B ) ROS ( DHE ) mean number ( N ) of yH2A . X foci in proliferating senescent ( 3 days after 20Gy X - ray ) MRC5 fibroblasts treated with or without 100nM Rapamycin / or 2 . 5mM of antioxidant NAC . Data are mean ± S . E . M of n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA .

O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O 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) 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 experimental design : human MRC5 fibroblasts were irradiated with 20Gy X - ray treated at day 3 after IR with 10µM of ATM inhibitor ( ATMi ) KU55933 / or 100nM Rapamycin ( Rap ) ; ( bottom ) Effect of single or combined inhibition of ATM mTORC1 on mean number ( N ) of γH2A . X foci , Sen - β - Gal activity p21 expression in proliferating 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 .

O O O O O O O O O O B-ORGANISM B-CELL_LINE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

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 ( DHE ) in proliferating senescent ( 3 days after 10Gy X - ray ) wild - type PGC - 1β - / - MEFs ( top ) 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 .

O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O O O O O O O O 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) 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 of immunofluorescence against FLAG - PGC - 1β ( red ) 53BP1 ( green ) in proliferating senescent ( 2 days after 10Gy X - ray ) PGC - 1β overexpressing MEFs cultured at 3 % O2 , with or without 250µM of 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 .

O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O 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) (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 ) ( bottom ) effect of 100nM rapamycin on mean number ( N ) of 53BP1 foci in proliferating senescent ( 3 days after 10Gy X - ray ) wild - type PGC - 1β - / - MEFs . Data are mean ± S . E . M of n = 3 independent experiments ( at least 125 were analyzed per condition ) . Asterisks denote statistical significant P < 0 . 05 One - way ANOVA .

O O O O O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O O O O 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) (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 for mitochondrial MT - CO1 DDR marker yH2A . X ( scale bar = 5µm ) ( bottom ) of MT - CO1 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 were analyzed per mouse ) ; Asterisks denote statistical significant P < 0 . 05 using Two - t - .

O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O B-ORGANISM O O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O

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 NDUFB8 expression in mouse liver tissue at 3 12 months of . Data are from n = 3 mice per group ;

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O B-ORGANISM O O O

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 ;

O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O

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 ) of mitochondrial number per cross section 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 were analyzed per mouse ) ; ( bottom ) Representative electron 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 B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O B-CELL_TYPE O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O O B-EXP_ASSAY O B-CELL_TYPE O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR 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 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 B-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-ORGANISM 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 presence of pyruvate / malate . State III was induced by injection of ADP . State IV was induced by inhibition of ATP synthase with oligomycin 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-TISSUE B-SUBCELLULAR O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM 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 of hepatocytes from 3 15 months old mice with or without rapamycin ( 12 months diet ) . Co - localizing foci are amplified in right panel ( amplified are from single Z - planes where co - localization was found ) ; ( bottom ) Dot plot graph of Telomere - associated foci ( TAF ) in 3 , 15 16 months old mice ( 15 16 months old mice were fed with rapamycin for 12 4 months respectively ) . Data are from n = 3 - 9 mice per group ( at least 50 were analyzed per mice ) ;

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM 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 15 months old micelivers [ control ( - ) or rapamycin ( + ) ] stained with Sen - - β - Gal solution ( Sen - - β - Gal activity is evidenced by blue ) . Data are from n = 3 mice per group ; ( bottom ) Representative image showing Sen - - β - Gal ( Sen - - β - Gal activity is evidenced by blue ) in hepatocytes 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 B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 effect of 4 months rapamycin feeding on p21 expression in 16 months old mice . Data are from n = 3 mice per group ;

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O

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 SASP components CXCL1 , CXCL5 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 - t - .

O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O

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 PGC - 1 - β - / - mice with 18 months of . Data are mean ± S . E . M of n = 4 mice per group ; Asterisks denote statistical significant P < 0 . 05 using Two - t - .

O O O O O O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O

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 next day , complete media were replaced with media containing serum ( 1 % ) 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 trastuzumab , 5 μg / ml each ) . Thereafter , were washed with cold saline extracted . were separated using gel electrophoresis transferred onto nitrocellulose membranes . After blocking , membranes were incubated overnight with indicated antibodies , followed by incubation with horseradish peroxidase - conjugated secondary antibodies ( 60 minutes ) , treatment with Clarity ™ Western ECL Blotting Substrates ( Bio - Rad ) . ECL signals were detected using ChemiDoc ™ Imaging System ( Bio - Rad ) were acquired using ImageLab software . Signals ( relative to Control ) were quantified normalized to signals of GAPDH ( numbers shown below each lane ) .

O O O B-DISEASE O O O O O B-GENEPROD O B-CELL_LINE O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD 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 trastuzumab , 5 μg / ml each ) . ( C ) PC9 were seeded on coverslips treated for 24 hours were washed in acidic buffer , fixed in paraformaldehyde ( 4 % ) incubated with specific antibodies , followed by an Alexa Fluor 555 - conjugated secondary antibody ( pseudo colored in green ) . DAPI ( blue ) was used to stain nuclei . were captured using a confocal microscope ( 40X magnification ) .

O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-CELL_LINE O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O

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 ( 3X106 / mouse ) were subcutaneously implanted in flanks of CD1 - nu / nu mice . When tumors became palpable , mice were randomized in groups of 5 - 9 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 ) two monoclonal antibodies . Following 60 days of treatment reduced frequency of erlotinib administration to once every other day ( underneath dotted ) . survival ( B ) are shown . Mice were euthanized when reached 1 , 500 mm3 .

B-CELL_LINE O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O 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-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-ORGANISM 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 ( exon 19 deletion ) were subcutaneously implanted in flank of CD1 - nu / nu mice ( 3X106 / mouse ) . When tumors became palpable , mice were randomized in groups of 5 - 9 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 two antibodies . Following 60 days of treatment , frequency of TKI administration was reduced to once every other day ( underneath dotted lines ) , while mAb treatment remained unaltered . survival ( B ) are shown . Mice were euthanized when reached 1 , 500 mm3 .

B-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O B-ORGANISM 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 ) 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 trastuzumab , each at 0 . 1 mg / mouse / injection ) . when singly applied used erlotinib at 50 mg / kg . ( B ) After treatment , mice were sacrificed tumors extracted . extracts were resolved by means of electrophoresis transfer to nitrocellulose membranes , which were later incubated overnight with indicated antibodies . This was followed by incubation with peroxidase - conjugated secondary antibodies . Signals were detected using ChemidocTM ( from Bio - Rad ) , quantified normalized to signals of GAPDH or tubulin ( numbers shown below each lane ) .

B-GENEPROD O O O O B-ORGANISM O B-CELL_LINE O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD 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 ( 3X106 / mouse ) were subcutaneously injected in 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 ; panel A ) or with osimertinib ( 5 mg / kg , grey ; panel B ) using oral gavage . Initially , tumors displayed stable disease or they regressed , but eventually started relapsing . Once relapsing tumors reached 800 mm3 , while under erlotinib or osimertinib treatment , we supplemented treatment with a combination of 2XmAbs ( cetuximab trastuzumab , each at 0 . 1 mg / mouse / injection ; twice a week ) plus either erlotinib ( 50 mg / kg ; A , red ) or osimertinib ( 5 mg / kg ; B , orange ) . volumes of mice are shown .

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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 fragments derived from two different PDX models : ( A ) TM00199 ( L858R - EGFR ; from Jackson Laboratory ) or ( B ) TM00193 ( E746 _ A750 del - EGFR ; from 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 either erlotinib or osimertinib , for either 32 days ( A ) or 42 days ( B ) . growth was monitored twice a week . antibodies were injected into peritoneum TKIs were delivered orally . figure shows volumes of mice respective survival curves ( right panels ) . grey areas mark time windows of treatment .

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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 ( 3X106 / mouse ) were subcutaneously injected in 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 reached 800 mm3 , we switched to a combination of 2XmAbs plus erlotinib ( 50 mg / kg ) . respective volumes were sacrificed when 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 volumes reached 800 mm3 whereas mice responding to erlotinib + 2XmAbs ( N = 2 ) were sacrificed after one week of treatment was extracted from 13 tumors utilized for - seq . Differentially expressed ( DE ) genes in Erlotinib ( Er ) + 2XmAbs arm compared to Control group are presented in .

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

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(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 B ) NIH3T3 stably expressing GFP - ULK1 ( A ) - 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 ) .

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

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(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 G ) Atg5 − / − ( F H ) MEFs were transfected with retroviral vectors encoding GFP - ULK1 - ULK2 . MEFs stably expressing GFP - ULK1 ( E F ) - ULK2 ( G H ) were cultured in complete or starvation medium for 120 min . were fixed examined by 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 of endogenous ULK1 . MEFs were cultured in complete or starvation medium for 60 min . ULK1 kinase activity was determined as described in Materials methods . Relative kinase activity is shown . Data are mean ± SE of five independent experiments .

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

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

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(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 were cotransfected with FLAG - FIP200 HA - ULKs . lysates were subjected to immunoprecipitation ( IP ) using antibodies against FLAG . resulting precipitates were examined by immunoblot ( IB ) with indicated antibodies . asterisk indicates nonspecific band .

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(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 , resulting precipitates were subjected to immunoblot with antibodies against ULK1 FIP200 .

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(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 were cotransfected with FLAG - FIP200 various ULK1 mutants analyzed as in A using anti - HA anti - FLAG antibodies .

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

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(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 stably expressing GFP - FIP200 were cultured in complete or starvation medium for 120 min GFP signal was observed .

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

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

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

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(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 FIP200 − / − MEFs were cultured in complete or starvation medium for indicated time with or without 100 nM bafilomycin A1 . lysates were subjected to immunoblot with anti - LC3 antibody .

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(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 FIP200 − / − MEFs were transfected with retroviral vectors encoding GFP - Atg5 or GFP - LC3 . Resulting were cultured in starvation medium for 120 min . were fixed examined by microscopy . Bar , 20 μm .

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

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

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

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(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 . were fixed subjected to immunofluorescence microscopy using anti - Atg16 antibody . Black squares indicate enlarged areas shown in insets . Bars , 20 μm .

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

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(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 FIP200 − / − MEFs stably expressing GFP - Atg5 or GFP - LC3 were cultured in presence of 100 ng / ml rapamycin for 120 min . formation of GFP - Atg5 ( top ) GFP - LC3 ( bottom ) was examined by 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 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 + / − FIP200 − / − MEFs stably expressing GFP - ULK1 or - ULK2 were cultured in starvation medium for 120 min . were fixed examined by 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 + / + FIP200 − / − MEFs were cultured in complete medium . lysates were subjected to immunoblot 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 + / + FIP200 − / − MEFs were cultured in complete medium . ULK1 was immunoprecipitated from lysates treated with λ phosphatase for 30 min at 30 ° C in absence or presence of phosphatase inhibitors ( 1 mM Na2VO4 , 50 mM KF , 15 mM Na4P2O7 , 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 of of WT 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 between WT 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 adenomas ( GSE65461 ) .

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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 ) 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 , - 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 - , 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 ) , HSPB1 ( * P = 0 . 0236 ) ( b ) in Ls174t - dnTCF4 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.


expression of HSP27 ( c ) in Ls174t - dnTCF4 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)


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

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


g , h , Representative of single Hspb1 KO clones passaged after loss of Apc ( g ) , 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 of Apc - / - organoids cultured in absence or presence of 60 μM BVDU ( i ) , 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 ) Lgr5 ( * P = 0 . 0142 ) in Apc - / - organoids cultured in 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 , - 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 in tissues incubated with EdU for 2 - hours ( b ) , of hyperproliferative zone ( * * P = 0 . 0057 , n = 3 mice ) ( c ) .

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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 , - ISH for Notum reveals Apc - mutant clones in crypt bottoms of control or BVDU - treated mice ( e ) , of 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 distributions of Notum + crypts in control ( n = 265 crypts ) or BVDU - treated ( n = 260 crypts ) mice , average clone number of fixed clones are included in 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 BVDU - treated mice ( P = 0 . 0351 , Mantel - Cox ) ( j )

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


macroscopic of adenoma development in distal small intestine ( k )

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


boxplots for 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.


upper panel shows strength of association of Affymetrix 500K SNPs in region , with combined P values shown in red for replicated SNPs . lower panel is a recombination map showing how 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 indicated time points , 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 9 post - wounding . B . Quantitative of 20 wound areas per group , expressed as of initial wound at day 0 . in each group represents mean value of 20 wounds from 5 mice . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t - 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 wound tissues were harvested at days 2 , 5 , 7 post - wounding . Depicted are representative of wound cryosections at days 2 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 was isolated from wound tissues , amount of human - specific Alu was quantified by real time PCR , then converted to number of AT - MSCs on a standard curve . Data are expressed as mean ± , n = 3 at each time point , * p < 0 . 05 by two - unpaired t - .

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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 ± , 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 . expression of human TGF - β1 in cultured AT - MSCs wound tissues of WT or CD18 - / - wounds received AT - MSCs injection was quantified by qPCR with primers specifically amplify human TGF - β1 , normalized on numbers of MSCs in each condition Data are expressed as mean ± , 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 7 post - wounding were immunostained for α - SMA ( red ) . Nuclei were stained with DAPI ( blue ) . dashed lines separate epidermis wound bed . e , epidermis ; wb , wound bed . Scale bars : 100 µm . B . 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 ) 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 of immunofluorescent for α - SMA ( red ) human β2M ( green ) on cultured human AT - MSCs ( A ) , murine dermal fibroblasts ( mFB ) ( B ) , mFB treated with 2 ng / ml recombinant human TGF - β1 ( C ) , 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 . 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 10 post - wounding , wound areas were analyzed using Adobe Photoshop . depicted result is quantitative of wound areas per group , expressed as of initial wound 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 , 7 post - wounding for of human TGF - β1 mRNA ( A ) at day 2 by qPCR , total TGF - β1 ( B ) active TGF - β1 ( C ) 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 7 by immunostaining on tissue sections . dashed lines indicate border of wound bed epidermis or eschar . e , epidermis ; es , eschar ; wb , wound bed . data are expressed as mean ± , 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


Expression of CD31 ( F , G ) at day 5 7 by immunostaining on tissue sections . dashed lines indicate border of wound bed epidermis or eschar . e , epidermis ; es , eschar ; wb , wound bed . data are expressed as mean ± , n = 3 wounds per

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Expression of CD31 (F, G) 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


A . AT - MSCs were cultured in labelling media containing [ 35S ] - Met / Cys increasing concentrations of r . h . TGF - β1 at 0 , 0 . 1 , 0 . 3 , 1 , 3 10 ng / ml for overnight . Supernatants were harvested incubated with TGF - β1 capture antibody . After washing captured TGF - β1 was transferred to glass filter , radioactivity was counted with a scintillation counter ( black bars ) . supernatants were incubated with new TGF - β1 capture antibody , radioactivity of captured TGF - β1 was counted again ( bars ) . data is given as mean ± SD of 5 repeated measurements . CPM , per minute .

O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O

A. AT-MSCs were cultured in the labelling media containing [35S]-Met/Cys and increasing concentrations of r.h. TGF-β1 at 0, 0.1, 0.3, 1, 3 and 10 ng/ml for overnight. Supernatants were harvested and incubated with TGF-β1 capture antibody. After washing the captured TGF-β1 was transferred to glass filter membrane, and the radioactivity was counted with a scintillation counter (black bars). The supernatants were incubated with new TGF-β1 capture antibody, and the radioactivity of captured TGF-β1 was counted again (white bars). The data is given as mean ± SD of 5 repeated measurements. CPM, count per minute.


AT - MSCs ( B ) were cultured in media containing increasing concentrations of r . h . TGF - β1 at 0 , 0 . 1 , 0 . 3 , 1 , 3 10 ng / ml for 24 h , before lysates or total RNAs were harvested . Western blot of Smad7 was performed with lysates semi - quantitatively analyzed with densitometry . β - actin served as loading control . Data is given as mean ± of three independent blots .

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O

AT-MSCs (B) were cultured in media containing increasing concentrations of r.h. TGF-β1 at 0, 0.1, 0.3, 1, 3 and 10 ng/ml for 24 h, before protein lysates or total RNAs were harvested. Western blot analysis of Smad7 was performed with protein lysates and semi-quantitatively analyzed with densitometry. β-actin served as loading control. Data is given as mean ± SEM of three independent blots.


HDFs ( C ) were cultured in media containing increasing concentrations of r . h . TGF - β1 at 0 , 0 . 1 , 0 . 3 , 1 , 3 10 ng / ml for 24 h , before lysates or total RNAs were harvested . Western blot of Smad7 was performed with lysates semi - quantitatively analyzed with densitometry . β - actin served as loading control . Data is given as mean ± of three independent blots . *

B-CELL_TYPE O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O

HDFs (C) were cultured in media containing increasing concentrations of r.h. TGF-β1 at 0, 0.1, 0.3, 1, 3 and 10 ng/ml for 24 h, before protein lysates or total RNAs were harvested. Western blot analysis of Smad7 was performed with protein lysates and semi-quantitatively analyzed with densitometry. β-actin served as loading control. Data is given as mean ± SEM of three independent blots. *


D . From total , mature miR - 21 endogenous control RNU6B were converted to cDNA , quantified by qPCR TaqMan microRNA assays . expression of miR - 21 was normalized on RNU6B . Data is given as mean ± of three independent experiments .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O

D. From total RNA, the mature miR-21 and endogenous control RNU6B were converted to cDNA, and quantified by qPCR based TaqMan microRNA assays. The expression of miR-21 was normalized on RNU6B. Data is given as mean ± SEM of three independent experiments.


A . AT - MSCs were pretreated with a TGFβRI inhibitor SB431542 at 10 µM , or control DMSO for 1 h , subsequently exposed to r . h . TGF - β1 at indicated concentrations . were cultured for another 24 h harvested for miR - 21 . expression of miR - 21 was normalized on RNU6B . Data is given as mean ± of three independent experiments .

O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O

A. AT-MSCs were pretreated with a TGFβRI inhibitor SB431542 at 10 µM, or control DMSO for 1 h, and subsequently exposed to r.h. TGF-β1 at indicated concentrations. The cells were cultured for another 24 h and harvested for miR-21 assay. The expression of miR-21 was normalized on RNU6B. Data is given as mean ± SEM of three independent experiments.


B - D . AT - MSCs were transfected with 30 nM Smad7 - siRNAs or control siRNA . Untransfected AT - MSCs served as control . were isolated 2 days after transfection for Smad7 expression by qPCR ( B ) western blot ( C ) . remaining were treated with r . h . TGF - β1 at indicated concentrations for another 24 h . expression of human TGF - β1 was analyzed by qPCR normalized on human GAPDH ( D ) . Data is given as mean ± of three independent experiments .

O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O

B-D. AT-MSCs were transfected with 30 nM Smad7-siRNAs or control siRNA. Untransfected AT-MSCs served as control. RNA and protein were isolated 2 days after transfection for Smad7 expression by qPCR (B) and western blot (C). The remaining cells were treated with r.h. TGF-β1 at indicated concentrations for another 24 h. The expression of human TGF-β1 was analyzed by qPCR and normalized on human GAPDH (D). Data is given as mean ± SEM of three independent experiments.


E - G . AT - MSCs were transfected with 5 nM miR - 21 mimic or control mimic , were exposed to r . h . TGF - β1 2 days after transfection at indicated concentrations for another 24 h . expression miR - 21 was analyzed by qPCR - miR - 21 normalized on RNU6B ( E ) , Samd7 level was shown by western blot ( F ) , human TGF - β1 expression was monitored by qPCR normalized on human GAPDH ( G ) . Data is given as mean ± of three independent experiments .

O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O

E-G. AT-MSCs were transfected with 5 nM miR-21 mimic or control mimic, and were exposed to r.h. TGF-β1 2 days after transfection at indicated concentrations for another 24 h. The expression miR-21 was analyzed by qPCR-based miR-21 assay normalized on RNU6B (E), Samd7 protein level was shown by western blot (F), and human TGF-β1 expression was monitored by qPCR normalized on human GAPDH (G). Data is given as mean ± SEM of three independent experiments.


H - J . AT - MSCs were transfected with 50 nM miR - 21 inhibitor or control inhibitor , were exposed to r . h . TGF - β1 2 days after transfection at indicated concentrations for another 24 h , subsequently subjected to of expression of miR - 21 ( H ) , Smad7 ( I ) human TGF - β1 mRNA ( J ) . Data is given as mean ± of three independent experiments .

O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O

H-J. AT-MSCs were transfected with 50 nM miR-21 inhibitor or control inhibitor, and were exposed to r.h. TGF-β1 2 days after transfection at indicated concentrations for another 24 h, and subsequently subjected to the analysis of expression of miR-21 (H), Smad7 protein (I) and human TGF-β1 mRNA (J). Data is given as mean ± SEM of three independent experiments.


K - L . Full - thickness excisional wounds were produced on CD18 - / - mice by 6 mm biopsy punches . One day after wounding , each wound received intradermal injection of 2 . 5x105 AT - MSCs that had been transfected with 50 nM miR - 21 inhibitor or control inhibitor 2 days after transfection . CD18 - / - wounds received mock injection with PBS served as control . Each wound region was digitally photographed at days 0 , 3 , 6 post - wounding , expressed as of initial wound at day 0 ( K ) . Wound tissues were harvested on day 6 post - wounding for of total TGF - β1 by ELISA ( L ) . Data is given as mean ± ; n = 5 wounds per group ; * , p < 0 . 05 ; * * , p < 0 . 01 , by one - way ANOVA with Tukey ' s . MSC _ miR - 21 - IN , miR - 21 inhibitor transfected AT - MSCs ; MSC _ ctrl - IN , control inhibitor transfected AT - MSCs .

O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O

K-L. Full-thickness excisional wounds were produced on CD18-/- mice by 6 mm biopsy punches. One day after wounding, each wound received intradermal injection of 2.5x105 AT-MSCs that had been transfected with 50 nM miR-21 inhibitor or control inhibitor 2 days after transfection. CD18-/- wounds received mock injection with PBS served as control. Each wound region was digitally photographed at days 0, 3, and 6 post-wounding, and expressed as percentage of the initial wound size at day 0 (K). Wound tissues were harvested on day 6 post-wounding for quantification of total TGF-β1 protein by ELISA (L). Data is given as mean ± SEM; n = 5 wounds per group; *, p<0.05; **, p<0.01, by one-way ANOVA with Tukey's test. MSC_miR-21-IN, miR-21 inhibitor transfected AT-MSCs; MSC_ctrl-IN, control inhibitor transfected AT-MSCs.


a , b , THP1 were stimulated with rotenone ( 10 µM ) , TTFA ( 10 µM ) or antimycin ( 40 µg ml − 1 ) for 6 h then stained with Mitotracker green Mitotracker deep red ( a ) or MitoSOX ( b ) for 30 min analysed by flow cytometry .

O O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O

a, b, THP1 cells were stimulated with rotenone (10 µM), TTFA (10 µM) or antimycin (40 µg ml−1) for 6 h and then stained with Mitotracker green and Mitotracker deep red (a) or MitoSOX (b) for 30 min and analysed by flow cytometry.


c , THP1 were stimulated for 6 h with indicated amounts of rotenone , TTFA or antimycin . Supernatants ( SN ) extracts ( input ) were analysed by western blotting as indicated .

O O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O

c, THP1 cells were stimulated for 6 h with the indicated amounts of rotenone, TTFA or antimycin. Supernatants (SN) and cell extracts (input) were analysed by western blotting as indicated.


d , LPS - primed bone - marrow - derived macrophages ( BMDMs ) from wild - type ( WT ) , Nlrp3 or Ipaf deficient mice were stimulated for 6 h with MSU ( 150 µg ml − 1 ) , rotenone ( 10 µM for THP1 40 µM for BMDMs ) or antimycin ( 40 µg ml − 1 for THP1 10 µg ml − 1 for BMDMs ) . release of caspase - 1 ( western blot ) was then determined . Data shown are representative of three independent experiments .

O O B-SMALL_MOLECULE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O O O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-CELL_LINE O O O O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O O O B-CELL_LINE O O O O O O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O

d, LPS-primed bone-marrow-derived macrophages (BMDMs) from wild-type (WT), Nlrp3 or Ipaf deficient mice were stimulated for 6 h with MSU (150 µg ml−1), rotenone (10 µM for THP1 cells and 40 µM for BMDMs) or antimycin (40 µg ml−1 for THP1 cells and 10 µg ml−1 for BMDMs). The release of caspase-1 (western blot) was then determined. Data shown are representative of three independent experiments.


a , BMDMs expressing GFP - LC3 were stimulated with rotenone ( 40 µM ) for 3 h co - localization of mitochondria GFP - LC3 dots were analysed using confocal microscopy .

O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O

a, BMDMs expressing GFP-LC3 were stimulated with rotenone (40 µM) for 3 h and the co-localization of mitochondria and GFP-LC3 dots were analysed using confocal microscopy.


b , THP1 stimulated with 3 - methyladenine ( 3 - MA , 10 mM ) for 24 h or THP1 stably expressing shRNA against beclin 1 or ATG5 were stained with Mitotracker green Mitotracker deep red for 30 min , analysed by flow cytometry .

O O B-CELL_LINE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-CELL_LINE O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O

b, THP1 cells stimulated with 3-methyladenine (3-MA, 10 mM) for 24 h or THP1 cells stably expressing shRNA against beclin 1 or ATG5 were stained with Mitotracker green and Mitotracker deep red for 30 min, and analysed by flow cytometry.


c , LPS - primed BMDMs from wild - type , Nlrp3 − / − , Asc − / − or Ipaf − / − knockout mice were stimulated with MSU ( 150 µg ml − 1 ) or 3 - MA ( 10 mM ) for 6 h release of active caspase - 1 IL - 1b was determined .

O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O

c, LPS-primed BMDMs from wild-type, Nlrp3−/−, Asc−/− or Ipaf −/− knockout mice were stimulated with MSU (150 µg ml−1) or 3-MA (10 mM) for 6 h and the release of active caspase-1 and IL-1b was determined.


d , THP1 stably expressing shRNA against beclin 1 or ATG5 were incubated for 24 h media supernatants ( SN ) inputs were analysed by western blotting .

O O B-CELL_LINE O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O

d, THP1 cells stably expressing shRNA against beclin 1 or ATG5 were incubated for 24 h and media supernatants (SN) and inputs were analysed by western blotting.


e , THP1 stably expressing shRNA against beclin 1 or ATG5 were stimulated with MSU or nigericin for 6 h . Data shown are representative of three independent experiments .

O O B-CELL_LINE O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O

e, THP1 cells stably expressing shRNA against beclin 1 or ATG5 were stimulated with MSU or nigericin for 6 h. Data shown are representative of three independent experiments.


a - d , THP1 expressing Flag - NLRP3 ( a , b ) or Flag - ASC ( c , d ) were analysed for co - localization of Flag - NLRP3 with endoplasmic reticulum ( ER ) marker calreticulin or mitochondria ( Mitotracker ) using confocal microscopy .

O O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O

a-d, THP1 cells expressing Flag-NLRP3 (a, b) or Flag-ASC (c, d) were analysed for the co-localization of Flag-NLRP3 with the endoplasmic reticulum (ER) marker calreticulin or mitochondria (Mitotracker) using confocal microscopy.


b , THP1 expressing Flag - NLRP3 were analysed for co - localization of Flag - NLRP3 with endoplasmic reticulum ( ER ) marker calreticulin using confocal microscopy .

O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O

b, THP1 cells expressing Flag-NLRP3 were analysed for the co-localization of Flag-NLRP3 with the endoplasmic reticulum (ER) marker calreticulin using confocal microscopy.


e , THP1 were stimulated with MSU or nigericin then fractionated . , ER , MAM mitochondrial fractions were analysed by western blotting as indicated in text .

O O B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O

e, THP1 cells were stimulated with MSU or nigericin and then fractionated. The cytosolic, ER, MAM and mitochondrial fractions were analysed by western blotting as indicated in the text.


f , THP1 stably expressing shRNA against NLRP3 were stimulated with nigericin subcellular fractions analysed as in e .

O O B-CELL_LINE O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O

f, THP1 cells stably expressing shRNA against NLRP3 were stimulated with nigericin and subcellular fractions analysed as in e.


g , TXNIP associates with mitochondria after NLRP3 inflammasome activation in a ROS - dependent manner . THP1 were stimulated with MSU nigericin , TXNIP localization was investigated in presence or absence of antioxidant APDC . Data shown are representative of two or three independent experiments . Scale bars : a - g , 20 µm .

O O B-GENEPROD O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O B-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

g, TXNIP associates with mitochondria after NLRP3 inflammasome activation in a ROS-dependent manner. THP1 cells were stimulated with MSU and nigericin, and TXNIP localization was investigated in the presence or absence of the antioxidant APDC. Data shown are representative of two or three independent experiments. Scale bars: a-g, 20 µm.


a , THP1 stably expressing shVDAC1 were stimulated with various NLRP inflammasome activators then analysed for caspase - 1 activation IL - 1b secretion .

O O B-CELL_LINE O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O

a, THP1 cells stably expressing shVDAC1 were stimulated with various NLRP inflammasome activators and then analysed for caspase-1 activation and IL-1b secretion.


b , THP1 expressing shRNA against VDAC2 were stimulated with MSU , R837 , silica , alum or nigericin analysed for IL - 1b secretion .

O O B-CELL_LINE O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O

b, THP1 cells expressing shRNA against VDAC2 were stimulated with MSU, R837, silica, alum or nigericin and analysed for IL-1b secretion.


c , THP1 expressing shRNA against VDAC1 were stimulated with MSU nigericin , NLRP3 localization was analysed by confocal microscopy . Scale bars : 20 µm .

O O B-CELL_LINE O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

c, THP1 cells expressing shRNA against VDAC1 were stimulated with MSU and nigericin, and NLRP3 localization was analysed by confocal microscopy. Scale bars: 20 µm.


B PCR performed on genomic with indicated primers as shown : o1 : oPAP8 _ rtpF , o2 : oPAP8 _ E3R , o3 : oPAP8 _ rtpR , oLB : oLBb1 . 3 , EF1α : ELONGATION FACTOR 1α , WT : wild type , pap8 - 1 : Homozygous albino , Ht : Heterozygous green ; T : T - , arrowhead : 670 - bp contaminant amplification product used as loading control .

O B-EXP_ASSAY O O O O O O O O O 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-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O 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 PCR performed on genomic DNA with indicated primers as shown: o1: oPAP8_rtpF, o2: oPAP8_E3R, o3: oPAP8_rtpR, oLB: oLBb1.3, EF1α: ELONGATION FACTOR 1α, WT: wild type, pap8-1: Homozygous albino plant, Ht: Heterozygous green plant; T: T-DNA, arrowhead: 670-bp contaminant amplification product used as loading control.


C RT - PCR on wild type pap8 - 1 homozygous grown in for 3 days followed with 72 - h growth under to allow greening of wild type ; EF1α used as control .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O

C RT-PCR on wild type and pap8-1 homozygous plants grown in the dark for 3 days followed with 72-h growth under white light to allow greening of the wild type; EF1α used as control.


E Half - open siliques of a heterozygous showing greening ; scale bar equals 250 µm . given number is position rank of silique from top to bottom of inflorescence presenting segregation of homozygous heterozygous seeds on their ability to transiently develop chloroplasts .

O O O O B-TISSUE O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O B-TISSUE O O O O O B-TISSUE O O O O O O B-SUBCELLULAR O

E Half-open siliques of a heterozygous plant showing the embryo greening; scale bar equals 250 µm. The given number is the position rank of the silique from top to bottom of the inflorescence presenting the segregation of homozygous and heterozygous seeds based on their ability to transiently develop chloroplasts.


F Mutant Rescue : WT two representative pap8 - 1 were grown in vitro using sucrose low ( of 10 µmol . m - 2 . s - 1 ) ; scale bar equals 20 mm .

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F Mutant Rescue: WT and two representative pap8-1 plants were grown in vitro using sucrose and low white light intensity (of 10 µmol.m-2.s-1); scale bar equals 20 mm.


B Two or six ( - 97m3 ) representative transformants expressing GUS under given PAP8 promoter version ; FC + , corresponding promoters were tested positive in functional complementation of mutant pap8 - 1 . Scale bar equals 3 mm .

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B Two or six (-97m3) representative primary transformants expressing GUS under the given PAP8 promoter version; FC+, the corresponding promoters were tested positive in functional complementation of the mutant pap8-1. Scale bar equals 3 mm.


D Dual luciferase reporter ; Renilla luciferase ( Rluc ) used as internal control GFPer used as control for transfected ; promoters driving Firefly luciferase ( Fluc ) were transfected in onion epidermis without or with constitutively expressed HY5 . Fluc / Rluc activity was set to 1 for minus - HY5 control ; mean ± standard error corresponding to 3 replicates ; photon are given in source data . * , ε - = 3 , 43 > 1 . 96 corresponding to p - value < 0 , 001 .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-ORGANISM B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O 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 Dual luciferase reporter assay; Renilla luciferase (Rluc) used as internal control and GFPer used as control for the transfected area; the promoters driving Firefly luciferase (Fluc) were transfected in onion epidermis cells without or with constitutively expressed HY5. The Fluc/Rluc activity was set to 1 for the minus-HY5 control; mean ± standard error corresponding to 3 replicates; photon counts are given in source data. *, ε-test = 3,43 > 1.96 corresponding to p-value < 0,001.


E Electro - mobility shift of a probe corresponding to near palindromic PAP8 element ( GAcGCTC ) with recombinant HY5 ; a probe containing a canonical G - box element ( CACGTG ) recognized by HY5 was used as cold competitor .

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E Electro-mobility shift assay of a probe corresponding to the near palindromic PAP8 element (GAcGCTC) with recombinant HY5 protein; a probe containing a canonical G-box element (CACGTG) recognized by HY5 was used as cold competitor.


F Integrative genomics viewer ( IGV ) of immunoprecipitation ( ChIP ) sequencing data30 at PAP8 locus ; TAIR10 , annotation according to Arabidopsis thaliana information resource orange box indicates PAP8 locus . ChIP on hy5 - ks50 ; 35S : HY5 - YFP exposed to blue or red using GFP antibody compared to mock corresponding to ChIP control experiment done without antibody . Each treatment is presented as track overlay of triplicates : read is given within " group autoscale " range in brackets . Close up on 5 ' - UTR region centred on - 95 - promoter element in yellow .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O B-ORGANISM I-ORGANISM O O O O O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O 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 Integrative genomics viewer (IGV) images of the Chromatin immunoprecipitation (ChIP) sequencing data30 at the PAP8 locus; TAIR10, annotation according to the Arabidopsis thaliana information resource orange box indicates the PAP8 locus. ChIP on hy5-ks50; 35S:HY5-YFP exposed to blue light or red light using GFP antibody and compared to mock corresponding to ChIP control experiment done without antibody. Each treatment is presented as track overlay of triplicates: the read count is given within the "group autoscale" range in brackets. Close up on the 5'-UTR region centred on the -95-promoter element in yellow.