( 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 O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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).


( 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 .

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(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 .

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(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 .

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(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.


( 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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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.


( 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 .

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(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 .

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(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.


B ) 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 ;

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B) 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 ) ;

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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 ;

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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 ;

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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 .

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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 ;

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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 .

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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.


A ) 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 ;

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A) 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 .

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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 ) ;

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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 .

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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 .

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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.


A ) 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 ;

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A) 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 ;

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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 ;

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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 .

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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 ) ;

O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O

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 ;

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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 .

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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.


A ) 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 .

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A) 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 .

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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 .

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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 .

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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 .

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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 .

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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.


A ) ( 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 .

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A) (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 ;

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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 ;

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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 ;

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

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 .

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

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 ;

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

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 ) ;

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

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 .

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

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 ;

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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 .

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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 .

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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.


( 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 ) .

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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).


PC9 cells ( 3X106 / mouse ) were subcutaneously implanted in the flanks of CD1 - nu / nu mice . When tumors became palpable , mice were randomized in groups of 5 - 9 animals and treated for 90 days ( grey areas ) with 2XmAbs ( cetuximab plus trastuzumab , 0 . 2 mg / mouse / injection ) , once every three days , or with erlotinib ( 50 mg / kg / day ) , once per day . Alternatively , mice were treated with combinations of erlotinib ( 50 , 20 , 10 , 5 or 1 mg / kg ) and the two monoclonal antibodies . Following 60 days of treatment reduced the frequency of erlotinib administration to once every other day ( underneath dotted line ) . animal survival ( B ) are shown . Mice were euthanized when tumor size reached 1 , 500 mm3 .

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

PC9 cells (3X106/mouse) were subcutaneously implanted in the flanks of CD1-nu/nu mice. When tumors became palpable, mice were randomized in groups of 5-9 animals and treated for 90 days (grey areas) with 2XmAbs (cetuximab plus trastuzumab, 0.2 mg/mouse/injection), once every three days, or with erlotinib (50 mg/kg/day), once per day. Alternatively, mice were treated with combinations of erlotinib (50, 20, 10, 5 or 1 mg/kg) and the two monoclonal antibodies. Following 60 days of treatment reduced the frequency of erlotinib administration to once every other day (underneath dotted line). animal survival (B) are shown. Mice were euthanized when tumor size reached 1,500 mm3.


PC9 cells ( exon 19 deletion ) were subcutaneously implanted in the flank of CD1 - nu / nu mice ( 3X106 / mouse ) . When tumors became palpable , mice were randomized in groups of 5 - 9 animals and treated for 90 days ( grey areas ) with 2XmAbs ( cetuximab plus trastuzumab , each at 0 . 1 mg / mouse / injection ) once every three days , or daily with different TKIs : osimertinib ( 5 mg / kg ) , afatinib ( 5 mg / kg ) or imatinib ( 100 mg / kg ) , either alone or in combination with the two antibodies . Following 60 days of treatment , the frequency of TKI administration was reduced to once every other day ( underneath dotted lines ) , while mAb treatment remained unaltered . animal survival ( B ) are shown . Mice were euthanized when tumor size reached 1 , 500 mm3 .

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

PC9 cells (exon 19 deletion) were subcutaneously implanted in the flank of CD1-nu/nu mice (3X106/mouse). When tumors became palpable, mice were randomized in groups of 5-9 animals and treated for 90 days (grey areas) with 2XmAbs (cetuximab plus trastuzumab, each at 0.1 mg/mouse/injection) once every three days, or daily with different TKIs: osimertinib (5 mg/kg), afatinib (5 mg/kg) or imatinib (100 mg/kg), either alone or in combination with the two antibodies. Following 60 days of treatment, the frequency of TKI administration was reduced to once every other day (underneath dotted lines), while mAb treatment remained unaltered. animal survival (B) are shown. Mice were euthanized when tumor size reached 1,500 mm3.


CD1 - nu / nu mice carrying PC9 xenografts were divided in groups ( 2 - 3 mice / group ) and treated for seven days with different TKIs : erlotinib ( 50 mg / kg or 20 mg / kg ) , osimertinib ( 5 mg / kg ) or afatinib ( 5 mg / kg ) , either alone or in combination with 2XmAbs ( cetuximab and trastuzumab , each at 0 . 1 mg / mouse / injection ) . when singly applied used erlotinib at 50 mg / kg . ( B ) After treatment , all mice were sacrificed and tumors extracted . Protein extracts were resolved by means of electrophoresis and transfer to nitrocellulose membranes , which were later incubated overnight with the indicated antibodies . This was followed by incubation with peroxidase - conjugated secondary antibodies . Signals were detected using ChemidocTM ( from Bio - Rad ) , quantified and normalized to the signals of GAPDH or tubulin ( numbers shown below each lane ) .

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

CD1-nu/nu mice carrying PC9 xenografts were divided in groups (2-3 mice/group) and treated for seven days with different TKIs: erlotinib (50 mg/kg or 20 mg/kg), osimertinib (5 mg/kg) or afatinib (5 mg/kg), either alone or in combination with 2XmAbs (cetuximab and trastuzumab, each at 0.1 mg/mouse/injection). when singly applied used erlotinib at 50 mg/kg. (B) After treatment, all mice were sacrificed and tumors extracted. Protein extracts were resolved by means of electrophoresis and transfer to nitrocellulose membranes, which were later incubated overnight with the indicated antibodies. This was followed by incubation with peroxidase-conjugated secondary antibodies. Signals were detected using ChemidocTM (from Bio-Rad), quantified and normalized to the signals of GAPDH or tubulin (numbers shown below each lane).


A , B PC9 cells ( 3X106 / mouse ) were subcutaneously injected in the flanks of CD1 - nu / nu mice . When tumors reached a volume of approximately 500 mm3 , mice were treated daily with erlotinib ( 50 mg / kg , blue area ; panel A ) or with osimertinib ( 5 mg / kg , grey area ; panel B ) using oral gavage . Initially , all tumors displayed stable disease or they regressed , but eventually all started relapsing . Once relapsing tumors reached 800 mm3 , while under erlotinib or osimertinib treatment , we supplemented the treatment with a combination of 2XmAbs ( cetuximab and trastuzumab , each at 0 . 1 mg / mouse / injection ; twice a week ) plus either erlotinib ( 50 mg / kg ; A , red area ) or osimertinib ( 5 mg / kg ; B , orange area ) . Tumor volumes of individual mice are shown .

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

A, B PC9 cells (3X106/mouse) were subcutaneously injected in the flanks of CD1-nu/nu mice. When tumors reached a volume of approximately 500 mm3, mice were treated daily with erlotinib (50 mg/kg, blue area; panel A) or with osimertinib (5 mg/kg, grey area; panel B) using oral gavage. Initially, all tumors displayed stable disease or they regressed, but eventually all started relapsing. Once relapsing tumors reached 800 mm3, while under erlotinib or osimertinib treatment, we supplemented the treatment with a combination of 2XmAbs (cetuximab and trastuzumab, each at 0.1 mg/mouse/injection; twice a week) plus either erlotinib (50 mg/kg; A, red area) or osimertinib (5 mg/kg; B, orange area). Tumor volumes of individual mice are shown.


A , B NSG mice were pre - implanted with tumor fragments derived from two different PDX models : ( A ) TM00199 ( L858R - EGFR ; from The Jackson Laboratory ) or ( B ) TM00193 ( E746 _ A750 del - EGFR ; from The Jackson Laboratory ) . Once tumors reached approximately 300 mm3 , mice were treated with monoclonal antibodies ( 2XmAbs , cetuximab plus trastuzumab , 200μg / injection ) twice a week , or with EGFR TKIs , erlotinib ( 50mg / kg ) or osimertinib ( 10mg / kg ) daily . Alternatively , mice were treated with a combination of 2XmAbs and either erlotinib or osimertinib , for either 32 days ( A ) or 42 days ( B ) . Tumor growth was monitored twice a week . the antibodies were injected into the peritoneum and the TKIs were delivered orally . The figure shows tumor volumes of individual mice and the respective survival curves ( right hand panels ) . The grey areas mark time windows of animal treatment .

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

A, B NSG mice were pre-implanted with tumor fragments derived from two different PDX models: (A) TM00199 (L858R-EGFR; from The Jackson Laboratory) or (B) TM00193 (E746_A750 del-EGFR; from The Jackson Laboratory). Once tumors reached approximately 300 mm3, mice were treated with monoclonal antibodies (2XmAbs, cetuximab plus trastuzumab, 200μg/injection) twice a week, or with EGFR TKIs, erlotinib (50mg/kg) or osimertinib (10mg/kg) daily. Alternatively, mice were treated with a combination of 2XmAbs and either erlotinib or osimertinib, for either 32 days (A) or 42 days (B). Tumor growth was monitored twice a week. the antibodies were injected into the peritoneum and the TKIs were delivered orally. The figure shows tumor volumes of individual mice and the respective survival curves (right hand panels). The grey areas mark time windows of animal treatment.


( B ) For RNAseq analyses , PC9 cells ( 3X106 / mouse ) were subcutaneously injected in the flanks of 13 CD1 - nu / nu mice . When tumors reached 500 mm3 , mice were treated daily with erlotinib ( 50 mg / kg ) . Once tumors that initially responded to erlotinib monotherapy started relapsing and reached 800 mm3 , we switched to a combination of 2XmAbs plus erlotinib ( 50 mg / kg ) . The respective tumor volumes were sacrificed when tumor volumes reached approximately 500 mm3 Mice responding to erlotinib ( N = 3 ) were sacrificed after one week of treatment Mice resistant to erlotinib ( N = 3 ) were sacrificed when tumor volumes reached 800 mm3 whereas mice responding to erlotinib + 2XmAbs ( N = 2 ) were sacrificed after one week of treatment RNA was extracted from all 13 tumors and utilized for RNA - seq analysis . Differentially expressed ( DE ) genes in the Erlotinib ( Er ) + 2XmAbs arm compared to the Control group are presented in the heatmap .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) For RNAseq analyses, PC9 cells (3X106/mouse) were subcutaneously injected in the flanks of 13 CD1-nu/nu mice. When tumors reached 500 mm3, mice were treated daily with erlotinib (50 mg/kg). Once tumors that initially responded to erlotinib monotherapy started relapsing and reached 800 mm3, we switched to a combination of 2XmAbs plus erlotinib (50 mg/kg). The respective tumor volumes were sacrificed when tumor volumes reached approximately 500 mm3 Mice responding to erlotinib (N=3) were sacrificed after one week of treatment Mice resistant to erlotinib (N=3) were sacrificed when tumor volumes reached 800 mm3 whereas mice responding to erlotinib+2XmAbs (N=2) were sacrificed after one week of treatment RNA was extracted from all 13 tumors and utilized for RNA-seq analysis. Differentially expressed (DE) genes in the Erlotinib (Er)+2XmAbs arm compared to the Control group are presented in the heatmap.


( C ) Protein extracts were prepared from all tumors and analyzed using immunoblotting , as indicated . Signals were quantified and normalized to the signals of GAPDH ( numbers shown below each lane ) .

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

(C) Protein extracts were prepared from all tumors and analyzed using immunoblotting, as indicated. Signals were quantified and normalized to the signals of GAPDH (numbers shown below each lane).


( A and B ) NIH3T3 cells stably expressing GFP - ULK1 ( A ) and - ULK2 ( B ) were cultured in complete or starvation medium for 30 min . They were then cultured in fresh complete medium for an additional 30 min ( starvation → complete ) .

O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A and B) NIH3T3 cells stably expressing GFP-ULK1 (A) and -ULK2 (B) were cultured in complete or starvation medium for 30 min. They were then cultured in fresh complete medium for an additional 30 min (starvation→complete).


( C and D ) NIH3T3 cells stably expressing GFP - ULK1 ( C ) and - ULK2 ( D ) were cultured in starvation medium for 120 min . The cells were fixed , permeabilized , and subjected to immunofluorescence microscopy using anti - Atg16L1 antibody and Alexa Fluor 660 - conjugated secondary antibody . More than 90 % of GFP - ULKs dots were positive for Atg16L1 .

O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O

(C and D) NIH3T3 cells stably expressing GFP-ULK1 (C) and -ULK2 (D) were cultured in starvation medium for 120 min. The cells were fixed, permeabilized, and subjected to immunofluorescence microscopy using anti-Atg16L1 antibody and Alexa Fluor 660-conjugated secondary antibody. More than 90% of GFP-ULKs dots were positive for Atg16L1.


( E - H ) Wild - type ( E and G ) and Atg5 − / − ( F and H ) MEFs were transfected with retroviral vectors encoding GFP - ULK1 and - ULK2 . MEFs stably expressing GFP - ULK1 ( E and F ) and - ULK2 ( G and H ) were cultured in complete or starvation medium for 120 min . The cells were fixed and examined by fluorescence microscopy . Bars , 20 μm .

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(E-H) Wild-type (E and G) and Atg5−/− (F and H) MEFs were transfected with retroviral vectors encoding GFP-ULK1 and -ULK2. MEFs stably expressing GFP-ULK1 (E and F) and -ULK2 (G and H) were cultured in complete or starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. Bars, 20 μm.


( A ) In vitro kinase assay of endogenous ULK1 . MEFs were cultured in complete or starvation medium for 60 min . ULK1 kinase activity was determined as described in Materials and methods . Relative kinase activity is shown . Data are the mean ± SE of five independent experiments .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) In vitro kinase assay of endogenous ULK1. MEFs were cultured in complete or starvation medium for 60 min. ULK1 kinase activity was determined as described in Materials and methods. Relative kinase activity is shown. Data are the mean ± SE of five independent experiments.


( B and C ) NIH3T3 cells were transiently transfected with HA - ULK1 , HA - ULK2 , or their kinase - dead mutants and subjected to immunofluoresence microscopy using monoclonal anti - HA and polyclonal anti - Atg16L1 antibodies for primary staining and Alexa Fluor 488 - conjugated goat anti - mouse IgG and Alexa Fluor 568 - conjugated goat anti - rabbit IgG antibodies for secondary antibodies . Transfected cells are indicated by arrows . Bars , 20 μm . ( D ) NIH3T3 cells stably expressing GFP - ULK1K46N and GFP - ULK2K39T were cultured in starvation medium for 120 min . The cells were subjected to immunofluorescence microscopy using anti - Atg16L1 antibody . Bar , 20 μm .

O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O

(B and C) NIH3T3 cells were transiently transfected with HA-ULK1, HA-ULK2, or their kinase-dead mutants and subjected to immunofluoresence microscopy using monoclonal anti-HA and polyclonal anti-Atg16L1 antibodies for primary staining and Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 568-conjugated goat anti-rabbit IgG antibodies for secondary antibodies. Transfected cells are indicated by arrows. Bars, 20 μm. (D) NIH3T3 cells stably expressing GFP-ULK1K46N and GFP-ULK2K39T were cultured in starvation medium for 120 min. The cells were subjected to immunofluorescence microscopy using anti-Atg16L1 antibody. Bar, 20 μm.


( E ) NIH3T3 cells were transfected with the retroviral vectors encoding HA - ULK1K46N or with the corresponding empty retrovirus ( mock ) . They were cultured in complete or starvation medium for 120 min . Cell lysates were then analyzed by immunoblot analysis with the indicated antibodies .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) NIH3T3 cells were transfected with the retroviral vectors encoding HA-ULK1K46N or with the corresponding empty retrovirus (mock). They were cultured in complete or starvation medium for 120 min. Cell lysates were then analyzed by immunoblot analysis with the indicated antibodies.


( A ) HEK293T cells were cotransfected with FLAG - FIP200 and HA - ULKs . Cell lysates were subjected to immunoprecipitation ( IP ) using antibodies against FLAG . The resulting precipitates were examined by immunoblot ( IB ) analysis with the indicated antibodies . The asterisk indicates nonspecific band .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) HEK293T cells were cotransfected with FLAG-FIP200 and HA-ULKs. Cell lysates were subjected to immunoprecipitation (IP) using antibodies against FLAG. The resulting precipitates were examined by immunoblot (IB) analysis with the indicated antibodies. The asterisk indicates nonspecific band.


( B ) Lysates of MEFs were immunoprecipitated with anti - ULK1 or anti - FIP200 antibody or preimmune rabbit serum , and the resulting precipitates were subjected to immunoblot analysis with antibodies against ULK1 and FIP200 .

O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O

(B) Lysates of MEFs were immunoprecipitated with anti-ULK1 or anti-FIP200 antibody or preimmune rabbit serum, and the resulting precipitates were subjected to immunoblot analysis with antibodies against ULK1 and FIP200.


( D ) HEK293T cells were cotransfected with FLAG - FIP200 and various ULK1 mutants and analyzed as in A using anti - HA and anti - FLAG antibodies .

O O O O O O O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

(D) HEK293T cells were cotransfected with FLAG-FIP200 and various ULK1 mutants and analyzed as in A using anti-HA and anti-FLAG antibodies.


( E ) NIH3T3 cells stably expressing GFP - ULK1 ( left ) and GFP - ULK1ΔC ( right ) were cultured in starvation medium for 120 min . Bar , 20 μm .

O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

(E) NIH3T3 cells stably expressing GFP-ULK1 (left) and GFP-ULK1ΔC (right) were cultured in starvation medium for 120 min. Bar, 20 μm.


( A ) NIH3T3 cells stably expressing GFP - FIP200 were cultured in complete or starvation medium for 120 min and the GFP signal was observed .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

(A) NIH3T3 cells stably expressing GFP-FIP200 were cultured in complete or starvation medium for 120 min and the GFP signal was observed.


( B ) NIH3T3 cells stably expressing GFP - FIP200 were cultured in starvation medium for 120 min and then subjected to immunofluorescence microscopy using anti - Atg16L1 antibody and Alexa Fluor 568 - conjugated secondary antibody . Bars , 20 μm . More than 90 % of GFP - FIP200 dots were positive for Atg16L1 .

O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O

(B) NIH3T3 cells stably expressing GFP-FIP200 were cultured in starvation medium for 120 min and then subjected to immunofluorescence microscopy using anti-Atg16L1 antibody and Alexa Fluor 568-conjugated secondary antibody. Bars, 20 μm. More than 90% of GFP-FIP200 dots were positive for Atg16L1.


( C ) NIH3T3 cells stably expressing GFP - ULK1 were starved for 120 min and then subjected to immunofluorescence microscopy using anti - FIP200 antibody and Alexa Fluor 568 - conjugated secondary antibody . Black squares indicate the enlarged areas shown in insets . Bar , 20 μm .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

(C) NIH3T3 cells stably expressing GFP-ULK1 were starved for 120 min and then subjected to immunofluorescence microscopy using anti-FIP200 antibody and Alexa Fluor 568-conjugated secondary antibody. Black squares indicate the enlarged areas shown in insets. Bar, 20 μm.


( A ) FIP200 + / + , FIP200 − / − , Atg5 + / + , and Atg5 − / − MEFs were cultured in complete or starvation medium for 60 min . In the recovery experiments , starved MEFs were cultured in fresh complete medium for an additional 60 min ( replenishment ) . The cell lysates were subjected to immunoblot analysis with the indicated antibodies .

O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) FIP200+/+, FIP200−/−, Atg5+/+, and Atg5−/− MEFs were cultured in complete or starvation medium for 60 min. In the recovery experiments, starved MEFs were cultured in fresh complete medium for an additional 60 min (replenishment). The cell lysates were subjected to immunoblot analysis with the indicated antibodies.


( B ) Wild - type and FIP200 − / − MEFs were cultured in the complete or starvation medium for indicated time with or without 100 nM bafilomycin A1 . The cell lysates were subjected to immunoblot analysis with anti - LC3 antibody .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O 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-MEASURED_VAR O O

(B) Wild-type and FIP200−/− MEFs were cultured in the complete or starvation medium for indicated time with or without 100 nM bafilomycin A1. The cell lysates were subjected to immunoblot analysis with anti-LC3 antibody.


( C ) Wild - type and FIP200 − / − MEFs were transfected with retroviral vectors encoding GFP - Atg5 or GFP - LC3 . Resulting cells were cultured in the starvation medium for 120 min . The cells were fixed and examined by fluorescence microscopy . Bar , 20 μm .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Wild-type and FIP200−/− MEFs were transfected with retroviral vectors encoding GFP-Atg5 or GFP-LC3. Resulting cells were cultured in the starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. Bar, 20 μm.


( D - G ) Wild - type ( D and E ) and FIP200 − / − ( F and G ) MEFs were cultured in complete ( D and F ) or starvation ( E and G ) medium for 120 min and then fixed and subjected to EM analysis . Autophagosome - like structures ( open arrowheads ) , and autolysosomes ( closed arrowheads ) are indicated . Bar , 1 μm . ( H ) The ratio of total area of autophagosomes ( AP ) and autolysosomes ( AL ) to total cytoplasmic area in D - G was determined by morphometric analysis .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D-G) Wild-type (D and E) and FIP200−/− (F and G) MEFs were cultured in complete (D and F) or starvation (E and G) medium for 120 min and then fixed and subjected to EM analysis. Autophagosome-like structures (open arrowheads), and autolysosomes (closed arrowheads) are indicated. Bar, 1 μm. (H) The ratio of total area of autophagosomes (AP) and autolysosomes (AL) to total cytoplasmic area in D-G was determined by morphometric analysis.


( A ) FAK + / − and FAK − / − MEFs were cultured in the complete or starvation medium for the indicated times with or without 100 nM bafilomycin A1 . The cell lysates were subjected to immunoblot analysis with the indicated antibodies .

O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) FAK+/− and FAK−/− MEFs were cultured in the complete or starvation medium for the indicated times with or without 100 nM bafilomycin A1. The cell lysates were subjected to immunoblot analysis with the indicated antibodies.


( B ) FAK + / − and FAK − / − MEFs stably expressing GFP - LC3 were cultured in complete or starvation medium for 120 min . The cells were fixed and examined by fluorescence microscopy .

O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

(B) FAK+/− and FAK−/− MEFs stably expressing GFP-LC3 were cultured in complete or starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy.


( C ) Wild - type MEFs stably expressing GFP - FAK were cultured in complete or starvation medium for 120 min . The cells were fixed and subjected to immunofluorescence microscopy using anti - Atg16 antibody . Black squares indicate the enlarged areas shown in insets . Bars , 20 μm .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

(C) Wild-type MEFs stably expressing GFP-FAK were cultured in complete or starvation medium for 120 min. The cells were fixed and subjected to immunofluorescence microscopy using anti-Atg16 antibody. Black squares indicate the enlarged areas shown in insets. Bars, 20 μm.


( A ) Wild - type and FIP200 − / − MEFs were treated with 100 ng / ml rapamycin ( rapa ) or vehicle ( DMSO ) for 120 min in the presence or absence of 100 nM bafilomycin A1 . The cell lysates were subjected to immunoblot analysis with anti - LC3 antibody .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O

(A) Wild-type and FIP200−/− MEFs were treated with 100 ng/ml rapamycin (rapa) or vehicle (DMSO) for 120 min in the presence or absence of 100 nM bafilomycin A1. The cell lysates were subjected to immunoblot analysis with anti-LC3 antibody.


( B ) Wild - type and FIP200 − / − MEFs stably expressing GFP - Atg5 or GFP - LC3 were cultured in the presence of 100 ng / ml rapamycin for 120 min . The formation of GFP - Atg5 ( top ) and GFP - LC3 ( bottom ) puncta was examined by fluorescence microscopy . Bar , 20 μm .

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(B) Wild-type and FIP200−/− MEFs stably expressing GFP-Atg5 or GFP-LC3 were cultured in the presence of 100 ng/ml rapamycin for 120 min. The formation of GFP-Atg5 (top) and GFP-LC3 (bottom) puncta was examined by fluorescence microscopy. Bar, 20 μm.


( C ) Wild - type and FIP200 − / − MEFs were treated with 10 mM lithium chloride for 24 h or 100 μM C2 - ceramide for 2 h .

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(C) Wild-type and FIP200−/− MEFs were treated with 10 mM lithium chloride for 24 h or 100 μM C2-ceramide for 2 h.


( A ) FIP200 + / − and FIP200 − / − MEFs stably expressing GFP - ULK1 or - ULK2 were cultured in starvation medium for 120 min . The cells were fixed and examined by fluorescence microscopy . Bar , 20 μm .

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(A) FIP200+/− and FIP200−/− MEFs stably expressing GFP-ULK1 or -ULK2 were cultured in starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. Bar, 20 μm.


( B ) FIP200 + / + and FIP200 − / − MEFs were cultured in complete medium . The cell lysates were subjected to immunoblot analysis with antibodies against ULK1 or HSP90 ( loading control ) .

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(B) FIP200+/+ and FIP200−/− MEFs were cultured in complete medium. The cell lysates were subjected to immunoblot analysis with antibodies against ULK1 or HSP90 (loading control).


( C ) Phosphatase sensitivity of ULK1 . FIP200 + / + and FIP200 − / − MEFs were cultured in complete medium . ULK1 was immunoprecipitated from cell lysates and treated with λ phosphatase for 30 min at 30 ° C in the absence or presence of phosphatase inhibitors ( 1 mM Na2VO4 , 50 mM KF , 15 mM Na4P2O7 , and 1 mM EGTA ) .

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(C) Phosphatase sensitivity of ULK1. FIP200+/+ and FIP200−/− MEFs were cultured in complete medium. ULK1 was immunoprecipitated from cell lysates and treated with λ phosphatase for 30 min at 30°C in the absence or presence of phosphatase inhibitors (1 mM Na2VO4, 50 mM KF, 15 mM Na4P2O7, and 1 mM EGTA).


b , Representative phase images of the morphology of WT and Apc - / - organoids before mRNA isolation .

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b, Representative phase images of the morphology of WT and Apc-/- organoids before mRNA isolation.


c , Heat map illustrating differentially expressed genes ( DEGs ) encoding heat shock proteins between WT and Apc - / - organoids .

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c, Heat map illustrating differentially expressed genes (DEGs) encoding heat shock proteins between WT and Apc-/- organoids.


d , Volcano plot of DEGs between normal mouse intestinal tissue and adenomas ( GSE65461 ) .

d, Volcano plot of DEGs between normal mouse intestinal tissue and adenomas (GSE65461).


e , f , Relative gene expression of Hspb1 ( * * * P = 0 . 0004 , n = 6 ) ( e ) and protein expression of HSP25 ( f ) in unrecombined versus recombined murine organoids .

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e, f, Relative gene expression of Hspb1 (***P=0.0004, n=6)(e) and protein expression of HSP25 (f) in unrecombined versus recombined murine organoids.


g , Gene expression of Hspb1 in normal ( N ) versus adenoma ( A ) tissue ( * * * P = 0 . 0002 , n = 4 ) .

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g, Gene expression of Hspb1 in normal (N) versus adenoma (A) tissue (***P=0.0002, n=4).


H , RNA - ISH for Hspb1 in murine intestinal adenoma . Scalebar , 100 μm . Zoom panel , 25

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H, RNA-ISH for Hspb1 in murine intestinal adenoma. Scalebar, 100 μm. Zoom panel, 25 μm


i , Relative gene expression of human HSPB1 in paired sets of normal versus adenoma tissue , ( * * * * P < 0 . 0001 , paired t - test , n = 59 pairs ) .

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i, Relative gene expression of human HSPB1 in paired sets of normal versus adenoma tissue, (****P<0.0001, paired t-test, n=59 pairs).


Relative gene expression of AXIN2 ( * P = 0 . 0129 ) , P21 ( * P = 0 . 0103 ) , and HSPB1 ( * P = 0 . 0236 ) ( b ) in Ls174t - dnTCF4 cells the presence or absence of doxycycline .

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Relative gene expression of AXIN2 (*P=0.0129), P21(*P=0.0103), and HSPB1(*P=0.0236) (b) in Ls174t-dnTCF4 cells the presence or absence of doxycycline.


protein expression of HSP27 ( c ) in Ls174t - dnTCF4 cells the presence or absence of doxycycline .

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protein expression of HSP27 (c) in Ls174t-dnTCF4 cells the presence or absence of doxycycline.


Relative gene expression of Hspb1 in murine WT organoids treated with different concentrations of CHIR - 99021 ( * * P = 0 . 0016 , one way ANOVA , n = 4 ) ( d )

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Relative gene expression of Hspb1 in murine WT organoids treated with different concentrations of CHIR-99021 (**P=0.0016, one way ANOVA, n=4) (d)


protein expression of HSP25 in WT and Apc - / - organoids ( e ) .

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protein expression of HSP25 in WT and Apc-/- organoids (e).


g , h , Representative images of single cell Hspb1 KO clones passaged after loss of Apc ( g ) , and quantification of their clonogenic potential ( * P = 0 . 0147 ( # 1 ) , * * P = 0 . 0035 ( # 2 ) , * * P = 0 . 0032 ( # 3 ) ) ( h ) .

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g, h, Representative images of single cell Hspb1 KO clones passaged after loss of Apc (g), and quantification of their clonogenic potential (*P=0.0147(#1), **P=0.0035(#2), **P=0.0032(#3)) (h).


i , j , Representative images of Apc - / - organoids cultured in the absence or presence of 60 μM BVDU ( i ) , and quantification of their clonogenic potential ( * * P = 0 . 0039 ) ( j ) .

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i, j, Representative images of Apc-/- organoids cultured in the absence or presence of 60 μM BVDU (i), and quantification of their clonogenic potential (**P=0.0039) (j).


k , l , Relative gene expression of Wnt target genes Axin2 ( * * P = 0 . 0080 ) and Lgr5 ( * P = 0 . 0142 ) in Apc - / - organoids cultured in the absence or presence of 60 μM BVDU .

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k, l, Relative gene expression of Wnt target genes Axin2 (**P=0.0080) and Lgr5 (*P=0.0142) in Apc-/- organoids cultured in the absence or presence of 60 μM BVDU.


m , RNA - ISH for Lgr5 in control or BVDU - treated Apc - / - organoids . Scale bar , 50 μm , zoom panel 20 μm .

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m, RNA-ISH for Lgr5 in control or BVDU-treated Apc-/- organoids. Scale bar, 50 μm, zoom panel 20 μm.


b , c , Visualization of proliferating cells in tissues incubated with EdU for 2 - hours ( b ) , and quantification of the hyperproliferative zone ( * * P = 0 . 0057 , n = 3 mice ) ( c ) .

b, c, Visualization of proliferating cells in tissues incubated with EdU for 2-hours (b), and quantification of the hyperproliferative zone (**P=0.0057, n=3 mice) (c).


e , f , RNA - ISH for Notum reveals Apc - mutant clones in crypt bottoms of control or BVDU - treated mice ( e ) , and quantification of the abundance of mutant crypts ( n = 2 mice per condition , n = 5 technical replicates per mouse ) ( f ) .

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e, f, RNA-ISH for Notum reveals Apc-mutant clones in crypt bottoms of control or BVDU-treated mice (e), and quantification of the abundance of mutant crypts (n=2 mice per condition, n=5 technical replicates per mouse) (f).


g , h , Clone size distributions of Notum + crypts in control ( n = 265 crypts ) or BVDU - treated ( n = 260 crypts ) mice , the average clone size and number of fixed clones are included in the figure .

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g, h, Clone size distributions of Notum+ crypts in control (n=265 crypts) or BVDU-treated (n=260 crypts) mice, the average clone size and number of fixed clones are included in the figure.


Survival curves for control and BVDU - treated mice ( P = 0 . 0351 , Mantel - Cox test ) ( j )

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Survival curves for control and BVDU-treated mice (P=0.0351, Mantel-Cox test) (j)


macroscopic images of adenoma development in the distal small intestine ( k )

macroscopic images of adenoma development in the distal small intestine (k)


boxplots for the number of adenomas per intestinal region ( * * P = 0 . 0013 ) ( l ) , n = 8 control , n = 11 treated .

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boxplots for the number of adenomas per intestinal region(**P=0.0013) (l), n=8 control, n=11 treated.


The upper panel shows strength of association of Affymetrix 500K SNPs in the region , with combined P values shown in red for replicated SNPs . The lower panel is a recombination map showing how the signals are clearly demarcated by recombination hotspots . Nearby genes are shown in yellow .

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The upper panel shows strength of association of Affymetrix 500K SNPs in the region, with combined P values shown in red for replicated SNPs. The lower panel is a recombination map showing how the signals are clearly demarcated by recombination hotspots. Nearby genes are shown in yellow.


A . Four full - thickness excisional wounds were produced on each of CD18 - / - or WT mice by 6 mm biopsy punches . Each wound received intradermal injection of 2 . 5x105 AT - MSCs or PBS control . Each wound region was digitally photographed at the indicated time points , and wound areas were analyzed using Adobe Photoshop . Depicted are representative macroscopic pictures of PBS injected or AT - MSCs injected WT or CD18 - / - wounds at days 0 , 3 , 5 , 7 and 9 post - wounding . B . Quantitative analysis of 20 wound areas per group , expressed as percentage of the initial wound size at day 0 . The line in each group represents the mean value of 20 wounds from 5 mice . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t - test with Welch ' s correction .

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A. Four full-thickness excisional wounds were produced on each of CD18-/- or WT mice by 6 mm biopsy punches. Each wound received intradermal injection of 2.5x105 AT-MSCs or PBS control. Each wound region was digitally photographed at the indicated time points, and wound areas were analyzed using Adobe Photoshop. Depicted are representative macroscopic pictures of PBS injected or AT-MSCs injected WT or CD18-/- wounds at days 0, 3, 5, 7 and 9 post-wounding. B. Quantitative analysis of 20 wound areas per group, expressed as percentage of the initial wound size at day 0. The line in each group represents the mean value of 20 wounds from 5 mice. *p<0.05, **p<0.01, ***p<0.001 by Student's t-test with Welch's correction.


A . AT - MSCs or PBS were intradermally injected to WT or CD18 - / - wounds and the wound tissues were harvested at days 2 , 5 , 7 post - wounding . Depicted are representative photographs of wound cryosections at days 2 and 5 post - wounding stained for human - specific β2M ( green ) . Nuclei were stained with DAPI ( blue ) . Scale bars : 100 µm .

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A. AT-MSCs or PBS were intradermally injected to WT or CD18-/- wounds and the wound tissues were harvested at days 2, 5, 7 post-wounding. Depicted are representative photographs of wound cryosections at days 2 and 5 post-wounding stained for human-specific β2M (green). Nuclei were stained with DAPI (blue). Scale bars: 100 µm.


B . Genomic DNA was isolated from the wound tissues , and the amount of human - specific Alu DNA was quantified by real time PCR , and then converted to the number of AT - MSCs based on a standard curve . Data are expressed as mean ± SEM , n = 3 at each time point , * p < 0 . 05 by two - tailed unpaired t - test .

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B. Genomic DNA was isolated from the wound tissues, and the amount of human-specific Alu DNA was quantified by real time PCR, and then converted to the number of AT-MSCs based on a standard curve. Data are expressed as mean ± SEM, n = 3 at each time point, *p<0.05 by two-tailed unpaired t-test.


C . Total TGF - β1 concentrations in wound lysates were measured by TGF - β1 specific ELISA . Results are expressed as mean ± SEM , n = 3 at each time

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C. Total TGF-β1 concentrations in wound lysates were measured by TGF-β1 specific ELISA. Results are expressed as mean ± SEM, n = 3 at each time point


D . The expression of human TGF - β1 in cultured AT - MSCs and wound tissues of WT or CD18 - / - wounds received AT - MSCs injection was quantified by qPCR with primers specifically amplify human TGF - β1 , and normalized on the numbers of MSCs in each condition Data are expressed as mean ± SEM , n = 8 wounds ( 2 wounds x 4 mice ) per time

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D. The expression of human TGF-β1 in cultured AT-MSCs and wound tissues of WT or CD18-/- wounds received AT-MSCs injection was quantified by qPCR with primers specifically amplify human TGF-β1, and normalized on the numbers of MSCs in each condition Data are expressed as mean ± SEM, n = 8 wounds (2 wounds x 4 mice) per time point


A . Cryosections of PBS or AT - MSCs injected CD18 - / - wounds at days 5 and 7 post - wounding were immunostained for α - SMA ( red ) . Nuclei were stained with DAPI ( blue ) . The dashed lines separate epidermis and wound bed . e , epidermis ; wb , wound bed . Scale bars : 100 µm . B . Quantification of α - SMA immunofluorescence shown in ( A )

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A. Cryosections of PBS or AT-MSCs injected CD18-/- wounds at days 5 and 7 post-wounding were immunostained for α-SMA (red). Nuclei were stained with DAPI (blue). The dashed lines separate epidermis and wound bed. e, epidermis; wb, wound bed. Scale bars: 100 µm. B. Quantification of α-SMA immunofluorescence shown in (A)


C . Cryosections of AT - MSCs injected CD18 - / - wounds at day 5 post - wounding were co - immunostained for human β2M ( green ) and TGF - β1 ( red , upper panel ) or α - SMA ( red , lower panel ) .

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C. Cryosections of AT-MSCs injected CD18-/- wounds at day 5 post-wounding were co-immunostained for human β2M (green) and TGF-β1 (red, upper panel) or α-SMA (red, lower panel).


A - F . Representative photographs of immunofluorescent staining for α - SMA ( red ) and human β2M ( green ) on cultured human AT - MSCs ( A ) , murine primary dermal fibroblasts ( mFB ) ( B ) , mFB treated with 2 ng / ml recombinant human TGF - β1 ( C ) , and co - cultures of mFB with AT - MSCs ( D ) or TGF - β1 siRNA transfected AT - MSCs ( E ) or control siRNA transfected AT - MSCs ( F ) after 48 h culture . Nuclei were counterstained with DAPI ( blue ) . G . Quantification of α - SMA immunofluorescence shown in ( A - F )

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A-F. Representative photographs of immunofluorescent staining for α-SMA (red) and human β2M (green) on cultured human AT-MSCs (A), murine primary dermal fibroblasts (mFB) (B), mFB treated with 2 ng/ml recombinant human TGF-β1 (C), and co-cultures of mFB with AT-MSCs (D) or TGF-β1 siRNA transfected AT-MSCs (E) or control siRNA transfected AT-MSCs (F) after 48 h culture. Nuclei were counterstained with DAPI (blue). G. Quantification of α-SMA immunofluorescence shown in (A-F)


H . Four full - thickness excisional wounds were produced on each of CD18 - / - mouse by 6 mm biopsy punches . Each wound received intradermal injection of 2 . 5x105 AT - MSCs or human dermal fibroblasts ( HDF ) or TGF - β1 siRNA transfected AT - MSCs or control siRNA transfected AT - MSCs or PBS . Each wound region was digitally photographed at days 0 , 3 , 5 , 7 and 10 post - wounding , and wound areas were analyzed using Adobe Photoshop . The depicted result is the quantitative analysis of all wound areas per group , expressed as percentage of the initial wound size at day 0 .

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H. Four full-thickness excisional wounds were produced on each of CD18-/- mouse by 6 mm biopsy punches. Each wound received intradermal injection of 2.5x105 AT-MSCs or human dermal fibroblasts (HDF) or TGF-β1 siRNA transfected AT-MSCs or control siRNA transfected AT-MSCs or PBS. Each wound region was digitally photographed at days 0, 3, 5, 7 and 10 post-wounding, and wound areas were analyzed using Adobe Photoshop. The depicted result is the quantitative analysis of all wound areas per group, expressed as percentage of the initial wound size at day 0.


A - C . 2 . 5x105 of TGF - β1 siRNA or control siRNA transfected AT - MSCs were intradermally injected around each of CD18 - / - murine wound . PBS mock injection served as negative control . Wound tissue were harvested at day 2 , 5 , and 7 post - wounding for quantification of human TGF - β1 mRNA ( A ) at day 2 by qPCR , total TGF - β1 ( B ) and active TGF - β1 ( C ) protein at day 5 by ELISA .

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A-C. 2.5x105 of TGF-β1 siRNA or control siRNA transfected AT-MSCs were intradermally injected around each of CD18-/- murine wound. PBS mock injection served as negative control. Wound tissue were harvested at day 2, 5, and 7 post-wounding for quantification of human TGF-β1 mRNA (A) at day 2 by qPCR, total TGF-β1 (B) and active TGF-β1 (C) protein at day 5 by ELISA.


Expression of α - SMA ( D , E ) at day 5 and 7 by immunostaining on tissue sections . The dashed lines indicate the border of the wound bed and epidermis or eschar . e , epidermis ; es , eschar ; wb , wound bed . Quantification data are expressed as mean ± SEM , n = 3 wounds per

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Expression of α-SMA (D, E) at day 5 and 7 by immunostaining on tissue sections. The dashed lines indicate the border of the wound bed and epidermis or eschar. e, epidermis; es, eschar; wb, wound bed. Quantification data are expressed as mean ± SEM, n = 3 wounds per group