; data represent the mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 ; Student ' s t - test ) . C The expression of Ttll10 ( the host gene of miR - 200b / a ) mRNA in the MOE of the NC and miR - 200b / a KD mice , as revealed by qPCR analysis ( n = 4 mice each group ; data represent the mean ± SEM ; p = 0 . 3540 ; Student ' s t - test ) . D The locomotor activity of the miR - 200b / a KD mice was similar to that of the NC mice ( NC : n = 13 mice , miR - 200b / a knockdown : n = 15 mice ; data represent the mean ± SEM ; p = 0 . 5030 ; Student ' s t - test ) . E The odorant habituation results shown as the ratio of odor sniffs / water of the miR - 200b / a KD mice were significantly reduced compared to those of the NC mice . These odorants included male urine ( 1 : 50 ) , female urine ( 1 : 50 ) , isopentyl acetate ( 50 μM ) , citral ( 50 μM ) , 2 - heptanone ( 50 μM ) and propyl propionate ( 50 μM ) ( n = 6 mice each group ; data represent the mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; Student ' s t - test ) . The resident / intruder experiment showed that the attack frequency ( F ) of the miR - 200b / a KD mice were impaired compared to those of the NC mice ( NC : n = 11 mice , miR - 200b / a KD : n = 10 mice ; data represent the mean ± SEM ; * * * * p < 0 . 0001 ; Student ' s t - test ) . G The resident / intruder experiment showed that the attack duration ( G ) of the miR - 200b / a KD mice were impaired compared to those of the NC mice ( NC : n = 11 mice , miR - 200b / a KD : n = 10 mice ; data represent the mean ± SEM ; * * * * p < 0 . 0001 ; Student ' s t - test ) . H , I The analysis of male - female mating behaviors showed that the mating frequency ( H ) and duration ( I ) of the miR - 200b / a KD mice were significantly reduced compared to those of the NC mice ( NC : n = 11 mice , miR - 200b / a KD : n = 10 mice ; data represent the mean ± SEM ; * * p < 0 . 01 , * * * p < 0 . 001 ; Student ' s t - test ) .

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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1B The expression of mature miR-200b and miR-200a in the MOE of the NC and miR-200b/a KD mice, as revealed by qPCR analysis (n=4 mice each group; data represent the mean±SEM; *p<0.05, **p<0.01; Student's t-test).C The expression of Ttll10 (the host gene of miR-200b/a) mRNA in the MOE of the NC and miR-200b/a KD mice, as revealed by qPCR analysis (n=4 mice each group; data represent the mean±SEM; p=0.3540; Student's t-test).D The locomotor activity of the miR-200b/a KD mice was similar to that of the NC mice (NC: n=13 mice, miR-200b/a knockdown: n=15 mice; data represent the mean±SEM; p=0.5030; Student's t-test).E The odorant habituation results shown as the ratio of odor sniffs/water of the miR-200b/a KD mice were significantly reduced compared to those of the NC mice. These odorants included male urine (1:50), female urine (1:50), isopentyl acetate (50 μM), citral (50 μM), 2-heptanone (50 μM) and propyl propionate (50 μM) (n=6 mice each group; data represent the mean±SEM; *p<0.05, **p<0.01, ***p<0.001; Student's t-test).The resident/intruder experiment showed that the attack frequency (F) of the miR-200b/a KD mice were impaired compared to those of the NC mice (NC: n=11 mice, miR-200b/a KD: n=10 mice; data represent the mean±SEM; ****p<0.0001; Student's t-test).G The resident/intruder experiment showed that the attack duration (G) of the miR-200b/a KD mice were impaired compared to those of the NC mice (NC: n=11 mice, miR-200b/a KD: n=10 mice; data represent the mean±SEM; ****p<0.0001; Student's t-test).H, I The analysis of male-female mating behaviors showed that the mating frequency (H) and duration (I) of the miR-200b/a KD mice were significantly reduced compared to those of the NC mice (NC: n=11 mice, miR-200b/a KD: n=10 mice; data represent the mean±SEM; **p<0.01, ***p<0.001; Student's t-test).


Figure 2B Representative IF staining with Ki67 antibody in the MOE of the NC and miR - 200b / a KD mice . Scale bars , 20 μm . C Quantification of the numbers of Ki67 + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; * * p < 0 . 01 , * * * * p < 0 . 0001 ; Student ' s t - test ) . D Representative IF costaining with both EdU and Ki67 antibody in the MOE of the NC and miR - 200b / a KD mice . The white arrows indicate the EdU + cells and the yellow arrows indicate the cells positive for both EdU and Ki67 . D ' is shown at higher magnification of the boxed region , as three channel merged images ( left image ) and the separated channel ( middle and right images ) . D : Scale bars , 20 μm ; D ' : Scale bars , 2 μm . E Quantification of the numbers of costained Ki67 + and EdU + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 3 mice each group ; data represent the mean ± SEM ; * p < 0 . 05 ; Student ' s t - test ) . F The proportion of GBCs that exited the cell cycle was significantly reduced in the miR - 200b / a KD mice compared with their NC controls [ the cell cycle exit index was calculated using the following formula : ( EdU + Ki67 − cells / total EdU + cells ) × 100 ; n = 3 mice each group ; data represent the mean ± SEM ; * * p < 0 . 01 ; Student ' s t - test ] . G Representative IF costaining with both EdU and BrdU antibody in the MOE of the NC and miR - 200b / a KD mice . The white arrows indicate the BrdU + cells , and the yellow arrows indicate the cells positive for both BrdU and EdU . G ' is shown at higher magnification of the boxed region , as three channel merged images ( left image ) and the separated channel ( middle and right images ) . G : Scale bars , 20 μm ; G ' : Scale bars , 2 μm . H The S phase length of the GBCs in the MOE of the NC and miR - 200b / a KD mice ( the S phase length was calculated using the following formula : 3 h × BrdU + / BrdU + EdU - ; n = 3 mice each group ; data represent the mean ± SEM ; * p < 0 . 05 ; Student ' s t - test ) . I Total cell cycle length of the GBCs in the MOE of the NC and miR - 200b / a KD mice ( total cell cycle length was calculated using the following formula : 20 h + ( Ts × EdU + BrdU - / Edu + ) ; n = 3 mice each group ; p = 0 . 9785 ; data represent the mean ± SEM ; Student ' s t - test ) .

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

Figure 2B Representative IF staining with Ki67 antibody in the MOE of the NC and miR-200b/a KD mice. Scale bars, 20 μm. C Quantification of the numbers of Ki67+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; **p<0.01, ****p<0.0001; Student's t-test). D Representative IF costaining with both EdU and Ki67 antibody in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the EdU+ cells and the yellow arrows indicate the cells positive for both EdU and Ki67. D' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (middle and right images). D: Scale bars, 20 μm; D': Scale bars, 2 μm. E Quantification of the numbers of costained Ki67+ and EdU+ cells in the MOE of the NC and miR-200b/a KD mice (n=3 mice each group; data represent the mean±SEM; *p<0.05; Student's t-test). F The proportion of GBCs that exited the cell cycle was significantly reduced in the miR-200b/a KD mice compared with their NC controls [the cell cycle exit index was calculated using the following formula: (EdU+ Ki67− cells/total EdU+ cells)×100; n=3 mice each group; data represent the mean±SEM; **p<0.01; Student's t-test]. G Representative IF costaining with both EdU and BrdU antibody in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the BrdU+ cells, and the yellow arrows indicate the cells positive for both BrdU and EdU. G' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (middle and right images). G: Scale bars, 20 μm; G': Scale bars, 2 μm.H The S phase length of the GBCs in the MOE of the NC and miR-200b/a KD mice (the S phase length was calculated using the following formula: 3 h×BrdU+/BrdU+EdU-; n=3 mice each group; data represent the mean±SEM; *p<0.05; Student's t-test).I Total cell cycle length of the GBCs in the MOE of the NC and miR-200b/a KD mice (total cell cycle length was calculated using the following formula: 20 h+(Ts×EdU+BrdU-/Edu+); n=3 mice each group; p=0.9785; data represent the mean±SEM; Student's t-test).


Figure 3B The mRNA levels of CK14 , Trp63 , Sox2 and Mash1 in the MOE of the NC and miR - 200b / a KD mice , as revealed by qPCR analysis ( n = 4 mice each group ; data represent the mean ± SEM ; CK14 : p = 0 . 2135 , Trp63 : p = 0 . 1930 , Sox2 : p = 0 . 5190 , * p < 0 . 05 ; Student ' s t - test ) . C Representative IF staining for CK14 , Sox2 , and Mash1 in the MOE of the NC and miR - 200b / a KD mice . Scale bars , 20 μm . D Quantification of the number of CK14 + , basal Sox2 + , apical Sox2 + , and Mash1 + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; CK14 : p = 0 . 8998 , basal Sox2 : p = 0 . 7056 , apical Sox2 : p = 0 . 4455 , * * p < 0 . 01 ; Student ' s t - test ) . E Western Blot analysis of the Sox2 and Mash1 protein levels in the MOE of the NC and miR - 200b / a KD mice ( n = 2 mice each group ) . GAPDH and Tubulin served as loading controls . The molecular weight of each band is indicated at the right . F Representative IF costaining with Sox2 antibody and EdU in the MOE of the NC and miR - 200b / a KD mice . The white arrows indicate the cells positive for both Sox2 and EdU . F ' is shown at higher magnification of the boxed region , as three channel merged images ( left image ) and the separated channel ( right images ) . F : Scale bars , 20 μm ; F ' : Scale bars , 2 μm . G Quantification of the number of Sox2 + EdU + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; p = 0 . 7988 ; Student ' s t - test ) . H Representative IF costaining with Mash1 antibody and EdU in the MOE of the NC and miR - 200b / a KD mice . The white arrows indicate the cells positive for both Mash1 and EdU . H ' is shown at higher magnification of the boxed region , as three channel merged images ( left image ) and the separated channel ( right images ) . H : Scale bars , 20 μm ; H ' : Scale bars , 2 μm . I Quantification of the number of Mash1 + EdU + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; * p < 0 . 05 ; Student ' s t - test ) .

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 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 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 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 3B The mRNA levels of CK14, Trp63, Sox2 and Mash1 in the MOE of the NC and miR-200b/a KD mice, as revealed by qPCR analysis (n=4 mice each group; data represent the mean±SEM; CK14: p=0.2135, Trp63: p=0.1930, Sox2: p=0.5190, *p<0.05; Student's t-test).C Representative IF staining for CK14, Sox2, and Mash1 in the MOE of the NC and miR-200b/a KD mice. Scale bars, 20 μm. D Quantification of the number of CK14+, basal Sox2+, apical Sox2+, and Mash1+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; CK14: p=0.8998, basal Sox2: p=0.7056, apical Sox2: p=0.4455, **p<0.01; Student's t-test). E Western Blot analysis of the Sox2 and Mash1 protein levels in the MOE of the NC and miR-200b/a KD mice (n=2 mice each group). GAPDH and Tubulin served as loading controls. The molecular weight of each band is indicated at the right. F Representative IF costaining with Sox2 antibody and EdU in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the cells positive for both Sox2 and EdU. F' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (right images). F: Scale bars, 20 μm; F': Scale bars, 2 μm. G Quantification of the number of Sox2+EdU+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; p=0.7988; Student's t-test). H Representative IF costaining with Mash1 antibody and EdU in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the cells positive for both Mash1 and EdU. H' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (right images). H: Scale bars, 20 μm; H': Scale bars, 2 μm. I Quantification of the number of Mash1+EdU+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; *p<0.05; Student's t-test).


Figure 4A Representative IF staining for Tuj1 , GAP43 , and OMP in the MOE of the NC and miR - 200b / a KD mice . Scale bars , 20 μm . B Quantification of the number of Tuj1 + , GAP43 + , and OMP + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; Student ' s t - test ) . C The mRNA levels of Ngn1 , GAP43 and OMP in the MOE of the NC and miR - 200b / a KD mice , as revealed by qPCR analysis ( n = 4 mice each group ; data represent the mean ± SEM ; * p < 0 . 05 ; Student ' s t - test ) . D Representative IF costaining with GAP43 and BrdU antibodies in the MOE of the NC and miR - 200b / a KD mice injected with BrdU at 3 d . The white arrows indicate the cells positive for both GAP43 and BrdU . D ' is shown at higher magnification of the boxed region , as three channel merged images ( left image ) and the separated channel ( middle and right images ) . D : Scale bars , 20 μm ; D ' : Scale bars , 2 μm . E Quantification of the ratio of the number of BrdU + GAP43 + cells and the number of total GAP43 + cells in the MOE of the NC and miR - 200b / a KD mice injected with BrdU at 3 d ( n = 3 mice each group ; data represent the mean ± SEM ; * * * * p < 0 . 0001 ; Student ' s t - test ) . F Representative IF staining with Cas3 antibody in the MOE of the NC and miR - 200b / a KD mice . Scale bars , 20 μm . G Quantification of the number of Cas3 + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; * * * p < 0 . 001 ; Student ' s t - test ) .

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 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 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

Figure 4A Representative IF staining for Tuj1, GAP43, and OMP in the MOE of the NC and miR-200b/a KD mice. Scale bars, 20 μm. B Quantification of the number of Tuj1+, GAP43+, and OMP+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; *p<0.05, **p<0.01, ***p<0.001; Student's t-test). C The mRNA levels of Ngn1, GAP43 and OMP in the MOE of the NC and miR-200b/a KD mice, as revealed by qPCR analysis (n=4 mice each group; data represent the mean±SEM; *p<0.05; Student's t-test). D Representative IF costaining with GAP43 and BrdU antibodies in the MOE of the NC and miR-200b/a KD mice injected with BrdU at 3 d. The white arrows indicate the cells positive for both GAP43 and BrdU. D' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (middle and right images). D: Scale bars, 20 μm; D': Scale bars, 2 μm. E Quantification of the ratio of the number of BrdU+ GAP43+ cells and the number of total GAP43+ cells in the MOE of the NC and miR-200b/a KD mice injected with BrdU at 3 d (n=3 mice each group; data represent the mean±SEM; ****p<0.0001; Student's t-test). F Representative IF staining with Cas3 antibody in the MOE of the NC and miR-200b/a KD mice. Scale bars, 20 μm. G Quantification of the number of Cas3+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; ***p<0.001; Student's t-test).