Figure 15A - B Confocal images showing staining of Olfactory Marker Protein ( OMP ) and ß3 - tubulin along the dorsal aspect of the mouse olfactory epithelium . While sections from both types of fixative show OMP signal in the olfactory sensory neuron somata , their dendrites , and axons , the axon bundles ( green arrow ) located above the olfactory epithelium exemplify the clear signal - to - noise ratio benefits of glyoxal fixation versus that of the PFA - fixative . Immunostaining with the ß3 - tubulin antibody stains the dendrites and axons in both PFA and glyoxal - fixed tissue , but strong staining of the cilia can only be observed in the glyoxal - fixed sections ( B ) . C - D Confocal images depicting bundles of axons belonging to olfactory sensory neurons on the path toward the olfactory bulb . Identities of the axons are in part defined by the neuropilin - 1 ( Nrp1 ) and neuropilin - 2 ( Nrp2 ) expression levels , visualized here with antibodies raised against the two proteins . While complementary expression of the two molecules can be seen in the PFA - fixed sections ( C ) , the glyoxal - fixed sections ( D ) exhibit profoundly improved signal - to - noise ratios for Nrp - 1 ( red arrows ) , and in the case of Nrp - 2 , also the segmentation of the axon bundle into varying levels of Nrp - 2 ( green arrows ) . E - F Confocal images of olfactory sensory neuron axons coalescing into glomeruli where they synapse with dendrites of olfactory bulb neurons . The axons of olfactory sensory neurons can be readily visualized with PFA staining ( green ) in the superficial olfactory nerve layer and terminating in glomeruli located below ( green arrows ) . While sections fixed with either PFA or glyoxal display adequate staining levels , the signal distribution of the Immunostaining - fixed tissue appears more irregular ( E ) , seemingly lacking the neurofilimentary morphology that appears preserved in the glyoxal - fixed sections ( F ) . The glomeruli themselves are neuropil structures comprised primarily of olfactory sensory neurons forming synapses with dendrites of mitral / tufted cells as well as PFA of periglomerular neurons . Immunostaining with Vesicular Glutamate Transporter 2 ( VGLUT2 ) allows visualization of these structures and is easily seen in the glyoxal - fixed section ( F ) , while it appears the antigen was masked by PFA fixation as no signal above background can be seen in the PFA - fixed panel ( red arrows in E ) . Note that a different polyclonal antibody for VGLUT2 from the same provider does provide signal with glyoxal , albeit weaker versus Glyoxal ( inset ) . Staining with ß3 - tubulin touts the benefits of glyoxal both due to the signal improvements in the case of the mild staining in the axons of the olfactory nerve layer that is only visible in the glyoxal - fixed tissue , but also in preserving tissue morphology as demonstrated by the dendritic processes inside glomeruli ( blue arrows ) and in the external plexiform layer located below the glomeruli .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 15A - B Confocal images showing staining of Olfactory Marker Protein (OMP) and ß3-tubulin along the dorsal aspect of the mouse olfactory epithelium. While sections from both types of fixative show OMP signal in the olfactory sensory neuron somata, their dendrites, and axons, the axon bundles (green arrow) located above the olfactory epithelium exemplify the clear signal-to-noise ratio benefits of glyoxal fixation versus that of the PFA-fixative. Immunostaining with the ß3-tubulin antibody stains the dendrites and axons in both PFA and glyoxal-fixed tissue, but strong staining of the cilia can only be observed in the glyoxal-fixed sections (B).C - D Confocal images depicting bundles of axons belonging to olfactory sensory neurons on the path toward the olfactory bulb. Identities of the axons are in part defined by the neuropilin-1 (Nrp1) and neuropilin-2 (Nrp2) expression levels, visualized here with antibodies raised against the two proteins. While complementary expression of the two molecules can be seen in the PFA-fixed sections (C), the glyoxal-fixed sections (D) exhibit profoundly improved signal-to-noise ratios for Nrp-1 (red arrows), and in the case of Nrp-2, also the segmentation of the axon bundle into varying levels of Nrp-2 (green arrows).E - F Confocal images of olfactory sensory neuron axons coalescing into glomeruli where they synapse with dendrites of olfactory bulb neurons. The axons of olfactory sensory neurons can be readily visualized with PFA staining (green) in the superficial olfactory nerve layer and terminating in glomeruli located below (green arrows). While sections fixed with either PFA or glyoxal display adequate staining levels, the signal distribution of the Immunostaining-fixed tissue appears more irregular (E), seemingly lacking the neurofilimentary morphology that appears preserved in the glyoxal-fixed sections (F). The glomeruli themselves are neuropil structures comprised primarily of olfactory sensory neurons forming synapses with dendrites of mitral/tufted cells as well as PFA of periglomerular neurons. Immunostaining with Vesicular Glutamate Transporter 2 (VGLUT2) allows visualization of these structures and is easily seen in the glyoxal-fixed section (F), while it appears the antigen was masked by PFA fixation as no signal above background can be seen in the PFA-fixed panel (red arrows in E). Note that a different polyclonal antibody for VGLUT2 from the same provider does provide signal with glyoxal, albeit weaker versus Glyoxal (inset). Staining with ß3-tubulin touts the benefits of glyoxal both due to the signal improvements in the case of the mild staining in the axons of the olfactory nerve layer that is only visible in the glyoxal-fixed tissue, but also in preserving tissue morphology as demonstrated by the dendritic processes inside glomeruli (blue arrows) and in the external plexiform layer located below the glomeruli.


Figure 2 ( A - C ) The developing tumor vasculature was imaged over time in an abdominal window chamber model with two - photon microscopy with ( A ) GFP - labeled tumor cells , ( B ) TdTomato labeled TECs in cyan and ( C ) perfusion . ( D ) Top : a representative image of MC38 tumor vasculature , bottom : segmentation with skeletonization . ( E - N ) The following parameters were quantified from the segmented image each day : ( E ) vessel tortuosity , ( F ) directional coherence , ( G ) perfusion , ( H ) number of sprouts , ( I ) fraction of branches < 80 µm , ( J ) fraction of branches > 400 µm , ( K ) length to diameter ratio , ( L ) nodes per mm2 , ( M ) vessel length , and ( N ) vessel diameter .

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

Figure 2(A-C) The developing tumor vasculature was imaged over time in an abdominal window chamber model with two-photon microscopy with (A) GFP-labeled tumor cells, (B) TdTomato labeled TECs in cyan and (C) perfusion. (D) Top: a representative image of MC38 tumor vasculature, bottom: segmentation with skeletonization. (E-N) The following parameters were quantified from the segmented image each day: (E) vessel tortuosity, (F) directional coherence, (G) perfusion, (H) number of sprouts,(I) fraction of branches <80 µm, (J) fraction of branches >400 µm, (K) length to diameter ratio, (L) nodes per mm2, (M) vessel length, and (N) vessel diameter.


Figure 3The developing MC38 tumor vasculature was imaged in an abdominal window chamber model with two - photon microscopy after IR at the indicated times . ( A ) ( top ) Representative two - photon images of a control MC38 tumor vasculature ( cyan ) and perfusion ( red ) at day 2 and 4 . ( Bottom ) zoom of the same tumor region from day 0 to day 4 of the tumor vasculature ( cyan ) and perfusion ( red ) . ( B - C ) Single dose 15 Gy IR ( B ) and 5 × 3 Gy ( C ) fractionated IR treated MC38 tumor vasculature ( top ) at day 2 and day 4 . ( Bottom ) zoom of the same tumor region from day 0 to day 4 of the tumor vasculature ( cyan ) and perfusion ( red ) . ( E - L ) Quantified vascular parameters , for each day of imaging the following parameters were quantified from segmented images and normalized to day one if indicated : ( E ) normalized node density , ( F ) normalized tortuosity , ( G ) normalized branch length < 80μm , ( H ) normalized branch length > 400μm , ( I ) vessel branch length , ( J ) vessel diameter , ( K ) normalized length to diameter ratio , ( L ) normalized perfused vessels . ( M - N ) Subcutaneous tumor growth measurements of MC38 ( M ) and B16F10 ( N ) tumors .

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

Figure 3The developing MC38 tumor vasculature was imaged in an abdominal window chamber model with two-photon microscopy after IR at the indicated times. (A) (top) Representative two-photon images of a control MC38 tumor vasculature (cyan) and perfusion (red) at day 2 and 4. (Bottom) zoom of the same tumor region from day 0 to day 4 of the tumor vasculature (cyan) and perfusion (red). (B-C) Single dose 15 Gy IR (B) and 5×3 Gy (C) fractionated IR treated MC38 tumor vasculature (top) at day 2 and day 4. (Bottom) zoom of the same tumor region from day 0 to day 4 of the tumor vasculature (cyan) and perfusion (red).(E-L) Quantified vascular parameters, for each day of imaging the following parameters were quantified from segmented images and normalized to day one if indicated: (E) normalized node density , (F) normalized tortuosity, (G) normalized branch length < 80μm, (H) normalized branch length > 400μm, (I) vessel branch length, (J) vessel diameter, (K) normalized length to diameter ratio, (L) normalized perfused vessels.(M-N) Subcutaneous tumor growth measurements of MC38 (M) and B16F10 (N) tumors.


Figure 4 ( A - B ) Representative immunofluorescent micrographs of control and treated tumor vasculature from MC38 tumors . Tumor vessels ( anti - CD31 ) and apoptosis ( anti - cleaved caspase 3 ) in ( A ) control and ( B ) 48 hours after 15 Gy IR . Arrows indicate apoptotic TEC . ( C ) quantification of apoptotic vessels after single dose 15 Gy IR from immunofluorescent images of whole MC38 tumor section ( n = 6 - 8 biological replicates per group ) . ( D ) Quantification of apoptotic vessels after fractionated 5 × 3 Gy IR from immunofluorescent images of whole MC38 tumor sections ( n = 6 - 8 biological replicates per group ) . ( E ) Quantification of apoptosis in TECs from B16F10 tumor sections ( n = 4 - 6 biological replicates per group ) . ( F ) Quantification of apoptotic vessels according to size on thin whole MC38 tumor sections ( Ctrl n = 4 and treatment n = 7 biological replicates ) . ( G ) Quantification of apoptotic vessels according to size in B16F10 tumor sections ( Ctrl n = 4 and treatment n = 10 biological replicates ) . ( H - I ) Area ( H ) and diameter ( I ) of apoptotic tumor vessels vs all tumor vessels from segmented thick 80 μm MC38 tumor sections ( n = 4 biological replicates ) . ( J - K ) Representative immunofluorescence image of control ( J ) and 15 Gy IR treated ( K ) MC38 and B16F10 tumors stained for proliferation ( EdU ) , EC nuclei ( anti - ERG ) and tumor vessels ( anti - CD31 ) . ( L - M ) Quantification of proliferating cells ( L ) and proliferating TECs ( M ) in MC38 tumors ( n = 8 biological replicates per group ) . ( N - O ) Quantification of proliferating cells ( N ) and proliferating TECs ( O ) in B16F10 tumors ( n = 4 to 9 biological replicates per group ) . ( P - Q ) Quantification of proliferating cells ( P ) and TECs ( Q ) in MC38 tumors after fractionated IR ( n = 5 - 7 biological replicates per group ) . ( R - S ) Quantification of proliferating cells ( R ) and TECs ( S ) in B16F10 tumors after fractionated IR ( n = 5 - 7 biological replicates per group ) . ( T - U ) Measurement of proliferating TECs in ( T ) MC38 tumors ( n = 9 biological replicates ) and ( U ) B16F10 tumors ( n = 5 biological replicates ) in small ( area < 250 µm2 ) and large ( area < 250 µm2 ) tumor blood vessels .

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 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

Figure 4(A-B) Representative immunofluorescent micrographs of control and treated tumor vasculature from MC38 tumors. Tumor vessels (anti-CD31) and apoptosis (anti-cleaved caspase 3) in (A) control and (B) 48 hours after 15 Gy IR. Arrows indicate apoptotic TEC. (C) quantification of apoptotic vessels after single dose 15 Gy IR from immunofluorescent images of whole MC38 tumor section (n= 6-8 biological replicates per group). (D) Quantification of apoptotic vessels after fractionated 5×3 Gy IR from immunofluorescent images of whole MC38 tumor sections (n= 6-8 biological replicates per group). (E) Quantification of apoptosis in TECs from B16F10 tumor sections (n= 4-6 biological replicates per group). (F) Quantification of apoptotic vessels according to size on thin whole MC38 tumor sections (Ctrl n=4 and treatment n=7 biological replicates). (G) Quantification of apoptotic vessels according to size in B16F10 tumor sections (Ctrl n=4 and treatment n= 10 biological replicates). (H-I) Area (H) and diameter (I) of apoptotic tumor vessels vs all tumor vessels from segmented thick 80 μm MC38 tumor sections (n=4 biological replicates).(J-K) Representative immunofluorescence image of control (J) and 15 Gy IR treated (K) MC38 and B16F10 tumors stained for proliferation (EdU), EC nuclei (anti-ERG) and tumor vessels (anti-CD31). (L-M) Quantification of proliferating cells (L) and proliferating TECs (M) in MC38 tumors (n= 8 biological replicates per group). (N-O) Quantification of proliferating cells (N) and proliferating TECs (O) in B16F10 tumors (n= 4 to 9 biological replicates per group). (P-Q) Quantification of proliferating cells (P) and TECs (Q) in MC38 tumors after fractionated IR (n=5-7 biological replicates per group). (R-S) Quantification of proliferating cells (R) and TECs (S) in B16F10 tumors after fractionated IR (n= 5-7 biological replicates per group). (T-U) Measurement of proliferating TECs in (T) MC38 tumors (n= 9 biological replicates) and (U) B16F10 tumors (n= 5 biological replicates) in small (area <250 µm2) and large (area <250 µm2) tumor blood vessels.


Figure 1A . The expression of EP subtypes in bone marrow myeloid cells and colon tumor myeloid cells . The short black lines indicate individual expressions , the blue and yellow lines indicate the average expression level in each condition . The grey dotted lines represent the overall average between Ptger1 and Ptger3 or Ptger2 and Ptger4 . B . The expression of EP subtypes in CD11b + cells isolated from tumor tissues and spleen of CT26 tumor - bearing mice by magnetic bead separation ( n = 3 ) . D . Representative flow cytometry plots revealing the effects of prostaglandin E2 ( PGE2 ) and EP1 - 4 antagonists on mouse bone marrow monocyte dendritic cell ( DC ) / macrophage differentiation ( n = 3 ) . E . The frequencies of F4 / 80 + CD11C − macrophages and F4 / 80 − CD11C + DCs under varying treatments as analyzed by flow cytometry analysis ( n = 3 ) . G . Representative flow cytometry plots revealing the effects of PGE2 and EP1 - 4 antagonists on mouse bone marrow derived - MDSC differentiation . Upper subpanel : vehicle group and PGE2 group . Lower subpanel : ONO - 8711 ( EP1 antagonist ) group , PF - 04418948 ( EP2 antagonist ) group , L - 798106 ( EP3 antagonist ) group , and E7046 ( EP4 antagonist ) group . ( n = 3 ) . H . The frequencies of Ly6C + Ly6G − mMDSC and Ly6CmidLy6G + PMN - MDSCs under varied treatments as analyzed by flow cytometry analysis ( n = 3 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O 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. The expression of EP subtypes in bone marrow myeloid cells and colon tumor myeloid cells. The short black lines indicate individual expressions, the blue and yellow lines indicate the average expression level in each condition. The grey dotted lines represent the overall average between Ptger1 and Ptger3 or Ptger2 and Ptger4.B. The expression of EP subtypes in CD11b+ cells isolated from tumor tissues and spleen of CT26 tumor-bearing mice by magnetic bead separation (n=3).D. Representative flow cytometry plots revealing the effects of prostaglandin E2 (PGE2) and EP1-4 antagonists on mouse bone marrow monocyte dendritic cell (DC)/macrophage differentiation (n = 3). E. The frequencies of F4/80+CD11C− macrophages and F4/80−CD11C+ DCs under varying treatments as analyzed by flow cytometry analysis (n = 3). G. Representative flow cytometry plots revealing the effects of PGE2 and EP1-4 antagonists on mouse bone marrow derived-MDSC differentiation. Upper subpanel: vehicle group and PGE2 group. Lower subpanel: ONO-8711 (EP1 antagonist) group, PF-04418948 (EP2 antagonist) group, L-798106 (EP3 antagonist) group, and E7046 (EP4 antagonist) group. (n = 3). H. The frequencies of Ly6C+Ly6G− mMDSC and Ly6CmidLy6G+ PMN-MDSCs under varied treatments as analyzed by flow cytometry analysis (n = 3).


Figure 2B . Dose - effect curve of TP - 16 in GloSensor ™ cAMP assay in EP4 - expressing 293 cells ( n = 3 ) . C . Dose - effect curves of TP - 16 in PGE2 - induced calcium flux assay ( n = 3 ) . D . The Schild plot of PGE2 in the presence of varying concentrations of TP - 16 . TP - 16 shifted the dose - response curve of PGE2 - induced intracellular cAMP levels in a dose - dependent manner ( n = 3 ) . E . The pA2 value and slope of the Schild plot .

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 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 O 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. Dose-effect curve of TP-16 in GloSensor™ cAMP assay in EP4- expressing 293 cells (n=3).C. Dose-effect curves of TP-16 in PGE2-induced calcium flux assay (n=3).D. The Schild plot of PGE2 in the presence of varying concentrations of TP-16. TP-16 shifted the dose-response curve of PGE2-induced intracellular cAMP levels in a dose-dependent manner (n=3). E. The pA2 value and slope of the Schild plot.


Figure 3B . The antitumor activities of E7046 ( 150 mg / kg ) and TP - 16 ( 37 . 5 , 75 , and150 mg / kg ) in CT26 tumor - bearing BALB / c mice ( n = 11 per group ) . Tumor growth curves in the CT26 model ( left panel ) . Bar graphs show the individual changes in tumor growth relative to after and before drug administration ( right panel ) . Tumor volumes reached 100 - 200 mm3 on day 7 . C . Representative graph showing that the antitumor activity of TP - 16 is lost in CT26 tumors engrafted into BALB / c nude mice ( left panel ) ( n = 8 ) . Bar graphs show the individual changes in tumor growth relative to after and before drug administration ( right panel ) . Tumor volumes reached 100 - 200 mm3 on day 7 . D . Single - cell suspensions of tumor tissues from CT26 tumor - bearing BALB / c mice treated with vehicle or TP - 16 for 2 weeks were analyzed for immune cell infiltration by flow cytometry analysis . Representative flow cytometry and dot plots of intratumoral frequencies of CD8 + T cells ( n = 5 ) . E . Growth curves ( left panel ) and relative change after 18 days treatment ( right panel ) in colon cancer ( MC38 ) xenograft tumors in C57BL / 6 mice ( n = 7 ) . Tumor volumes reached 100 - 200 mm3 on day 7 . F . Single - cell suspensions of tumor tissues from MC38 tumor - bearing C57BL / 6 mice treated with vehicle or TP - 16 for 2 weeks were analyzed for immune cell infiltration by flow cytometry analysis . Representative flow cytometry and dot plots of intratumoral frequencies of CD8 + T cells ( n = 5 ) . G . The representative bioluminescence images ( left panel ) and quantitative growth curves ( right panel ) of an orthotopic colorectal cancer mouse model , CT26 - Luc model ( n = 4 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 3B. The antitumor activities of E7046 (150 mg/kg) and TP-16 (37.5, 75, and150 mg/kg) in CT26 tumor-bearing BALB/c mice (n = 11 per group). Tumor growth curves in the CT26 model (left panel). Bar graphs show the individual changes in tumor growth relative to after and before drug administration (right panel). Tumor volumes reached 100-200 mm3 on day 7.C. Representative graph showing that the antitumor activity of TP-16 is lost in CT26 tumors engrafted into BALB/c nude mice (left panel) (n = 8). Bar graphs show the individual changes in tumor growth relative to after and before drug administration (right panel). Tumor volumes reached 100-200 mm3 on day 7.D. Single-cell suspensions of tumor tissues from CT26 tumor-bearing BALB/c mice treated with vehicle or TP-16 for 2 weeks were analyzed for immune cell infiltration by flow cytometry analysis. Representative flow cytometry and dot plots of intratumoral frequencies of CD8+ T cells (n = 5).E. Growth curves (left panel) and relative change after 18 days treatment (right panel) in colon cancer (MC38) xenograft tumors in C57BL/6 mice (n = 7). Tumor volumes reached 100-200 mm3 on day 7.F. Single-cell suspensions of tumor tissues from MC38 tumor-bearing C57BL/6 mice treated with vehicle or TP-16 for 2 weeks were analyzed for immune cell infiltration by flow cytometry analysis. Representative flow cytometry and dot plots of intratumoral frequencies of CD8+ T cells (n = 5).G. The representative bioluminescence images (left panel) and quantitative growth curves (right panel) of an orthotopic colorectal cancer mouse model, CT26-Luc model (n = 4).


Figure 4A , B . Tumors from CT26 tumor - bearing BALB / c mice treated with vehicle or TP - 16 for 2 weeks were harvested , and tumor single - cell suspensions were analyzed for tumor - associated myeloid cells by flow cytometry analysis . Representative graphs and quantification of F4 / 80 + MHC - II + immunosuppressive myeloid cells ( IMCs ) - M1 macrophages ( A ) and F4 / 80 + CD206 + IMCs - M2 macrophages ( B ) gated on CD45 + CD11b + myeloid cells ( n = 5 ) . C . Representative flow cytometry analysis and quantification of Ly6C + Ly6G - IMCs - monocytic myeloid - derived suppressor cell ( mMDSC ) and Ly6CmidLy6G + IMCs - PMN - MDSC frequencies gated on CD45 + CD11b + myeloid cells ( n = 5 ) . Relative expression of selected M1 , M2 markers on flow - sorted M1 macrophages ( D ) , M2 macrophages ( E ) from CT26 tumors . Data are normalized to β - actin expression levels ( n = 5 ) . Relative expression of selected MDSC markers on flow - sorted mMDSCs ( F ) , and PMN - MDSCs ( G ) from CT26 tumors . Data are normalized to β - actin expression levels ( n = 5 ) . H - K . Representative flow analysis and quantification of CD8 + T cell activation markers : Granzyme B ( H ) , IFN - γ ( I ) , TNF - α ( J ) , and PD - 1 ( K ) ( n = 5 ) .

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

Figure 4A, B. Tumors from CT26 tumor-bearing BALB/c mice treated with vehicle or TP-16 for 2 weeks were harvested, and tumor single-cell suspensions were analyzed for tumor-associated myeloid cells by flow cytometry analysis. Representative graphs and quantification of F4/80+MHC-II+ immunosuppressive myeloid cells (IMCs)-M1 macrophages (A) and F4/80+CD206+ IMCs-M2 macrophages (B) gated on CD45+CD11b+ myeloid cells (n = 5).C. Representative flow cytometry analysis and quantification of Ly6C+Ly6G- IMCs-monocytic myeloid-derived suppressor cell (mMDSC) and Ly6CmidLy6G+ IMCs-PMN-MDSC frequencies gated on CD45+CD11b+ myeloid cells (n = 5).Relative expression of selected M1, M2 markers on flow-sorted M1 macrophages (D), M2 macrophages (E) from CT26 tumors. Data are normalized to β-actin expression levels (n = 5).Relative expression of selected MDSC markers on flow-sorted mMDSCs (F), and PMN-MDSCs (G) from CT26 tumors. Data are normalized to β-actin expression levels (n = 5).H-K. Representative flow analysis and quantification of CD8+ T cell activation markers: Granzyme B (H), IFN-γ (I), TNF-α (J), and PD-1 (K) (n = 5).


Figure 5M - CSF induced mouse bone marrow derived macrophages ( BMMs ) were stimulated with 50 ng / mL mouse recombinant IFN - γ ( A ) in the absence or presence of PGE2 ± varying concentrations of TP - 16 for 12 hr . The mRNA levels of selected M1 markers ( Cxcl10 and Tnfa ) were measured by q - PCR . Data are normalized to β - actin expression levels ( n = 3 ) . B . M - CSF induced mouse bone marrow derived macrophages ( BMMs ) were stimulated with 20 ng / mL mouse recombinant IL - 4 ( B ) in the absence or presence of PGE2 ± varying concentrations of TP - 16 for 12 hr . The mRNA levels of selected M2 markers ( Arg - 1 and Ym - 1 ) were measured by q - PCR . Data are normalized to β - actin expression levels ( n = 3 ) . C . Bone marrow cells were cultured with 40 ng / mL GM - CSF / IL - 6 or co - stimulated with PGE2 ± varying concentrations of TP - 16 for 6 days . Representative quantification of Ly6C + Ly6G − monocytic myeloid - derived suppressor cells ( mMDSCs ) and Ly6CmidLy6G + PMN - MDSCs ( n = 3 ) . D . Bone marrow cells were treated with GM - CSF / IL - 6 for 6 days to induce MDSCs and thereafter , with PGE2 ± varying concentrations of TP - 16 for 12 hr . The mRNA levels of selected MDSC markers ( Arg - 1 , Ptgs2 , Il - 4 , and Il - 10 ) were measured by qPCR . Data are normalized to β - actin expression levels ( n = 3 ) . E , F . In vitro T cell suppression activity of CT26 - tumor infiltrating CD11b + myeloid cells collected at 2 weeks post - treatment with vehicle or 75 mg / kg TP - 16 . CD8 + T cells were labeled with CFSE and then stimulated with CD3 / CD28 antibodies for 3 days in the absence or presence of tumor - infiltrating CD11b + myeloid cells from vehicle - or 75 mg / kg TP - 16 - treated CT26 tumors . Representative flow cytometry histograms ( E ) and percentages ( F ) of proliferating CD8 + T cells when plated in a ratio of 2 : 1 , 1 : 1 , and 1 : 2 CD8 + T cells to CD11b + myeloid cells ( n = 3 ) .

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 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 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 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

Figure 5M-CSF induced mouse bone marrow derived macrophages (BMMs) were stimulated with 50 ng/mL mouse recombinant IFN-γ (A) in the absence or presence of PGE2 ± varying concentrations of TP-16 for 12 hr. The mRNA levels of selected M1 markers (Cxcl10 and Tnfa) were measured by q-PCR. Data are normalized to β-actin expression levels (n=3).B. M-CSF induced mouse bone marrow derived macrophages (BMMs) were stimulated with 20 ng/mL mouse recombinant IL-4 (B) in the absence or presence of PGE2 ± varying concentrations of TP-16 for 12 hr. The mRNA levels of selected M2 markers (Arg-1 and Ym-1) were measured by q-PCR. Data are normalized to β-actin expression levels (n=3).C. Bone marrow cells were cultured with 40 ng/mL GM-CSF/IL-6 or co-stimulated with PGE2 ± varying concentrations of TP-16 for 6 days. Representative quantification of Ly6C+Ly6G− monocytic myeloid-derived suppressor cells (mMDSCs) and Ly6CmidLy6G+ PMN-MDSCs (n = 3).D. Bone marrow cells were treated with GM-CSF/IL-6 for 6 days to induce MDSCs and thereafter, with PGE2 ± varying concentrations of TP-16 for 12 hr. The mRNA levels of selected MDSC markers (Arg-1, Ptgs2, Il-4, and Il-10) were measured by qPCR. Data are normalized to β-actin expression levels (n=3).E, F. In vitro T cell suppression activity of CT26-tumor infiltrating CD11b+ myeloid cells collected at 2 weeks post-treatment with vehicle or 75 mg/kg TP-16. CD8+ T cells were labeled with CFSE and then stimulated with CD3/CD28 antibodies for 3 days in the absence or presence of tumor-infiltrating CD11b+ myeloid cells from vehicle- or 75 mg/kg TP-16-treated CT26 tumors. Representative flow cytometry histograms (E) and percentages (F) of proliferating CD8+ T cells when plated in a ratio of 2:1, 1:1, and 1:2 CD8+ T cells to CD11b+ myeloid cells (n = 3).


Figure 6A . The growth of CT26 tumor volume when treated with vehicle , TP - 16 ( 75 mg / kg , p . o . , daily ) , anti - PD - 1 antibody ( 50 μg , i . p . , twice weekly ) or their combination ( left ) and percent change in tumor volumes between days 0 and 20 ( right ) ( n = 12 ) . B . BALB / c mice with subcutaneous CT26 tumors were treated with vehicle , TP - 16 ( 75 mg / kg , p . o . , daily ) , PD - 1 antibody ( 50 μg , i . p . , twice weekly ) or combined therapies for 2 weeks . Animal survival ( time to tumor burden reaching 2000 mm3 ) was analyzed by the Kaplan - Meier method using GraphPad Prism ( n = 10 ) . P values were calculated using the Log - rank test . C . Representative immunofluorescence staining images of tumor sections from CT26 tumor - bearing mice stained for CD8 and DAPI . Scale bars , 50 μm . D . Representative immunostaining images of tumor sections from CT26 tumor - bearing mice stained for p - STAT3 , p - AKT and PD - L1 . Scale bars , 100 μm . F . The heatmap of T cell activation biomarkers for RNA - seq analysis . The unit of heatmap scale is expression value after centering and scaling by genes .

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 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

Figure 6A. The growth of CT26 tumor volume when treated with vehicle, TP-16 (75 mg/kg, p.o., daily), anti-PD-1 antibody (50 μg, i.p., twice weekly) or their combination (left) and percent change in tumor volumes between days 0 and 20 (right) (n=12).B. BALB/c mice with subcutaneous CT26 tumors were treated with vehicle, TP-16 (75 mg/kg, p.o., daily), PD-1 antibody (50 μg, i.p., twice weekly) or combined therapies for 2 weeks. Animal survival (time to tumor burden reaching 2000 mm3) was analyzed by the Kaplan-Meier method using GraphPad Prism (n=10). P values were calculated using the Log-rank test.C. Representative immunofluorescence staining images of tumor sections from CT26 tumor-bearing mice stained for CD8 and DAPI. Scale bars, 50 μm. D. Representative immunostaining images of tumor sections from CT26 tumor-bearing mice stained for p-STAT3, p-AKT and PD-L1. Scale bars, 100 μm. F. The heatmap of T cell activation biomarkers for RNA-seq analysis. The unit of heatmap scale is expression value after centering and scaling by genes.


Figure 7Representative resected colons ( B ) after 5 weeks of indicated treatment ( n = 7 ) . total number of colon tumors per mouse ( C , left ) and tumor size distribution ( C , right ) , and colon length ( D ) after 5 weeks of indicated treatment ( n = 7 ) . E . Representative hematoxylin and eosin ( H & E ) staining and immunostaining for CD8 , p - STAT3 , p - AKT , and ARG - 1 of tumors treated with vehicle , TP - 16 , anti - PD - 1 antibody , or TP - 16 and anti - PD - 1 combined . Scale bars , 500 μm ( left images ) , 50 μm ( right images ) . F . Quantitative polymerase chain reaction ( q - PCR ) analysis of the mRNA level of genes associated with immunosuppression ( n = 6 ) .

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Figure 7Representative resected colons (B) after 5 weeks of indicated treatment (n=7).total number of colon tumors per mouse (C, left) and tumor size distribution (C, right), and colon length (D) after 5 weeks of indicated treatment (n=7).E. Representative hematoxylin and eosin (H&E) staining and immunostaining for CD8, p-STAT3, p-AKT, and ARG-1 of tumors treated with vehicle, TP-16, anti-PD-1 antibody, or TP-16 and anti-PD-1 combined. Scale bars, 500 μm (left images), 50 μm (right images).F. Quantitative polymerase chain reaction (q-PCR) analysis of the mRNA level of genes associated with immunosuppression (n = 6).


Figure 1 ( A ) Detection of Fstl1 protein in ischemic myocardium of WT mice . After MI induction , the heart was harvested at the indicated time points , and separated to ischemic and remote ( non - ischemic ) areas . Fstl1 protein was detected by western blotting ( n = 3 , each time point ) . Statistical analysis was performed by ordinary one way ANOVA . Post Hoc test was performed by Dunnett test . ( B ) Serum Fstl1 was detected by western blotting at the indicated time points . Ponceau S staining of serum reveals equivalent amount of loaded protein ( n = 4 , each time point ) . Error bars represent mean ± SEM . Statistical analysis was performed by ordinary one way ANOVA . Post Hoc test was performed by Dunnett test .

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Figure 1(A) Detection of Fstl1 protein in ischemic myocardium of WT mice. After MI induction, the heart was harvested at the indicated time points, and separated to ischemic and remote (non-ischemic) areas. Fstl1 protein was detected by western blotting (n=3, each time point). Statistical analysis was performed by ordinary one way ANOVA. Post Hoc test was performed by Dunnett test.(B) Serum Fstl1 was detected by western blotting at the indicated time points. Ponceau S staining of serum reveals equivalent amount of loaded protein (n=4, each time point). Error bars represent mean ± SEM. Statistical analysis was performed by ordinary one way ANOVA. Post Hoc test was performed by Dunnett test.


Figure 2 ( A ) DABimmunohistochemistry staining of Fstl1 with ischemic heart at day 3 after LAD ligation . Arrows indicate a subset of Fstl1 positive cells . Counter staining was performed with hematoxylin . Scale bar indicates 50μm . ( B ) Immunofluorescent microscopy staining of Fstl1 ( green ) and sarcomeric actin ( red ) . Fstl1 protein was detected in interstitial areas of the ischemic and border zone but not in cardiomyocytes . Scale bar indicates 100μm . ( C ) Immunofluorescent microscopy staining of Fstl1 ( green ) and S100a4 ( red ) in the ischemic area . Arrows indicate Fstl1 and S100a4 double positive cells . Scale bar indicates 20μm . ( D ) Immunofluorescent microscopy staining of Fstl1 ( green ) and α - smooth muscle actin ( red ) . Arrows indicate Fstl1 and α - SMA double positive cells . Scale bar indicates 20μm . Nuclei were stained by DAPI .

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Figure 2(A) DABimmunohistochemistry staining of Fstl1 with ischemic heart at day 3 after LAD ligation. Arrows indicate a subset of Fstl1 positive cells. Counter staining was performed with hematoxylin. Scale bar indicates 50μm.(B) Immunofluorescent microscopy staining of Fstl1 (green) and sarcomeric actin (red). Fstl1 protein was detected in interstitial areas of the ischemic and border zone but not in cardiomyocytes. Scale bar indicates 100μm.(C) Immunofluorescent microscopy staining of Fstl1 (green) and S100a4 (red) in the ischemic area. Arrows indicate Fstl1 and S100a4 double positive cells. Scale bar indicates 20μm.(D) Immunofluorescent microscopy staining of Fstl1 (green) and α-smooth muscle actin (red). Arrows indicate Fstl1 and α-SMA double positive cells. Scale bar indicates 20μm. Nuclei were stained by DAPI.


Figure 3Genotypes are indicated as WT for S100a4cre - / - x Fstl1flox + / flox + mice and cfKO for S100a4cre + / - x Fstl1flox + / flox + . ( A ) S100a4 expression is induced in the ischemic area ( IA ) . qPCR analysis of mRNA expression of S100a4 in ischemic heart and sham operated heart of WT mice and cfKO mice . Heart samples were harvested at 7 days after the surgery . Statistical analysis was performed by two - way ANOVA . Post Hoc test was performed by Tukey test . Error bars represent mean ± SEM ( n = 16 and 15 for WT and cfKO sham group , n = 15 and 14 for WT and cfKO MI group , respectively ) . Genotypes are indicated as WT for S100a4cre - / - x Fstl1flox + / flox + mice and cfKO for S100a4cre + / - x Fstl1flox + / flox + . ( B ) qPCR analysis of mRNA expression of Fstl1 and Tgfβ1 in sham and post - MI heart . Error bars represent mean ± SEM ( n = 16 and 15 for WT and cfKO sham group , n = 15 and 14 for WT and cfKO MI group , respectively ) . Statistical analysis was performed by two - way ANOVA . Post Hoc test was performed by Tukey test . Genotypes are indicated as WT for S100a4cre - / - x Fstl1flox + / flox + mice and cfKO for S100a4cre + / - x Fstl1flox + / flox + . ( C ) Western blot analysis of Fstl1 protein expression in ischemic and sham - operated hearts at day 7 after the surgery . Quantified values of Fstl1 protein in WT and cfKO mouse hearts normalized by GAPDH band intensity are shown . Statistical analysis was performed by two - way ANOVA . Post Hoc test was performed by Tukey test . Error bars represent mean ± SEM ( n = 5 for each sham group and n = 6 for each MI group ) . Genotypes are indicated as WT for S100a4cre - / - x Fstl1flox + / flox + mice and cfKO for S100a4cre + / - x Fstl1flox + / flox + . ( D ) Fstl1 protein expression in isolated cardiac fibroblasts from cfKO and littermate WT neonatal mice . Cell lysate and its media from cells cultured for 24 hours without FBS were assessed by western blotting . Error bars represent mean ± SEM ( n = 3 per group ) . Statistical analysis was performed by unpaired t - test ( 2 - tailed ) . The experiments were performed twice independently .

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Figure 3Genotypes are indicated as WT for S100a4cre-/- x Fstl1flox+/flox+ mice and cfKO for S100a4cre+/- x Fstl1flox+/flox+. (A) S100a4 expression is induced in the ischemic area (IA). qPCR analysis of mRNA expression of S100a4 in ischemic heart and sham operated heart of WT mice and cfKO mice. Heart samples were harvested at 7 days after the surgery. Statistical analysis was performed by two-way ANOVA. Post Hoc test was performed by Tukey test. Error bars represent mean ± SEM (n=16 and 15 for WT and cfKO sham group, n=15 and 14 for WT and cfKO MI group, respectively).Genotypes are indicated as WT for S100a4cre-/- x Fstl1flox+/flox+ mice and cfKO for S100a4cre+/- x Fstl1flox+/flox+. (B) qPCR analysis of mRNA expression of Fstl1 and Tgfβ1 in sham and post-MI heart. Error bars represent mean ± SEM (n=16 and 15 for WT and cfKO sham group, n=15 and 14 for WT and cfKO MI group, respectively). Statistical analysis was performed by two-way ANOVA. Post Hoc test was performed by Tukey test.Genotypes are indicated as WT for S100a4cre-/- x Fstl1flox+/flox+ mice and cfKO for S100a4cre+/- x Fstl1flox+/flox+. (C) Western blot analysis of Fstl1 protein expression in ischemic and sham-operated hearts at day 7 after the surgery. Quantified values of Fstl1 protein in WT and cfKO mouse hearts normalized by GAPDH band intensity are shown. Statistical analysis was performed by two-way ANOVA. Post Hoc test was performed by Tukey test. Error bars represent mean ± SEM (n=5 for each sham group and n=6 for each MI group).Genotypes are indicated as WT for S100a4cre-/- x Fstl1flox+/flox+ mice and cfKO for S100a4cre+/- x Fstl1flox+/flox+. (D) Fstl1 protein expression in isolated cardiac fibroblasts from cfKO and littermate WT neonatal mice. Cell lysate and its media from cells cultured for 24 hours without FBS were assessed by western blotting. Error bars represent mean ± SEM (n=3 per group). Statistical analysis was performed by unpaired t-test (2-tailed). The experiments were performed twice independently.


Figure 4 ( A ) Mouse survival curve after MI and sham surgery . The mortality of the WT and Fstl1 - cfKO mice after MI surgery was 27 . 1 % and 46 . 7 % , respectively . Log - rank ( Mantel - Cox ) test was used for statistical analysis ( n = 16 for WT sham , n = 15 for Fstl1 - cfKO sham , n = 59 for WT MI and n = 30 for Fstl1 - cfKO MI ) . ( B ) Representative macroscopic images of cardiac rupture . Left ; impending LV rupture found in a cfKO mouse post - MI when sampling . Right ; evidence of hemothorax that was diagnosed as cardiac rupture in deceased mice . ( C ) Representative microscopic images of ruptured heart stained by H & amp ; amp ; E . Penetrating hematoma due to a tear from the LV endocardium to the epicardium . Left ; an image obtained with a low magnification lens . Scale bar indicates 200μm . Right ; higher magnification image . Scale bar indicates 100μm . Arrow indicates the tear site . H indicates hematoma .

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Figure 4(A) Mouse survival curve after MI and sham surgery. The mortality of the WT and Fstl1-cfKO mice after MI surgery was 27.1% and 46.7%, respectively. Log-rank (Mantel-Cox) test was used for statistical analysis (n=16 for WT sham, n=15 for Fstl1-cfKO sham, n=59 for WT MI and n=30 for Fstl1-cfKO MI).(B) Representative macroscopic images of cardiac rupture. Left; impending LV rupture found in a cfKO mouse post-MI when sampling. Right; evidence of hemothorax that was diagnosed as cardiac rupture in deceased mice.(C) Representative microscopic images of ruptured heart stained by H&amp;E. Penetrating hematoma due to a tear from the LV endocardium to the epicardium. Left; an image obtained with a low magnification lens. Scale bar indicates 200μm. Right; higher magnification image. Scale bar indicates 100μm. Arrow indicates the tear site. H indicates hematoma.


Figure 5 ( A ) Transcript mRNA expression of Col1a1 and Fn1 in sham and post - MI hearts . Hearts were obtained at day 7 after surgery . Error bars represent mean ± SEM ( n = 13 - 16 for each group ) . Statistical analysis was performed by two - way ANOVA . Post Hoc test was performed by Tukey test . ( B ) Protein expression of collagen I , fibronectin and αSMA in the infarcted and sham hearts at day 7 after surgery , as assessed by western blotting . Quantified values of proteins of interest were normalized relative to the tubulin band intensity . Error bars represent mean ± SEM . Statistical analysis was performed by unpaired t - test ( 2 - tailed ) for fibronectin and αSMA and non - parametric t - test ( 2 - tailed ) for collagen I ( fibronectin and collagen I : n = 5 per group , αSMA : n = 3 and 4 for WT - IA and cfKO - IA , respectively ) . ( C ) α - smooth muscle actin - positive myofibroblasts ( red ) were stained in histology sections from infarcted hearts of WT and cfKO mice . Samples were harvested at day 7 after MI . Counter staining was performed by hematoxylin . Scale bar indicates 100μm for top and middle images , 50µm for bottom images . Error bars represent mean ± SEM . Statistical analysis was performed by Mann - Whitney test ( n = 16 for WT and n = 14 for cfKO ) . ( D ) Representative images of Picro Sirius Red staining of infarcted heart at 7 days after MI surgery . Images were taken by standard light ( top ) and polarized light ( bottom ) microscopes . Scale bar indicates 100μm . Polarized collagen fibers were sorted to orange - red ( middle ) and green - yellow ( bottom ) fibers by Image J software . Error bars represent mean ± SEM . Statistical analysis was performed by unpaired t - test ( 2 - tailed ) ( n = 11 for WT and n = 13 for cfKO ) .

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 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 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

Figure 5(A) Transcript mRNA expression of Col1a1 and Fn1 in sham and post-MI hearts. Hearts were obtained at day 7 after surgery. Error bars represent mean ± SEM (n=13-16 for each group). Statistical analysis was performed by two-way ANOVA. Post Hoc test was performed by Tukey test.(B) Protein expression of collagen I, fibronectin and αSMA in the infarcted and sham hearts at day 7 after surgery, as assessed by western blotting. Quantified values of proteins of interest were normalized relative to the tubulin band intensity. Error bars represent mean ± SEM. Statistical analysis was performed by unpaired t-test (2-tailed) for fibronectin and αSMA and non-parametric t-test (2-tailed) for collagen I (fibronectin and collagen I: n=5 per group, αSMA: n=3 and 4 for WT-IA and cfKO-IA, respectively).(C) α-smooth muscle actin-positive myofibroblasts (red) were stained in histology sections from infarcted hearts of WT and cfKO mice. Samples were harvested at day 7 after MI. Counter staining was performed by hematoxylin. Scale bar indicates 100μm for top and middle images, 50µm for bottom images. Error bars represent mean ± SEM. Statistical analysis was performed by Mann-Whitney test (n=16 for WT and n=14 for cfKO). (D) Representative images of Picro Sirius Red staining of infarcted heart at 7 days after MI surgery. Images were taken by standard light (top) and polarized light (bottom) microscopes. Scale bar indicates 100μm. Polarized collagen fibers were sorted to orange-red (middle) and green-yellow (bottom) fibers by Image J software. Error bars represent mean ± SEM. Statistical analysis was performed by unpaired t-test (2-tailed) (n=11 for WT and n=13 for cfKO).


Figure 6 ( A ) Immunofluorescence microscopy staining of Fst1 ( green ) and α - SMA ( red ) in NRCFbs at 24 hours after recombinant TGF - β1 ( 10ng / ml ) stimulation . Nuclei were stained by DAPI . Scale bar indicates 50μm . ( B ) Time course changes of Fstl1 protein expression in NRCFbs after stimulation of recombinant TGF - β1 ( 10ng / ml ) . Protein expression of Fstl1 was detected by immunoblotting . Tubulin was used as a loading control ( n = 3 for each time point ) . Two independent experiments were performed . ( C ) Transcript level of Fstl1 , S100a4 and Acta2 mRNA in NRCFbs was determined by qPCR analysis . The samples were harvested at 24 hours after stimulation with recombinant TGF - β1 ( 10ng / ml ) or control vehicle . Error bars represent mean ± SEM ( Fstl1 and S100a4 : n = 6 for each group , Acta2 : n = 3 for each group ) . Statistical analysis was performed by unpaired t - test ( 2 - tailed ) for Fstl1 and S100a4 , and non - parametric unpaired t - test ( 2 - tailed ) for Acta2 . Two independent experiments were performed .

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

Figure 6(A) Immunofluorescence microscopy staining of Fst1 (green) and α-SMA (red) in NRCFbs at 24 hours after recombinant TGF-β1 (10ng/ml) stimulation. Nuclei were stained by DAPI. Scale bar indicates 50μm.(B) Time course changes of Fstl1 protein expression in NRCFbs after stimulation of recombinant TGF-β1 (10ng/ml). Protein expression of Fstl1 was detected by immunoblotting. Tubulin was used as a loading control (n=3 for each time point). Two independent experiments were performed.(C) Transcript level of Fstl1, S100a4 and Acta2 mRNA in NRCFbs was determined by qPCR analysis. The samples were harvested at 24 hours after stimulation with recombinant TGF-β1 (10ng/ml) or control vehicle. Error bars represent mean ± SEM (Fstl1 and S100a4: n=6 for each group, Acta2: n=3 for each group). Statistical analysis was performed by unpaired t-test (2-tailed) for Fstl1 and S100a4, and non-parametric unpaired t-test (2-tailed) for Acta2. Two independent experiments were performed.


Figure 7 ( A ) NRCFbs at passage 1 were stimulated with recombinant Fstl1 ( 50ng / ml ) or vehicle after cultured in serum - reduced conditions ( FBS 0 . 5 % ) for 24 hours . The samples were harvested at the indicated time points after stimulation . The expression of ERK1 / 2 and tubulin were detected by immunoblotting . Error bars represent mean ± SEM . Statistical analysis was performed by one - way ANOVA and Tukey multi comparison test ( n = 3 for each time point ) . Three independent experiments were performed . ( B ) Fstl1 induced phosphorylation of ERK1 / 2 was ablated by pretreatment with MEK inhibitor PD98059 . NRCFbs were treated by PD98059 ( 5nM ) for 30min and then stimulated with recombinant Fstl1 protein ( 50ng / ml ) for 15min . Error bars represent mean ± SEM . Statistical analysis was performed by one - way ANOVA and Tukey multi comparison test ( n = 3 for each time point ) . Two independent experiments were performed . ( C ) Representative images of NRCFbs scratch assay ( left ) and quantified cell migration ( right ) . NRCFbs were transfected by Fstl1 siRNA or siRNA non - targeting negative control for 12 hours followed by culturing in 0 . 5 % FBS media for 24 hours . The confluent cell sheet was scratched and cell migration was assessed at 6 hours after the scratch . Scale bar indicates 100µm . Error bars represent mean ± SEM ( n = 10 for each group ) . Statistical analysis was performed by unpaired t - test ( 2 - tailed ) . Two independent experiments were performed . ( D ) Fstl1 - stimulation of cell migration was reversed by PD98059 . Endogenous Fstl1 in NRCFbs was ablated by Fstl1 siRNA . Serum - deprived NRCFbs were treated with PD98059 ( 5nM ) for 30 min and then stimulated by Fstl1 ( 50ng / ml ) or vehicle . Cell migration was assessed at 6 hours after Fstl1 stimulation . Error bars represent mean ± SEM ( n = 7 - 9 for each group ) . Statistical analysis was performed by Kruskal - Wallis test and Dunnett ' s T3 test . Two independent experiments were performed . ( E ) Morphological changes of NRCFbs after recombinant Fstl1stimulation was assessed by immunocytochemistry . 24 hours serum starved NRCFbs was stimulated by Fstl1 ( 50ng / ml ) or vehicle for 3 hours . PD98059 ( 5nM ) or DMSO was added 30min prior to Fstl1 stimulation . Cells were stained with Alexa Fluor 488 - conjugated phalloidin antibody and nuclei were stained by DAPI . Arrow shows lamellipodium of the cell . Scale bar indicates 50μm .

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

Figure 7(A) NRCFbs at passage 1 were stimulated with recombinant Fstl1 (50ng/ml) or vehicle after cultured in serum-reduced conditions (FBS 0.5%) for 24 hours. The samples were harvested at the indicated time points after stimulation. The expression of ERK1/2 and tubulin were detected by immunoblotting. Error bars represent mean ± SEM. Statistical analysis was performed by one-way ANOVA and Tukey multi comparison test (n=3 for each time point). Three independent experiments were performed.(B) Fstl1 induced phosphorylation of ERK1/2 was ablated by pretreatment with MEK inhibitor PD98059. NRCFbs were treated by PD98059 (5nM) for 30min and then stimulated with recombinant Fstl1 protein (50ng/ml) for 15min. Error bars represent mean ± SEM. Statistical analysis was performed by one-way ANOVA and Tukey multi comparison test (n=3 for each time point). Two independent experiments were performed.(C) Representative images of NRCFbs scratch assay (left) and quantified cell migration (right). NRCFbs were transfected by Fstl1 siRNA or siRNA non-targeting negative control for 12 hours followed by culturing in 0.5% FBS media for 24 hours. The confluent cell sheet was scratched and cell migration was assessed at 6 hours after the scratch. Scale bar indicates 100µm. Error bars represent mean ± SEM (n=10 for each group). Statistical analysis was performed by unpaired t-test (2-tailed). Two independent experiments were performed.(D) Fstl1-stimulation of cell migration was reversed by PD98059. Endogenous Fstl1 in NRCFbs was ablated by Fstl1 siRNA. Serum-deprived NRCFbs were treated with PD98059 (5nM) for 30 min and then stimulated by Fstl1 (50ng/ml) or vehicle. Cell migration was assessed at 6 hours after Fstl1 stimulation. Error bars represent mean ± SEM (n=7-9 for each group). Statistical analysis was performed by Kruskal-Wallis test and Dunnett's T3 test. Two independent experiments were performed.(E) Morphological changes of NRCFbs after recombinant Fstl1stimulation was assessed by immunocytochemistry. 24 hours serum starved NRCFbs was stimulated by Fstl1 (50ng/ml) or vehicle for 3 hours. PD98059 (5nM) or DMSO was added 30min prior to Fstl1 stimulation. Cells were stained with Alexa Fluor 488-conjugated phalloidin antibody and nuclei were stained by DAPI. Arrow shows lamellipodium of the cell. Scale bar indicates 50μm.


Figure 8 ( A , B ) Fibroblast proliferation was assessed by Edu incorporation assay . Endogenous Fstl1 was ablated by siRNA . NRCFbs were cultured in 0 . 5 % FBS condition for 48 hours to synchronize the cell cycle . EdU ( 10μM as the final concentration ) was added into media at 4 hours before harvest . Error bars represent mean ± SEM ( n = 5 , each group ) . Statistical analysis was performed by unpaired t - test ( 2 - tailed ) . Two independent experiments were performed . ( B ) Effect of exogenous Fstl1 on cardiac fibroblast proliferation . Cells were cultured in FBS 0 . 5 % media for 24 hours . Recombinant Fstl1 ( 50ng / ml ) or vehicle were added to 2 % FBS - containing media and cultured for 48 hours . EdU was added into media at 4 hours before harvest . Error bars represent mean ± SEM ( n = 7 , each group ) . Statistical analysis was performed by unpaired t - test ( 2 - tailed ) . Two independent experiments were performed . ( C ) Fstl1 promoted fibroblasts proliferation was diminished by PD98059 . PD98059 ( 5nM ) was added 30 min prior to recombinant Fstl1 stimulation ( 50ng / ml ) . The cells were cultured for 48 hours , and EdU was added into media at 4 hours before harvest . Error bars represent mean ± SEM ( n = 4 - 10 , per treatment group ) . Statistical analysis was performed by one - way ANOVA and Tukey multi comparison test . Two independent experiments were performed .

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 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

Figure 8(A, B) Fibroblast proliferation was assessed by Edu incorporation assay. Endogenous Fstl1 was ablated by siRNA. NRCFbs were cultured in 0.5% FBS condition for 48 hours to synchronize the cell cycle. EdU (10μM as the final concentration) was added into media at 4 hours before harvest. Error bars represent mean ± SEM (n=5, each group). Statistical analysis was performed by unpaired t-test (2-tailed). Two independent experiments were performed. (B) Effect of exogenous Fstl1 on cardiac fibroblast proliferation. Cells were cultured in FBS 0.5% media for 24 hours. Recombinant Fstl1 (50ng/ml) or vehicle were added to 2% FBS-containing media and cultured for 48 hours. EdU was added into media at 4 hours before harvest. Error bars represent mean ± SEM (n=7, each group). Statistical analysis was performed by unpaired t-test (2-tailed). Two independent experiments were performed.(C) Fstl1 promoted fibroblasts proliferation was diminished by PD98059. PD98059 (5nM) was added 30 min prior to recombinant Fstl1 stimulation (50ng/ml). The cells were cultured for 48 hours, and EdU was added into media at 4 hours before harvest. Error bars represent mean ± SEM (n=4-10, per treatment group). Statistical analysis was performed by one-way ANOVA and Tukey multi comparison test. Two independent experiments were performed.


Figure 1B ) Western blots showing absence of mitochondrial proteins from the different mitochondrial complexes : NDUFB8 ( complex I ) , SDHA ( complex II ) , UQCRC2 ( complex III ) and MTCO - 1 ( complex IV ) , in senescent ( 10 days after 20Gy X - ray ) Control and Parkin - expressing MRC5 fibroblasts . Data is representative of 3 independent experiments ; C ) Cellular oxygen consumption rates ( OCR ) in senescent ( 10 days after 20Gy X - ray ) Control and Parkin - expressing MRC5 fibroblasts . Data were obtained using Seahorse XF24 analyzer and shows mean ± S . D . n = 4 technical repeats ( representative of 2 independent experiments ) ; D ) Representative 3D EM pictures of senescent ( 20 days after 20Gy X - ray ) Parkin - expressing MRC5 fibroblasts with or without CCCP . Mitochondria are in red , the nucleus in blue ; E ) Representative images of cell size , Sen - β - Gal activity ( blue cytoplasmic staining ) , macro - H2A foci and SDHA immunofluorescence in proliferating and senescent ( 10 days after 20Gy X - ray ) Parkin - expressing MRC5 fibroblasts ; F ) Quantification of ROS levels ( DHE fluorescence ) , Sen - β - Gal positive cells and Senescence - associated heterochromatin foci ( SAHF ) observed by DAPI in proliferating and senescent ( 10 days after 20Gy X - ray ) Control and Parkin - expressing MRC5 fibroblasts . Data are mean ± S . E . M of n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA . G ) Representative western blots showing p21 and p16 expression in proliferating and senescent ( 10 days after 20Gy X - ray ) Control and Parkin - expressing MRC5 fibroblasts . Data are representative of 3 independent experiments ; H ) Secreted protein measured in a cytokine array ( RayBiotech ) of a variety of inflammatory proteins in proliferating and senescent ( 10 days after 20Gy X - ray ) Parkin - expressing MRC5 fibroblasts . Data are mean ± S . E . M of n = 3 independent experiments . Asterisks denote statistical significant P < 0 . 05 One - way ANOVA .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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) Western blots showing absence of mitochondrial proteins from the different mitochondrial complexes: NDUFB8 (complex I), SDHA (complex II), UQCRC2 (complex III) and MTCO-1 (complex IV), in senescent (10 days after 20Gy X-ray) Control and Parkin-expressing MRC5 fibroblasts. Data is representative of 3 independent experiments;C) Cellular oxygen consumption rates (OCR) in senescent (10 days after 20Gy X-ray) Control and Parkin-expressing MRC5 fibroblasts. Data were obtained using Seahorse XF24 analyzer and shows mean±S.D. n=4 technical repeats (representative of 2 independent experiments);D) Representative 3D EM pictures of senescent (20 days after 20Gy X-ray) Parkin-expressing MRC5 fibroblasts with or without CCCP. Mitochondria are in red, the nucleus in blue;E) Representative images of cell size, Sen-β-Gal activity (blue cytoplasmic staining), macro-H2A foci and SDHA immunofluorescence in proliferating and senescent (10 days after 20Gy X-ray) Parkin- expressing MRC5 fibroblasts;F) Quantification of ROS levels (DHE fluorescence), Sen-β-Gal positive cells and Senescence-associated heterochromatin foci (SAHF) observed by DAPI in proliferating and senescent (10 days after 20Gy X-ray) Control and Parkin-expressing MRC5 fibroblasts. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.G) Representative western blots showing p21 and p16 expression in proliferating and senescent (10 days after 20Gy X-ray) Control and Parkin-expressing MRC5 fibroblasts. Data are representative of 3 independent experiments;H) Secreted protein measured in a cytokine array (RayBiotech) of a variety of inflammatory proteins in proliferating and senescent (10 days after 20Gy X-ray) Parkin-expressing MRC5 fibroblasts. Data are mean±S.E.M of n=3 independent experiments. Asterisks denote statistical significant P<0.05 One-way ANOVA.


Figure 3A ) Representative images of colony assays of wild - type and PGC - 1β - / - MEFs grown at 3 % or 21 % O2 ( 10 days after seeding 5 , 000 cells / well ) . Data are representative of 3 independent experiments ; B ) Effect of 3 % or 21 % O2 and X - ray irradiation ( at 3 % O2 ) on the percentage of Ki67 ( at day 6 ) and Sen - β - Gal positive cells ( at day 10 ) and number ( N ) of 53BP1 foci ( at day 6 ) in wild - type and PGC - 1β - / - MEFs . Data are mean ± S . E . M of n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA . C ) Representative images of Sen - β - Gal activity ( blue cytoplasmatic staining ) , Ki - 67 and 53BP1 foci in proliferating and senescent wild - type and PGC - 1β - / - MEFs ( scale bar = 10µm ) ; D ) mRNA expression of PGC - 1β CXCL - 1 and p16 in proliferating and senescent ( 10 days after 10Gy X - ray ) wild type and PGC - 1β - / - MEFs . Data are mean ± S . E . M . of n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA . E ) Effects of overexpression of PGC - 1β on the percentage of Ki67 and Sen - β - Gal positive cells , number ( N ) of 53BP1 foci and percentage change of mitochondrial mass ( NAO intensity ) in proliferating and senescent ( 2 days after 10Gy X - ray ) MEFs cultured at 3 % O2 . Data are mean ± S . E . M of n = 3 independent experiments . Asterisks denote statistical significant P < 0 . 05 One - way ANOVA .

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

Figure 3A) Representative images of colony assays of wild-type and PGC-1β -/- MEFs grown at 3% or 21% O2 (10 days after seeding 5,000 cells/well). Data are representative of 3 independent experiments;B) Effect of 3% or 21% O2 and X-ray irradiation (at 3% O2) on the percentage of Ki67 (at day 6) and Sen-β-Gal positive cells (at day 10) and number (N) of 53BP1 foci (at day 6) in wild-type and PGC-1β -/- MEFs. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.C) Representative images of Sen-β-Gal activity (blue cytoplasmatic staining), Ki-67 and 53BP1 foci in proliferating and senescent wild-type and PGC-1β-/- MEFs (scale bar=10µm);D) mRNA expression of PGC-1β CXCL-1 and p16 in proliferating and senescent (10 days after 10Gy X-ray) wild type and PGC-1β-/- MEFs. Data are mean±S.E.M. of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.E) Effects of overexpression of PGC-1β on the percentage of Ki67 and Sen-β-Gal positive cells, number (N) of 53BP1 foci and percentage change of mitochondrial mass (NAO intensity) in proliferating and senescent (2 days after 10Gy X-ray) MEFs cultured at 3% O2. Data are mean±S.E.M of n=3 independent experiments. Asterisks denote statistical significant P<0.05 One-way ANOVA.


Figure 4A ) Representative western blot of mTORC1 activity measured by phosphorylated p70S6K ( T389 ) from 6 to 72 hours after 20Gy in MRC5 fibroblasts . Data are representative of 3 independent experiments ; B ) Representative western blots of the mitochondrialproteins TOMM20 , NDUFB8 ( complex I ) , SDHA ( complex II ) , UQCRC2 ( complex III ) and MT - CO1 ( complex IV ) following 20Gy irradiation with or without 100nM Rapamycin treatment in MRC5fibroblasts . Data are representative of 4 independent experiments ; C ) Effect of 100nM rapamycin on mitochondrial mass ( measured by NAO fluorescence ) 2 - 4 days following replication exhaustion ( RS ) or genotoxic stress ( generated by X - ray irradiation , Etoposide , Neocarcinostatin ( NCS ) , H2O2 or telomere dysfunction ( TRF2ΔBΔM ) ) in a variety of cell lines . Data are mean from 3 independent experiments per cell line or treatment ; D ) ( top ) Representative transmission electron microscopy ( T . E . M . ) micrographs of proliferating and senescent ( 3 days after 20Gy X - ray ) MRC5 fibroblasts treated with or without 100nM rapamycin . Mitochondria are labeled in pink . Scale bar = 2µm ; ( bottom left and middle ) Quantification of mitochondrial volume fraction ( % Vv ) and mitochondrial number per cross section in proliferating and senescent ( 3 days after 20Gy X - ray ) MRC5 fibroblasts treated with or without 100nM rapamycin . T . E . M . mitochondrial analysis are mean ± S . E . M of 24 electron micrographs per condition ; ( bottom right ) mtDNA copy number analysis by q - PCR in proliferating and senescent ( 3 days after 20Gy X - ray ) MRC5 fibroblasts treated with or without 100nM Rapamycin . Data are mean ± S . E . M of n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA . E ) Representative western blots showing expression of phosphorylated p70S6K ( T389 ) and AKT ( S473 ) , the mitochondrial protein NDUFB8 and the DDR downstream target p21 in MRC5 fibroblasts treated with or without 10µM of the ATM inhibitor KU55933 and in fibroblasts from a patient with Ataxia Telangiectasis ( AT ) at different time points after 20Gy X - ray . Data are representative of 3 independent experiments ( ATM inhibitor ) and 1 experiment ( AT patient ) ; F ) Western blots showing the effect of the ATM inhibitor KU55933 on γH2A . X , AKT phosphorylation and p21 expression in MRC5fibroblasts after 20Gy X - ray . Data is from one experiment ; G ) Effect of rapamycin and / or ATM inhibitor ( KU55933 ) on mitochondrial mass ( NAO intensity ) in proliferating and senescent ( 3 days after 20Gy X - ray ) MRC5 fibroblasts . Data are mean ± S . E . M of n = 3 independent experiments . Asterisks denote statistical significant P < 0 . 05 One - way ANOVA .

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

Figure 4A) Representative western blot of mTORC1 activity measured by phosphorylated p70S6K (T389) from 6 to 72 hours after 20Gy in MRC5 fibroblasts. Data are representative of 3 independent experiments;B) Representative western blots of the mitochondrialproteins TOMM20, NDUFB8 (complex I), SDHA (complex II), UQCRC2 (complex III) and MT-CO1 (complex IV) following 20Gy irradiation with or without 100nM Rapamycin treatment in MRC5fibroblasts. Data are representative of 4 independent experiments;C) Effect of 100nM rapamycin on mitochondrial mass (measured by NAO fluorescence) 2-4 days following replication exhaustion (RS) or genotoxic stress (generated by X-ray irradiation, Etoposide, Neocarcinostatin (NCS), H2O2 or telomere dysfunction (TRF2ΔBΔM)) in a variety of cell lines. Data are mean from 3 independent experiments per cell line or treatment;D) (top) Representative transmission electron microscopy (T.E.M.) micrographs of proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts treated with or without 100nM rapamycin. Mitochondria are labeled in pink. Scale bar=2µm; (bottom left and middle) Quantification of mitochondrial volume fraction (%Vv) and mitochondrial number per cross section in proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts treated with or without 100nM rapamycin. T.E.M. mitochondrial analysis are mean±S.E.M of 24 electron micrographs per condition; (bottom right) mtDNA copy number analysis by q-PCR in proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts treated with or without 100nM Rapamycin. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.E) Representative western blots showing expression of phosphorylated p70S6K (T389) and AKT (S473), the mitochondrial protein NDUFB8 and the DDR downstream target p21 in MRC5 fibroblasts treated with or without 10µM of the ATM inhibitor KU55933 and in fibroblasts from a patient with Ataxia Telangiectasis (AT) at different time points after 20Gy X-ray. Data are representative of 3 independent experiments (ATM inhibitor) and 1 experiment (AT patient);F) Western blots showing the effect of the ATM inhibitor KU55933 on γH2A.X, AKT phosphorylation and p21 expression in MRC5fibroblasts after 20Gy X-ray. Data is from one experiment;G) Effect of rapamycin and/or ATM inhibitor (KU55933) on mitochondrial mass (NAO intensity) in proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts. Data are mean±S.E.M of n=3 independent experiments. Asterisks denote statistical significant P<0.05 One-way ANOVA.


Figure 5A ) ROS levels ( DHE intensity ) and mean number ( N ) of yH2A . X foci after mTOR knockdown ( 72 hours ) in proliferating and senescent ( 2 days after 20Gy X - ray ) MRC5 fibroblasts . Data are mean ± S . E . M of n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA . B ) ROS levels ( DHE intensity ) and mean number ( N ) of yH2A . X foci in proliferating and senescent ( 3 days after 20Gy X - ray ) MRC5 fibroblasts treated with or without 100nM Rapamycin and / or 2 . 5mM of the antioxidant NAC . Data are mean ± S . E . M of n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA . C ) ( top ) Scheme illustrating the experimental design : human MRC5 fibroblasts were irradiated with 20Gy X - ray and treated at day 3 after IR with 10µM of the ATM inhibitor ( ATMi ) KU55933 and / or 100nM Rapamycin ( Rap ) ; ( bottom ) Effect of single or combined inhibition of ATM and mTORC1 on the mean number ( N ) of γH2A . X foci , Sen - β - Gal activity and p21 expression in proliferating and senescent ( 10 days after 20Gy X - ray ) MRC5fibroblasts . Data are mean ± S . E . M of n = 3 independent experiments . Western blots are representative of 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA . D ) ROS levels ( DHE intensity ) in proliferating and senescent ( 3 days after 10Gy X - ray ) wild - type and PGC - 1β - / - MEFs ( top ) and PGC - 1β overexpressing MEFs ( bottom ) . Data are mean ± S . E . M . of n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA . E ) ( top ) Representative images and quantification of immunofluorescence staining against FLAG - PGC - 1β ( red ) and 53BP1 ( green ) in proliferating and senescent ( 2 days after 10Gy X - ray ) PGC - 1β overexpressing MEFs cultured at 3 % O2 , with or without 250µM of the antioxidant Trolox ( scale bar = 10µm ) . Data are mean ± S . E . M , n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA . F ) ( top ) Effect of overexpression of mutated Rheb ( N153T ) and ( bottom ) effect of 100nM rapamycin on the mean number ( N ) of 53BP1 foci in proliferating and senescent ( 3 days after 10Gy X - ray ) wild - type and PGC - 1β - / - MEFs . Data are mean ± S . E . M of n = 3 independent experiments ( at least 125 cells were analyzed per condition ) . Asterisks denote statistical significant P < 0 . 05 One - way ANOVA .

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

Figure 5A) ROS levels (DHE intensity) and mean number (N) of yH2A.X foci after mTOR knockdown (72 hours) in proliferating and senescent (2 days after 20Gy X-ray) MRC5 fibroblasts. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.B) ROS levels (DHE intensity) and mean number (N) of yH2A.X foci in proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts treated with or without 100nM Rapamycin and/or 2.5mM of the antioxidant NAC. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.C) (top) Scheme illustrating the experimental design: human MRC5 fibroblasts were irradiated with 20Gy X-ray and treated at day 3 after IR with 10µM of the ATM inhibitor (ATMi) KU55933 and/or 100nM Rapamycin (Rap); (bottom) Effect of single or combined inhibition of ATM and mTORC1 on the mean number (N) of γH2A.X foci, Sen-β-Gal activity and p21 expression in proliferating and senescent (10 days after 20Gy X-ray) MRC5fibroblasts. Data are mean±S.E.M of n=3 independent experiments. Western blots are representative of 3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.D) ROS levels (DHE intensity) in proliferating and senescent (3 days after 10Gy X-ray) wild-type and PGC-1β-/- MEFs (top) and PGC-1β overexpressing MEFs (bottom). Data are mean±S.E.M. of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.E) (top) Representative images and quantification of immunofluorescence staining against FLAG-PGC-1β (red) and 53BP1 (green) in proliferating and senescent (2 days after 10Gy X-ray) PGC-1β overexpressing MEFs cultured at 3% O2, with or without 250µM of the antioxidant Trolox (scale bar=10µm). Data are mean±S.E.M, n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.F) (top) Effect of overexpression of mutated Rheb (N153T) and (bottom) effect of 100nM rapamycin on the mean number (N) of 53BP1 foci in proliferating and senescent (3 days after 10Gy X-ray) wild-type and PGC-1β -/- MEFs. Data are mean±S.E.M of n=3 independent experiments (at least 125 cells were analyzed per condition). Asterisks denote statistical significant P<0.05 One-way ANOVA.


Figure 6A ) ( top ) Representative image of immunofluorescence double staining for the mitochondrial protein MT - CO1 and the DDR marker yH2A . X ( scale bar = 5µm ) and ( bottom ) quantification of MT - CO1 intensity vs number of yH2A . X foci in hepatocytes from 12 months old mice . Data are mean ± S . E . M of n = 3 mice ( at least 30 cells were analyzed per mouse ) ; Asterisks denote statistical significant P < 0 . 05 using Two - tailed t - test . B ) Representative western blots of pS6 , S6 , p21 , PGC - 1β , MT - CO1 and NDUFB8 protein expression in mouse liver tissue at 3 and 12 months of age . Data are from n = 3 mice per group ; C ) Effect of 4 months of rapamycin supplemented diet on PGC - 1β expression in liver tissue of 16 months old mice . Data are from n = 3 mice per group ; D ) ( top ) Quantification of mitochondrial number per cross section and mitochondrial volume fraction ( % Vv ) in hepatocytes from 16 months old mice with or without 4 months rapamycin diet . Data are mean ± S . E . M of n = 3 mice per group ( at least 20 cells were analyzed per mouse ) ; ( bottom ) Representative electron micrographs of hepatocytes from 16 months old mice with or without 4 months rapamycin diet , ( mitochondria are labelled in pink ) . Scale bar = 5µm ; E ) mtDNA copy number ( measured by q - PCR ) in mice liver tissue at 3 , 12 , 16 months and at 16 months after 4 months rapamycin diet . Data are mean ± S . E . M of n = 3 - 4 mice per group ; Asterisks denote statistical significant P < 0 . 05 using One - way ANOVA . F ) Oxygen consumption rates ( OCR ) in isolated liver mitochondria from 16 months old mice fed with or without rapamycin for 4 months , in the presence of pyruvate / malate . State III was induced by injection of ADP . State IV was induced by inhibition of the ATP synthase with oligomycin and uncoupled respiration rates were determined by injection of FCCP . Antimycin A ( AA ) was used to determine background , non - mitochondrial OXPHOS , OCR . Data are mean ± S . E . M of n = 5 mice per group ; G ) ( top ) Representative immuno - TeloFISH images of hepatocytes from 3 and 15 months old mice with or without rapamycin ( 12 months diet ) . Co - localizing foci are amplified in the right panel ( amplified images are from single Z - planes where co - localization was found ) ; ( bottom ) Dot plot graph of Telomere - associated foci ( TAF ) in 3 , 15 and 16 months old mice ( 15 and 16 months old mice were fed with rapamycin for 12 and 4 months respectively ) . Data are from n = 3 - 9 mice per group ( at least 50 cells were analyzed per mice ) ; H ) ( top ) 4 months old and 15 months old micelivers [ control ( - ) or rapamycin ( + ) ] stained with Sen - - β - Gal solution ( Sen - - β - Gal activity is evidenced by blue staining ) . Data are from n = 3 mice per group ; ( bottom ) Representative image showing Sen - - β - Gal staining ( Sen - - β - Gal activity is evidenced by blue staining ) in hepatocytes and corresponding immuno - TeloFISH ( arrows represent co - localizing foci ) ; Asterisks denote statistical significant P < 0 . 05 using One - way ANOVA . I ) Representative western blot showing the effect of 4 months rapamycin feeding on p21 expression in 16 months old mice . Data are from n = 3 mice per group ; j ) Effect of 4 months rapamycin feeding on mRNA expression of the SASP components CXCL1 , CXCL5 and Inhibin A in liver tissue of 16 months old mice with or without 4 months rapamycin treatment . Data are from n = 5 mice per condition ; Asterisks denote statistical significant P < 0 . 05 using Two - tailed t - test . K ) Mean number of TAF in hepatocytes of wild type and PGC - 1 - β - / - mice with 18 months of age . Data are mean ± S . E . M of n = 4 mice per group ; Asterisks denote statistical significant P < 0 . 05 using Two - tailed 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 6A) (top) Representative image of immunofluorescence double staining for the mitochondrial protein MT-CO1 and the DDR marker yH2A.X (scale bar=5µm) and (bottom) quantification of MT-CO1 intensity vs number of yH2A.X foci in hepatocytes from 12 months old mice. Data are mean±S.E.M of n=3 mice (at least 30 cells were analyzed per mouse); Asterisks denote statistical significant P<0.05 using Two-tailed t-test.B) Representative western blots of pS6, S6, p21, PGC-1β, MT-CO1 and NDUFB8 protein expression in mouse liver tissue at 3 and 12 months of age. Data are from n=3 mice per group;C) Effect of 4 months of rapamycin supplemented diet on PGC-1β expression in liver tissue of 16 months old mice. Data are from n=3 mice per group;D) (top) Quantification of mitochondrial number per cross section and mitochondrial volume fraction (%Vv) in hepatocytes from 16 months old mice with or without 4 months rapamycin diet. Data are mean±S.E.M of n=3 mice per group (at least 20 cells were analyzed per mouse); (bottom) Representative electron micrographs of hepatocytes from 16 months old mice with or without 4 months rapamycin diet, (mitochondria are labelled in pink). Scale bar=5µm;E) mtDNA copy number (measured by q-PCR) in mice liver tissue at 3, 12, 16 months and at 16 months after 4 months rapamycin diet. Data are mean±S.E.M of n=3-4 mice per group; Asterisks denote statistical significant P<0.05 using One-way ANOVA.F) Oxygen consumption rates (OCR) in isolated liver mitochondria from 16 months old mice fed with or without rapamycin for 4 months, in the presence of pyruvate/malate. State III was induced by injection of ADP. State IV was induced by inhibition of the ATP synthase with oligomycin and uncoupled respiration rates were determined by injection of FCCP. Antimycin A (AA) was used to determine background, non-mitochondrial OXPHOS, OCR. Data are mean±S.E.M of n=5 mice per group;G) (top) Representative immuno-TeloFISH images of hepatocytes from 3 and 15 months old mice with or without rapamycin (12 months diet). Co-localizing foci are amplified in the right panel (amplified images are from single Z-planes where co-localization was found); (bottom) Dot plot graph of Telomere-associated foci (TAF) in 3, 15 and 16 months old mice (15 and 16 months old mice were fed with rapamycin for 12 and 4 months respectively). Data are from n=3-9 mice per group (at least 50 cells were analyzed per mice);H) (top) 4 months old and 15 months old micelivers [control (-) or rapamycin (+)] stained with Sen--β-Gal solution (Sen--β-Gal activity is evidenced by blue staining). Data are from n=3 mice per group; (bottom) Representative image showing Sen--β-Gal staining (Sen--β-Gal activity is evidenced by blue staining) in hepatocytes and corresponding immuno-TeloFISH (arrows represent co-localizing foci); Asterisks denote statistical significant P<0.05 using One-way ANOVA.I) Representative western blot showing the effect of 4 months rapamycin feeding on p21 expression in 16 months old mice. Data are from n=3 mice per group;j) Effect of 4 months rapamycin feeding on mRNA expression of the SASP components CXCL1, CXCL5 and Inhibin A in liver tissue of 16 months old mice with or without 4 months rapamycin treatment. Data are from n=5 mice per condition; Asterisks denote statistical significant P<0.05 using Two-tailed t-test.K) Mean number of TAF in hepatocytes of wild type and PGC-1-β-/- mice with 18 months of age. Data are mean±S.E.M of n=4 mice per group; Asterisks denote statistical significant P<0.05 using Two-tailed t-test.


Figure 1 ( A ) NSCLC expressing single site mutants of EGFR , PC9 ( 3X106 ) or H3255 ( 8X106 ) , were seeded in 10 - cm dishes . On the next day , complete media were replaced with media containing serum ( 1 % ) and the cells were treated for 24 hours with different EGFR - specific TKIs ( erlotinib , 50 nM ; osimertinib , 50 nM , or afatinib , 10 nM ) , either alone or in combination with 2XmAbs ( cetuximab and trastuzumab , 5 μg / ml each ) . Thereafter , cells were washed with cold saline and extracted . Proteins were separated using gel electrophoresis and transferred onto nitrocellulose membranes . After blocking , membranes were incubated overnight with the indicated primary antibodies , followed by incubation with horseradish peroxidase - conjugated secondary antibodies ( 60 minutes ) , and treatment with Clarity ™ Western ECL Blotting Substrates ( Bio - Rad ) . ECL signals were detected using the ChemiDoc ™ Imaging System ( Bio - Rad ) and images were acquired using the ImageLab software . Signals ( relative to Control ) were quantified and normalized to the signals of GAPDH ( numbers shown below each lane ) . with different EGFR - specific TKIs ( erlotinib , 50 nM ; osimertinib , 50 nM , or afatinib , 10 nM ) , either alone or in combination with 2XmAbs ( cetuximab and trastuzumab , 5 μg / ml each ) . ( C ) PC9 cells were seeded on coverslips and treated for 24 hours Cells were washed in acidic buffer , fixed in paraformaldehyde ( 4 % ) and incubated with specific primary antibodies , followed by an Alexa Fluor 555 - conjugated secondary antibody ( pseudo colored in green ) . DAPI ( blue ) was used to stain nuclei . Images were captured using a confocal microscope ( 40X magnification ) .

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 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

Figure 1(A) NSCLC expressing single site mutants of EGFR, PC9 (3X106) or H3255 (8X106), were seeded in 10-cm dishes. On the next day, complete media were replaced with media containing serum (1%) and the cells were treated for 24 hours with different EGFR-specific TKIs (erlotinib, 50 nM; osimertinib, 50 nM, or afatinib, 10 nM), either alone or in combination with 2XmAbs (cetuximab and trastuzumab, 5 μg/ml each). Thereafter, cells were washed with cold saline and extracted. Proteins were separated using gel electrophoresis and transferred onto nitrocellulose membranes. After blocking, membranes were incubated overnight with the indicated primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (60 minutes), and treatment with Clarity™ Western ECL Blotting Substrates (Bio-Rad). ECL signals were detected using the ChemiDoc™ Imaging System (Bio-Rad) and images were acquired using the ImageLab software. Signals (relative to Control) were quantified and normalized to the signals of GAPDH (numbers shown below each lane).with different EGFR-specific TKIs (erlotinib, 50 nM; osimertinib, 50 nM, or afatinib, 10 nM), either alone or in combination with 2XmAbs (cetuximab and trastuzumab, 5 μg/ml each). (C) PC9 cells were seeded on coverslips and treated for 24 hours Cells were washed in acidic buffer, fixed in paraformaldehyde (4%) and incubated with specific primary antibodies, followed by an Alexa Fluor 555-conjugated secondary antibody (pseudo colored in green). DAPI (blue) was used to stain nuclei. Images were captured using a confocal microscope (40X magnification).