D Real - time RT - PCR measurement of MpFGMYB transcript levels . Three biological replicates are analyzed for each line . Data information bars represent mean ± SD . Asterisks indicate significant differences from WT [ X ] ( D ) ( p < 0 . 05 , two - tailed Student ' s t - test ) .

D Real-time RT-PCR measurement of MpFGMYB transcript levels. Three biological replicates are analyzed for each line. Data information bars represent mean ± SD. Asterisks indicate significant differences from WT [X] (D) (p <0.05, two-tailed Student's t-test).


E - H Gametangiophores ( E , G ) and gametangia ( F , H ) of gMpFGMYB - S [ Y ] ( E , F ) and gMpFGMYB - L [ Y ] ( G , H ) . Scale bars , 1 mm ( E , G ) , 50 μm ( F ) , 100 μm ( H ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H Gametangiophores (E, G) and gametangia (F, H) of gMpFGMYB-S [Y] (E, F) and gMpFGMYB-L [Y] (G, H). Scale bars, 1 mm (E, G), 50 μm (F), 100 μm (H).


( a ) Representative confocal micrographs of mitochondrial morphology in MEFs of the indicated genotype 24 h after transfection with mtYFP . Where indicated , cells were starved for 2 . 5 h . Scale bar , 20 μm . ( b ) Morphometric analysis of mitochondrial shape . Experiments were carried out as in a . Data represent mean ± s . e . m . of three independent experiments ( n = 100 cells per condition in each experiment ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative confocal micrographs of mitochondrial morphology in MEFs of the indicated genotype 24 h after transfection with mtYFP. Where indicated, cells were starved for 2.5 h. Scale bar, 20 μm. (b) Morphometric analysis of mitochondrial shape. Experiments were carried out as in a. Data represent mean ± s.e.m. of three independent experiments (n=100 cells per condition in each experiment).


( c ) Top left , 48 h after transfection with the indicated siRNA , MEFs were lysed and 25 μg samples of proteins were separated by DS - polyacrylamide gel electrophoresis ( PAGE ) and immunoblotted with the indicated antibodies . Top right and bottom , representative images show mitochondrial morphology of MEFs transfected with the indicated siRNA and after 24 h with mtYFP . After a further 24 h , confocal micrographs were acquired . Scale bar , 20 μm . Uncropped images of blots are shown in Supplementary Fig . S8 . ( d ) Morphometric analysis of mitochondrial shape . Experiments were carried out as in c . Data represent mean ± s . e . m . of five independent experiments ( n = 100 cells per condition in each experiment ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top left, 48 h after transfection with the indicated siRNA, MEFs were lysed and 25 μg samples of proteins were separated by DS-polyacrylamide gel electrophoresis (PAGE) and immunoblotted with the indicated antibodies. Top right and bottom, representative images show mitochondrial morphology of MEFs transfected with the indicated siRNA and after 24 h with mtYFP. After a further 24 h, confocal micrographs were acquired. Scale bar, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S8. (d) Morphometric analysis of mitochondrial shape. Experiments were carried out as in c. Data represent mean ± s.e.m. of five independent experiments (n=100 cells per condition in each experiment).


( e ) Representative images of mitochondrial fusion over time ( indicated in min ) . MEFs were co - transfected with mtPAGFP and mtRFP and after 24 h , mtPAGFP was photoactivated in a region of interest and cells were imaged by real - time confocal microscopy . Where indicated , MEFs were starved for 2 . 5 h . Scale bar , 20 μm . See also Supplementary Movies S1 and S2 . ( f , g ) Quantification of mitochondrial fusion in MEFs of the indicated genotype . Experiments were carried out as in e . Data represent mean ± s . e . m . of four independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of mitochondrial fusion over time (indicated in min). MEFs were co-transfected with mtPAGFP and mtRFP and after 24 h, mtPAGFP was photoactivated in a region of interest and cells were imaged by real-time confocal microscopy. Where indicated, MEFs were starved for 2.5 h. Scale bar, 20 μm. See also Supplementary Movies S1 and S2. (f,g) Quantification of mitochondrial fusion in MEFs of the indicated genotype. Experiments were carried out as in e. Data represent mean ± s.e.m. of four independent experiments.


( h ) Representative electron micrographs of muscle ( longitudinal sections ; four top micrographs ) and liver ( four bottom micrographs ) from CD1 mice . Where indicated , mice were fasted for 12 h . The outlined regions of micrographs from muscle samples are magnified × 7 in the images below . Scale bars , 2 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative electron micrographs of muscle (longitudinal sections; four top micrographs) and liver (four bottom micrographs) from CD1 mice. Where indicated, mice were fasted for 12 h. The outlined regions of micrographs from muscle samples are magnified ×7 in the images below. Scale bars, 2 μm.


( a - d ) Levels of mitochondria - shaping proteins during starvation . Protein samples ( 20 μg ) from MEFs of the indicated genotype were separated by SDS - PAGE and immunoblotted with the indicated antibodies . Cells were starved for the indicated times .

O O O O O O O O O O O O O O O O O O O O O O O O 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-d) Levels of mitochondria-shaping proteins during starvation. Protein samples (20 μg) from MEFs of the indicated genotype were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Cells were starved for the indicated times.


( e ) Association of DRP1 with mitochondria during starvation . Mitochondria were isolated from MEFs starved for the indicated times and 25 μg samples of proteins were separated by SDS - PAGE and immunoblotted with the indicated antibodies . ( f ) Levels of Ser 637 phosphorylation of DRP1 during starvation . Equal amounts of cell lysates from wild - type MEFs starved for the indicated times were immunoprecipitated with the indicated antibody and the immunoprecipitated proteins were separated by SDS - PAGE and immunoblotted with the indicated antibodies . ( g ) Quantitative analysis of Ser 637 phosphorylation of DRP1 during starvation . Experiments were carried out as in f . Data are normalized to total levels of DRP1 and represent the mean ± s . e . m . of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Association of DRP1 with mitochondria during starvation. Mitochondria were isolated from MEFs starved for the indicated times and 25 μg samples of proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (f) Levels of Ser 637 phosphorylation of DRP1 during starvation. Equal amounts of cell lysates from wild-type MEFs starved for the indicated times were immunoprecipitated with the indicated antibody and the immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (g) Quantitative analysis of Ser 637 phosphorylation of DRP1 during starvation. Experiments were carried out as in f. Data are normalized to total levels of DRP1 and represent the mean ± s.e.m. of three independent experiments.


( h ) MEFs were treated for the indicated times with 100 nM rapamycin or with 25 μM forskolin ( FRSK ) for 0 . 5 h , lysed and equal amounts ( 50 μg ) of proteins were separated by SDS - PAGE and immunoblotted using 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 B-CONTROLLED_VAR 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

(h) MEFs were treated for the indicated times with 100 nM rapamycin or with 25 μM forskolin (FRSK) for 0.5 h, lysed and equal amounts (50 μg) of proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies.


( i ) HeLa cells were transfected for 2 days with the indicated siRNA or treated with 25 μM forskolin for 0 . 5 h and lysed . Equal amounts ( 50 μg ) of proteins were separated by SDS - PAGE and immunoblotted using the indicated antibodies . Uncropped images of all blots in this figure are shown in Supplementary Fig . S8 .

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

(i) HeLa cells were transfected for 2 days with the indicated siRNA or treated with 25 μM forskolin for 0.5 h and lysed. Equal amounts (50 μg) of proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8.


( a ) Pseudocolour - coded images of EPACI - camps FRET from real - time imaging of wild - type MEFs transfected with EPACI - camps . Where indicated , cells were perfused with the starvation solution for the indicated times ( in min ) or with 25 μM forskolin ( FRSK ) . Scale bar , 20 μm . Colour scale indicates F1 / F0 ratio . See also Supplementary Movie S3 . ( b ) Quantitative analysis of CFP / YFP FRET ratio . Experiments were carried out as in a . Where indicated , cells were perfused with the starvation solution or with 25 μM forskolin . Data represent mean ± s . e . m . of 13 independent experiments .

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

(a) Pseudocolour-coded images of EPACI-camps FRET from real-time imaging of wild-type MEFs transfected with EPACI-camps. Where indicated, cells were perfused with the starvation solution for the indicated times (in min) or with 25 μM forskolin (FRSK). Scale bar, 20 μm. Colour scale indicates F1/F0 ratio. See also Supplementary Movie S3. (b) Quantitative analysis of CFP/YFP FRET ratio. Experiments were carried out as in a. Where indicated, cells were perfused with the starvation solution or with 25 μM forskolin. Data represent mean ± s.e.m. of 13 independent experiments.


( c ) Lysate samples ( 50 μg ) from MEFs of the indicated genotypes were analysed by SDS - PAGE and immunoblotting using the indicated antibodies . Where indicated , MEFs were starved , or treated with 25 μM forskolin . Where indicated , 20 μM H89 was added during starvation . Uncropped images of all blots in this figure are shown in Supplementary Fig . S8 .

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

(c) Lysate samples (50 μg) from MEFs of the indicated genotypes were analysed by SDS-PAGE and immunoblotting using the indicated antibodies. Where indicated, MEFs were starved, or treated with 25 μM forskolin. Where indicated, 20 μM H89 was added during starvation. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8.


( d ) Representative images of the effect of H89 on mitochondrial morphology on starvation . Wild - type and Mfn2 − / − MEFs were transfected with mtYFP , and after 24 h confocal micrographs were acquired . Where indicated , cells were starved for 2 . 5 h and 20 μM H89 was added . Scale bar , 20 μm . ( e ) Morphometric analysis . Experiments were carried out as in d . Data represent mean ± s . e . m . of five independent experiments ( n = 100 cells per condition ) .

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 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

(d) Representative images of the effect of H89 on mitochondrial morphology on starvation. Wild-type and Mfn2−/−MEFs were transfected with mtYFP, and after 24 h confocal micrographs were acquired. Where indicated, cells were starved for 2.5 h and 20 μM H89 was added. Scale bar, 20 μm. (e) Morphometric analysis. Experiments were carried out as in d. Data represent mean ± s.e.m. of five independent experiments (n=100 cells per condition).


( f ) Starvation - induced mitochondrial elongation depends on Ser 637 of DRP1 . Representative confocal micrographs of mitochondrial morphology of wild - type MEFs co - transfected with mtRFP and the indicated plasmids . At 24 h after transfection , where indicated cells were starved for 2 . 5 h and imaged . Where indicated , 20 μM H89 was present during starvation . Scale bar , 20 μm . ( g ) Morphometric analysis of mitochondrial shape . Experiments were carried out as in f . Data represent mean ± s . e . m . of five independent experiments ( n = 50 cells per condition ) .

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

(f) Starvation-induced mitochondrial elongation depends on Ser 637 of DRP1. Representative confocal micrographs of mitochondrial morphology of wild-type MEFs co-transfected with mtRFP and the indicated plasmids. At 24 h after transfection, where indicated cells were starved for 2.5 h and imaged. Where indicated, 20 μM H89 was present during starvation. Scale bar, 20 μm. (g) Morphometric analysis of mitochondrial shape. Experiments were carried out as in f. Data represent mean ± s.e.m. of five independent experiments (n=50 cells per condition).


( h ) Representative confocal micrographs of mitochondrial morphology of Drp1 − / − MEFs co - transfected with mtRFP and the indicated plasmids . At 24 h after transfection , where indicated cells were starved for 2 . 5 h and imaged . Where indicated , 20 μM H89 was present during starvation . Scale bar , 20 μm . ( i ) Morphometric analysis of mitochondrial shape . Experiments were carried out as in h . Data represent mean ± s . e . m . of five independent experiments ( n = 50 cells per condition ) .

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

(h) Representative confocal micrographs of mitochondrial morphology of Drp1−/− MEFs co-transfected with mtRFP and the indicated plasmids. At 24 h after transfection, where indicated cells were starved for 2.5 h and imaged. Where indicated, 20 μM H89 was present during starvation. Scale bar, 20 μm. (i) Morphometric analysis of mitochondrial shape. Experiments were carried out as in h. Data represent mean ± s.e.m. of five independent experiments (n=50 cells per condition).


( a - c ) MEFs of the indicated genotype were treated as indicated , counted and 2 . 7 × 105 cells were lysed . Lysates were separated by SDS - PAGE and immunoblotted using the indicated antibodies . ( d ) Ratio between the densitometric levels of cyclophilin D and those of PMP70 in MEFs of the indicated genotype . One representative experiment of five independent repetitions performed as in a - c is 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

(a-c) MEFs of the indicated genotype were treated as indicated, counted and 2.7×105 cells were lysed. Lysates were separated by SDS-PAGE and immunoblotted using the indicated antibodies. (d) Ratio between the densitometric levels of cyclophilin D and those of PMP70 in MEFs of the indicated genotype. One representative experiment of five independent repetitions performed as in a-c is shown.


( e ) MEFs of the indicated genotype starved for 5 h were treated where indicated with 0 . 5 μM wortmannin ( Wortm . ) . Lysates from 2 . 7 × 105 cells were separated by SDS - PAGE and immunoblotted with the indicated antibodies . Uncropped images of all blots in this figure are shown in Supplementary Fig . S8 .

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

(e) MEFs of the indicated genotype starved for 5 h were treated where indicated with 0.5 μM wortmannin (Wortm.). Lysates from 2.7×105 cells were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8.


( a , b ) Quantitative analysis of TMRM fluorescence changes over mitochondrial regions in MEFs of the indicated genotype . Where indicated , cells were starved for 5 h before TMRM loading . Where indicated ( arrows ) , 2 . 5 μg ml − 1 oligomycin and 2 μM carbonyl cyanide - p - trifluoromethoxyphenylhydrazone ( FCCP ) were added . Data represent mean ± s . e . m . of seven independent experiments .

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

(a,b) Quantitative analysis of TMRM fluorescence changes over mitochondrial regions in MEFs of the indicated genotype. Where indicated, cells were starved for 5 h before TMRM loading. Where indicated (arrows), 2.5 μg ml−1 oligomycin and 2 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) were added. Data represent mean ± s.e.m. of seven independent experiments.


( c ) Total cellular ATP levels were measured in cells of the indicated genotype starved for the indicated times . Data represent mean ± s . e . m . of five independent experiments .

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

(c) Total cellular ATP levels were measured in cells of the indicated genotype starved for the indicated times. Data represent mean ± s.e.m. of five independent experiments.


( d ) Mitochondrial ATP measured in situ by mitochondrially targeted luciferase in cells of the indicated genotype starved for the indicated times . Data represent mean ± s . e . m . of five independent experiments and are normalized to the initial value .

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

(d) Mitochondrial ATP measured in situ by mitochondrially targeted luciferase in cells of the indicated genotype starved for the indicated times. Data represent mean ± s.e.m. of five independent experiments and are normalized to the initial value.


( e , f ) Cells of the indicated genotype were starved for the indicated times . Viability was determined by flow cytometry as the percentage of annexin - V - and propidium iodide ( PI ) - negative events . Data represent mean ± s . e . m . of five independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,f) Cells of the indicated genotype were starved for the indicated times. Viability was determined by flow cytometry as the percentage of annexin-V- and propidium iodide (PI)-negative events. Data represent mean ± s.e.m. of five independent experiments.


( g ) MEFs of the indicated genotype were starved for 2 . 5 h . Where indicated , cells were treated with 20 μM H89 . Viability was determined as in e , f . Data represent mean ± s . e . m . of five independent experiments .

O O O 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

(g) MEFs of the indicated genotype were starved for 2.5 h. Where indicated, cells were treated with 20 μM H89. Viability was determined as in e,f. Data represent mean ± s.e.m. of five independent experiments.


( h ) Cells of the indicated genotype were starved for the indicated times . Viability was determined as in e , f . Data represent mean ± s . e . m . of five independent experiments .

O O O O O O O O O O O O O O O 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) Cells of the indicated genotype were starved for the indicated times. Viability was determined as in e,f. Data represent mean ± s.e.m. of five independent experiments.


( i ) Drp1 − / − MEFs were transfected with the indicated plasmids and after 24 h starved for 5 h where indicated . Cell death was determined by flow cytometry as the percentage of YFP - and propidium - iodide - positive events . Data represent mean ± s . e . m . of four independent experiments .

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

(i) Drp1−/− MEFs were transfected with the indicated plasmids and after 24 h starved for 5 h where indicated. Cell death was determined by flow cytometry as the percentage of YFP- and propidium-iodide-positive events. Data represent mean ± s.e.m. of four independent experiments.


( j , k ) MEFs of the indicated genotype were starved for 5 h in the presence of 2 . 5 μg ml − 1 oligomycin where indicated . Viability was determined as in e , f . Data represent mean ± s . e . m . of five independent experiments .

O O O O O 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

(j,k) MEFs of the indicated genotype were starved for 5 h in the presence of 2.5 μg ml−1 oligomycin where indicated. Viability was determined as in e,f. Data represent mean ± s.e.m. of five independent experiments.


( a ) Blue - native electrophoresis analysis of ATPase dimerization and activity . Cells of the indicated genotype were treated as indicated and 500 μg of total cell extract was solubilized with 4 % digitonin and proteins were separated by blue - native PAGE . ATPase activity was measured in - gel ( top ) and ATPase levels were measured by immunoblotting for the indicated antibody ( bottom ) . ( b , c ) Quantitative analysis of the ratio of levels ( b ) and activity ( c ) between dimeric and monomeric forms of ATPase , assessed by densitometric analysis of gels from experiments performed as in a . Data represent mean ± s . e . m . of five independent experiments . ( d ) Experiments were carried out as in a , but with cells of the indicated genotype .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Blue-native electrophoresis analysis of ATPase dimerization and activity. Cells of the indicated genotype were treated as indicated and 500 μg of total cell extract was solubilized with 4% digitonin and proteins were separated by blue-native PAGE. ATPase activity was measured in-gel (top) and ATPase levels were measured by immunoblotting for the indicated antibody (bottom). (b,c) Quantitative analysis of the ratio of levels (b) and activity (c) between dimeric and monomeric forms of ATPase, assessed by densitometric analysis of gels from experiments performed as in a. Data represent mean ± s.e.m. of five independent experiments. (d) Experiments were carried out as in a, but with cells of the indicated genotype.


( a ) Representative electron micrographs of cells of the indicated genotype starved where indicated for 5 h , fixed and processed for electron microscopy . Scale bar , 2 μm . ( b ) Representative electron micrographs of randomly selected mitochondria from cells of the indicated genotype . Where indicated , cells were starved for 5 h . Scale bars , 0 . 5 μm . ( c ) Morphometric analysis of cristae density in cells of the indicated genotype . Experiments were carried out as in a . The number of cristae in 50 randomly selected mitochondria of the indicated genotype was normalized for the calculated surface of the organelle . Data represent mean ± s . e . m . of five independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative electron micrographs of cells of the indicated genotype starved where indicated for 5 h, fixed and processed for electron microscopy. Scale bar, 2 μm. (b) Representative electron micrographs of randomly selected mitochondria from cells of the indicated genotype. Where indicated, cells were starved for 5 h. Scale bars, 0.5 μm. (c) Morphometric analysis of cristae density in cells of the indicated genotype. Experiments were carried out as in a. The number of cristae in 50 randomly selected mitochondria of the indicated genotype was normalized for the calculated surface of the organelle. Data represent mean ± s.e.m. of five independent experiments.


p53 has been show to response with a series of undamped pulse to ionizing irradiation leading to cell cycle arrest while intrinsic DNA damage during cell cycle does not induce regular pulsatile p53 and subsequent gene expression programs . Schematic representations of p53 dynamics in both cellular conditions 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

p53 has been show to response with a series of undamped pulse to ionizing irradiation leading to cell cycle arrest while intrinsic DNA damage during cell cycle does not induce regular pulsatile p53 and subsequent gene expression programs. Schematic representations of p53 dynamics in both cellular conditions are shown.


Induction of selected p53 target genes after DNA damage induction in A549 wild - type and p53 knockdown cells . RNA levels were measured by qRT - PCR before and 3h after treatment with 10 Gy R . Fold changes relative to basal levels are shown for each cell line .

Induction of selected p53 target genes after DNA damage induction in A549 wild-type and p53 knockdown cells. RNA levels were measured by qRT-PCR before and 3h after treatment with 10 Gy R. Fold changes relative to basal levels are shown for each cell line.


Fluorescence microscopy images of smFISH probes labelled with CAL - Fluor 610 ( grey ) overlayed with Hoechst 33342 stainings ( blue ) for the indicated target genes in untreated A549 cells . Scale bar corresponds to 10 µm distance , images were contrast and brightness enhanced for better visualization .

Fluorescence microscopy images of smFISH probes labelled with CAL-Fluor 610 (grey) overlayed with Hoechst 33342 stainings (blue) for the indicated target genes in untreated A549 cells. Scale bar corresponds to 10 µm distance, images were contrast and brightness enhanced for better visualization.


Histograms of quantitative analysis of RNAs per cell for each target gene in the absence of DNA damage ( basal ) . smFISH staining and quantitative analysis of p53 targets shows broad variability of RNA counts per cell for all genes in basal conditions .

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

Histograms of quantitative analysis of RNAs per cell for each target gene in the absence of DNA damage (basal). smFISH staining and quantitative analysis of p53 targets shows broad variability of RNA counts per cell for all genes in basal conditions.


Fluorescence microscopy images of smFISH probes CAL - Fluor 610 ( grey ) overlayed with Hoechst 33342 staining ( blue ) at 3 h after 10 Gy IR or the indicated target genes in A549 cells . Scale bar corresponds to 10 µm distance , images were contrast and brightness enhanced for better visualization .

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

Fluorescence microscopy images of smFISH probes CAL-Fluor 610 (grey) overlayed with Hoechst 33342 staining (blue) at 3 h after 10 Gy IR or the indicated target genes in A549 cells. Scale bar corresponds to 10 µm distance, images were contrast and brightness enhanced for better visualization.


We quantified RNAs per cell for the indicated target genes before ( basal , grey ) and 3 h ( red ) , 6 h ( blue ) and 9 h ( orange ) after DNA damage ( 10 Gy IR ) . smFISH based single cell analysis of gene expression patterns highlights distinct RNA counts for p53 targets . RNA counts per cell are displayed as boxplots ( see Data Visualization section ) ; n : number of analyzed cells , fc : median fold of induction relative to time point basal ( indicated by grey lines ) Distributions of RNAs per cell for the indicated target genes before ( basal , grey ) and 3 h ( red ) , 6 h ( blue ) and 9 h ( orange ) after DNA damage ( 10 Gy IR ) . Despite a clear change in median levels ( m : median ) , single cell analysis reveals a strong dispersion that overlaps for the different conditions , as shown by the strongly overlapping distributions of RNA counts per cells . For better visualization , probability density estimates ( PDF ) based on a normal kernel are

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

We quantified RNAs per cell for the indicated target genes before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR). smFISH based single cell analysis of gene expression patterns highlights distinct RNA counts for p53 targets. RNA counts per cell are displayed as boxplots (see Data Visualization section); n: number of analyzed cells, fc: median fold of induction relative to time point basal (indicated by grey lines) Distributions of RNAs per cell for the indicated target genes before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR). Despite a clear change in median levels (m: median), single cell analysis reveals a strong dispersion that overlaps for the different conditions, as shown by the strongly overlapping distributions of RNA counts per cells. For better visualization, probability density estimates (PDF) based on a normal kernel are shown


Quantification of stochastic gene expression for the indicated p53 target genes before ( basal , grey ) and 3 h ( red ) , 6 h ( blue ) and 9 h ( orange ) after DNA damage ( 10 Gy IR ) . The fraction ( f ) of active promoters ( proxy for burst frequency ) increases , while the transcription rate ( µ ) ( proxy for burst size ) at active TSS remains similar upon DNA damage for all time points . Left panel : The percentage of cells with active TSS is shown as stacked bar graphs . We subdivided the population in cells with strong TSS activity ( > 75 % of TSS active , solid colors ) and those with partial TSS activity ( at least one , but less than 75 % of TSS active , shaded colors ) . The mean fraction of active promoters ( ratio of all active TSS to the total number of genomic loci analyzed ) is indicated above each bar . Right panel : Distributions of calculated transcription rates µ [ RNAs / h ] at active TSS are presented for each time point as probability density estimates ( PDF , see Data Visualization section ) . The number of TSS analyzed is indicated in each plot ( compare Fig EV2C ) .

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

Quantification of stochastic gene expression for the indicated p53 target genes before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR). The fraction (f) of active promoters (proxy for burst frequency) increases, while the transcription rate (µ) (proxy for burst size) at active TSS remains similar upon DNA damage for all time points. Left panel: The percentage of cells with active TSS is shown as stacked bar graphs. We subdivided the population in cells with strong TSS activity (> 75% of TSS active, solid colors) and those with partial TSS activity (at least one, but less than 75% of TSS active, shaded colors). The mean fraction of active promoters (ratio of all active TSS to the total number of genomic loci analyzed) is indicated above each bar. Right panel: Distributions of calculated transcription rates µ [RNAs/h] at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). The number of TSS analyzed is indicated in each plot (compare Fig EV2C).


Mean degradation rates of indicated RNAs in transcriptionally active cells before ( basal , grey ) and 3 h ( red ) , 6 h ( blue ) and 9 h ( orange ) after DNA damage ( 10 Gy IR ) as calculated from smFISH data . RNA stability is not changing in the measured time frame upon DNA damage . The plot displays the average RNA degradation rate per cell [ 1 / h ] over time after DNA damage , calculated from model ( C ) in actively transcribing cells for each gene .

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Mean degradation rates of indicated RNAs in transcriptionally active cells before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR) as calculated from smFISH data. RNA stability is not changing in the measured time frame upon DNA damage. The plot displays the average RNA degradation rate per cell [1/h] over time after DNA damage, calculated from model (C) in actively transcribing cells for each gene.


Amount of p53 bound to indicated target gene promoters before ( basal , grey ) and 3 h ( red ) , 6 h ( blue ) and 9 h ( orange ) after DNA damage ( 10 Gy IR ) as measured by ChIP . The amount of bound p53 was calculated as percentage of input and normalized to the time - point of the first p53 peak at 3 h . Individual data points ( mean values of triplicate quantification in qRT - PCR measurements ) from 3 - 4 biological repeats are shown as dots , mean values are displayed as black horizontal lines . Dashed lines serve as guide to the eyes . We could not detect p53 binding above IgG controls at the published p53 response element in the PPM1D promoter ( indicated by n . d . )

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

Amount of p53 bound to indicated target gene promoters before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR) as measured by ChIP. The amount of bound p53 was calculated as percentage of input and normalized to the time-point of the first p53 peak at 3 h. Individual data points (mean values of triplicate quantification in qRT-PCR measurements) from 3-4 biological repeats are shown as dots, mean values are displayed as black horizontal lines. Dashed lines serve as guide to the eyes. We could not detect p53 binding above IgG controls at the published p53 response element in the PPM1D promoter (indicated by n.d.)


Chk2 inhibition with the small molecule BML - 277 induces transient p53 dynamics with only one pulse after 10 Gy IR . A schematic illustration of the experimental setup and quantification of p53 levels in A549 wild type cells after irradiation with 10 Gy IR and addition of 10 µM BML - 277 by immunofluorescence staining ( see Methods section for details ) are shown as box plots ( see Data Visualization section ) .

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

Chk2 inhibition with the small molecule BML-277 induces transient p53 dynamics with only one pulse after 10 Gy IR . A schematic illustration of the experimental setup and quantification of p53 levels in A549 wild type cells after irradiation with 10 Gy IR and addition of 10 µM BML-277 by immunofluorescence staining (see Methods section for details) are shown as box plots (see Data Visualization section).


We quantified promoter activity of BAX ( B , sustained archetype ) and PPM1D ( C , pulsatile archetype ) before ( basal , grey ) and 3 h ( red ) , 6 h ( blue ) and 9 h ( orange ) after irradiation with 10 Gy IR and inhibition of the second p53 pulse by Chk2 inhibition . Left panel : The percentage of cells with active TSS , subdivided into populations with strong ( > 75 % of TSS , solid colors ) and weak ( < 75 % of TSS , shaded colors ) activity , is shown as stacked bar graphs , the mean fraction of active promoters is indicated above each bar . Right panel : Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates ( PDF , see Data Visualization section ) . The fraction of active promoters was reduced at 6 h and 9 h after irradiation ; the transcription rate was not notably affected .

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

We quantified promoter activity of BAX (B, sustained archetype) and PPM1D (C, pulsatile archetype) before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after irradiation with 10 Gy IR and inhibition of the second p53 pulse by Chk2 inhibition. Left panel: The percentage of cells with active TSS, subdivided into populations with strong (> 75% of TSS, solid colors) and weak (<75% of TSS, shaded colors) activity, is shown as stacked bar graphs, the mean fraction of active promoters is indicated above each bar. Right panel: Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). The fraction of active promoters was reduced at 6 h and 9 h after irradiation; the transcription rate was not notably affected.


Sequential treatment with Nutlin - 3 converts pulsatile p53 dynamics into sustained nuclear levels . A schematic illustration of the experimental setup and quantification of p53 levels in A549 wild type cells after irradiation with 10 Gy IR and sequential treatment with 0 . 75 µM Nutlin - 3 at 2 . 5 h , with 2 . 25 µM at 3 . 5 h and 4 µM at 5 . 5 h post IR based on immunofluorescence staining ( see Methods section for details ) are shown as box plots ( see Data Visualization section ) .

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

Sequential treatment with Nutlin-3 converts pulsatile p53 dynamics into sustained nuclear levels. A schematic illustration of the experimental setup and quantification of p53 levels in A549 wild type cells after irradiation with 10 Gy IR and sequential treatment with 0.75 µM Nutlin-3 at 2.5 h, with 2.25 µM at 3.5 h and 4 µM at 5.5 h post IR based on immunofluorescence staining (see Methods section for details) are shown as box plots (see Data Visualization section).


We quantified promoter activity of MDM2 ( E , transient archetype ) and CDKN1A ( F , transient archetype ) before ( basal , grey ) and 3 h ( red ) , 6 h ( blue ) and 9 h ( orange ) after irradiation with 10 Gy IR and sequential Nutlin - 3 treatment . Left panel : The percentage of cells with active TSS , subdivided into populations with strong ( > 75 % of TSS , solid colors ) and weak ( < 75 % of TSS , shaded colors ) activity , is shown as stacked bar graphs , the mean fraction of active promoters is indicated above each bar . Right panel : Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates ( PDF , see Data Visualization section ) . The relative fraction of active promoters strongly increased , changing transient to sustained archetypes . The transcription rate increased as well both compared to basal levels and to previous experiments with pulsatile p53 dynamics ( inset , fold change relative to IR alone for each time point ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-CONTROLLED_VAR I-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

We quantified promoter activity of MDM2 (E, transient archetype) and CDKN1A (F, transient archetype) before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after irradiation with 10 Gy IR and sequential Nutlin-3 treatment. Left panel: The percentage of cells with active TSS, subdivided into populations with strong (> 75% of TSS, solid colors) and weak (<75% of TSS, shaded colors) activity, is shown as stacked bar graphs, the mean fraction of active promoters is indicated above each bar. Right panel: Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). The relative fraction of active promoters strongly increased, changing transient to sustained archetypes. The transcription rate increased as well both compared to basal levels and to previous experiments with pulsatile p53 dynamics (inset, fold change relative to IR alone for each time point).


Total p53 , p53 acetylated at K382 and K370 as well as GAPDH were measured by Western Blot at indicated time points in the context of different p53 dynamics : pulsing p53 ( 10 Gy IR ) , transient p53 ( 10 Gy IR + BML - 277 , central lanes ) and sustained p53 ( 10 Gy IR + Nutlin - 3 , right lanes ) . See Figure 3 and Methods section for details . The relative change in p53 acetylation at K370 ( light green ) and K382 ( dark green ) was quantified from Western Blot and normalized to the abundance 3 h post IR . Means and propagated standard errors from 3 independent experiments are indicated . Acetylation increased over time in the context of sustained p53 . See also Appendix Fig S12 .

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

Total p53, p53 acetylated at K382 and K370 as well as GAPDH were measured by Western Blot at indicated time points in the context of different p53 dynamics: pulsing p53 (10 Gy IR), transient p53 (10 Gy IR + BML-277, central lanes) and sustained p53 (10 Gy IR + Nutlin-3, right lanes). See Figure 3 and Methods section for details. The relative change in p53 acetylation at K370 (light green) and K382 (dark green) was quantified from Western Blot and normalized to the abundance 3 h post IR. Means and propagated standard errors from 3 independent experiments are indicated. Acetylation increased over time in the context of sustained p53. See also Appendix Fig S12.


The p53 - K370 methylase Smyd2 was down - regulated in a clonal stable A549 cell line expressing a corresponding shRNA . Transcript levels were measured in wild type and knockdown cells by qRT - PCR . Mean levels and standard deviation from technical triplicates are indicated .

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The p53-K370 methylase Smyd2 was down-regulated in a clonal stable A549 cell line expressing a corresponding shRNA. Transcript levels were measured in wild type and knockdown cells by qRT-PCR. Mean levels and standard deviation from technical triplicates are indicated.


Promoter activity of CDKN1A ( E ) and MDM2 ( F ) were quantified in Smyd2 knockdown cells before ( basal , grey ) and 3 h ( red ) , 6 h ( blue ) and 9 h ( orange ) after DNA damage ( 10 Gy IR ) . Left panel : The percentage of cells with active TSS , subdivided into populations with strong ( > 75 % of TSS , solid colors ) and weak ( < 75 % of TSS , shaded colors ) activity , is shown as stacked bar graphs , the mean fraction of active promoters is indicated above each bar . Right panel : Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates ( PDF , see Data Visualization section ) . We measured a higher fraction of active promoters upon damage compared to A549 wild type cells ( Fig 3 ) , while transcription rates remained unchanged .

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

Promoter activity of CDKN1A (E) and MDM2 (F) were quantified in Smyd2 knockdown cells before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR). Left panel: The percentage of cells with active TSS, subdivided into populations with strong (> 75% of TSS, solid colors) and weak (<75% of TSS, shaded colors) activity, is shown as stacked bar graphs, the mean fraction of active promoters is indicated above each bar. Right panel: Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). We measured a higher fraction of active promoters upon damage compared to A549 wild type cells (Fig 3), while transcription rates remained unchanged.


The p53 - K382 methylase Set8 was down - regulated in a clonal stable A549 cell line expressing a corresponding shRNA . Transcript levels were measured in wild type and knockdown cells by qRT - PCR . Mean levels and standard deviation from technical triplicates are indicated .

The p53-K382 methylase Set8 was down-regulated in a clonal stable A549 cell line expressing a corresponding shRNA. Transcript levels were measured in wild type and knockdown cells by qRT-PCR. Mean levels and standard deviation from technical triplicates are indicated.


Promoter activity of CDKN1A ( E ) and MDM2 ( F ) were quantified in Set8 knock - down cells before ( basal , grey ) and 3 h ( red ) , 6 h ( blue ) and 9 h ( orange ) after DNA damage ( 10 Gy IR ) . Left panel : The percentage of cells with active TSS , subdivided into populations with strong ( > 75 % of TSS , solid colors ) and weak ( < 75 % of TSS , shaded colors ) activity , is shown as stacked bar graphs , the mean fraction of active promoters is indicated above each bar . Right panel : Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates ( PDF , see Data Visualization section ) . We measured a higher fraction of active promoters upon damage compared to A549 wild type cells ( Figure 3 ) , while transcription rates remained unchanged .

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

Promoter activity of CDKN1A (E) and MDM2 (F) were quantified in Set8 knock-down cells before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR). Left panel: The percentage of cells with active TSS, subdivided into populations with strong (> 75% of TSS, solid colors) and weak (<75% of TSS, shaded colors) activity, is shown as stacked bar graphs, the mean fraction of active promoters is indicated above each bar. Right panel: Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). We measured a higher fraction of active promoters upon damage compared to A549 wild type cells (Figure 3), while transcription rates remained unchanged.


a , Western blot analysis of yeast cell lysates with polyclonal anti - flag antibody to detect flag - tagged human Beclin 1 expression . Two - hundred micrograms of protein per lane was loaded . SEY6210 , wild - type yeast ; JCY3000 , SEY6210 disrupted in apg6 / vps30 ; ( + ) control , lysates of mammalian cells transfected with flag - beclin 1 .

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a, Western blot analysis of yeast cell lysates with polyclonal anti-flag antibody to detect flag-tagged human Beclin 1 expression. Two-hundred micrograms of protein per lane was loaded. SEY6210, wild-type yeast; JCY3000, SEY6210 disrupted in apg6/vps30; (+) control, lysates of mammalian cells transfected with flag-beclin 1.


b , Autophagicbody formation in yeast deprived of nitrogen in the presence ( + ) and absence ( - ) of 1 mM PMSF . Arrows denote cells that would be scored as positive in the experiment shown in c .

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

b, Autophagicbody formation in yeast deprived of nitrogen in the presence (+) and absence (-) of 1 mM PMSF. Arrows denote cells that would be scored as positive in the experiment shown in c.


c , Quantitative effects of apg6 / vps30 and beclin 1 transformation on autophagicbody formation in apg6 / vps30 - disrupted yeast in the presence of PMSF . Cells with one or more autophagic bodies within the vacuole were scored as positive ( see arrows in b ) . A minimum of 100 cells was counted for each sample . Results represent the mean ( ± s . e . m . ) percentage of cells with autophagic bodies within the vacuole for triplicate samples . Similar results were obtained in five independent experiments .

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c, Quantitative effects of apg6/vps30 and beclin 1 transformation on autophagicbody formation in apg6/vps30-disrupted yeast in the presence of PMSF. Cells with one or more autophagic bodies within the vacuole were scored as positive (see arrows in b). A minimum of 100 cells was counted for each sample. Results represent the mean (±s.e.m.) percentage of cells with autophagic bodies within the vacuole for triplicate samples. Similar results were obtained in five independent experiments.


d , Sorting of vacuolar protein carboxypeptidase Y , ( CPY ) . p2CPY , precursor form ; mCPY , sorted mature form . Mr , relative molecular mass .

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d, Sorting of vacuolar protein carboxypeptidase Y, (CPY). p2CPY, precursor form; mCPY, sorted mature form. Mr, relative molecular mass.


a - f , Electron micrographs of MCF7 . control ( clone 38 ) ( a , b ) and MCF7 . beclin1 ( clone 17 ) cells ( c - f ) grown in nutrient - rich media ( a , c ) or subjected to 4 h of serum and amino - acid deprivation ( b , d - f ) . Asterisks in e and f denote autophagic vacuoles that would be counted in the experiment shown in g . Autophagic vacuoles were defined as double - membrane vacuolar structures containing recognizable cytoplasmic contents . Scale bars , 1 µm .

O O O O O O O O O O O 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-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-f, Electron micrographs of MCF7.control (clone 38) (a, b) and MCF7.beclin1 (clone 17) cells (c- f) grown in nutrient-rich media (a, c) or subjected to 4 h of serum and amino-acid deprivation (b, d-f). Asterisks in e and f denote autophagic vacuoles that would be counted in the experiment shown in g. Autophagic vacuoles were defined as double-membrane vacuolar structures containing recognizable cytoplasmic contents. Scale bars, 1 µm.


g , Quantitative effects of beclin 1 on basal and nutrient - deprivation - induced autophagy of MCF7 cells . Bars indicate mean ( ± s . e . m . ) number of autophagic vacuoles per cell for cells growing in normal media ( black ) , for cells subjected to 4 h of serum and amino - acid deprivation ( grey ) , and for cells pre - treated for 30 min with 10 mM 3 - MA and subjected to 4 h of serum and amino - acid deprivation in the presence of 3 - MA ( open ) . Mean was determined by counting the total number of autophagic vacuoles in each cell for 100 cells per clone per treatment .

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

g, Quantitative effects of beclin 1 on basal and nutrient-deprivation-induced autophagy of MCF7 cells. Bars indicate mean (±s.e.m.) number of autophagic vacuoles per cell for cells growing in normal media (black), for cells subjected to 4 h of serum and amino-acid deprivation (grey), and for cells pre-treated for 30 min with 10 mM 3-MA and subjected to 4 h of serum and amino-acid deprivation in the presence of 3-MA (open). Mean was determined by counting the total number of autophagic vacuoles in each cell for 100 cells per clone per treatment.


h , Comparison of the rates of degradation of long - lived proteins in MCF7 . control cells ( squares , clone 38 ) , MCF7 . beclin1 cells ( circles , clone 17 ) and MCF7 . beclin1stop cells ( triangles , clone 70 ) , in EBSS + 10 % serum and complete amino acids ( open symbols , solid lines ) in EBSS alone ( closed symbols , solid lines ) , and in EBSS + 10 mM 3 - MA ( closed symbols , dotted lines ) . Results are mean ( ± s . e . m . ) of triplicate wells . Similar results were obtained in three independent experiments . Results shown with these clones are representative of results obtained with other clones analysed by electron microscopy in g .

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

h, Comparison of the rates of degradation of long-lived proteins in MCF7.control cells (squares, clone 38), MCF7.beclin1 cells (circles, clone 17) and MCF7.beclin1stop cells (triangles, clone 70), in EBSS + 10% serum and complete amino acids (open symbols, solid lines) in EBSS alone (closed symbols, solid lines), and in EBSS + 10 mM 3-MA (closed symbols, dotted lines). Results are mean (±s.e.m.) of triplicate wells. Similar results were obtained in three independent experiments. Results shown with these clones are representative of results obtained with other clones analysed by electron microscopy in g.


a , Western blot analysis of flag - tagged beclin - 1 - transfected MCF7 clones . C , MCF7 . control cells ( clone 38 ) ; St , MCF7 . beclin1stop cells ( clone 70 ) . Mr , relative molecular mass .

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a, Western blot analysis of flag-tagged beclin-1-transfected MCF7 clones. C, MCF7.control cells (clone 38); St, MCF7.beclin1stop cells (clone 70). Mr, relative molecular mass.


b , Western blot analysis of flag - tagged beclin1stop - transfected MCF7 clones . C , MCF . control cells ( clone 38 ) ; B , MCF7 . beclin1 cells ( clone 1 ) .

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b, Western blot analysis of flag-tagged beclin1stop-transfected MCF7 clones. C, MCF.control cells (clone 38); B, MCF7.beclin1 cells (clone 1).


c , Photomicrographs of MCF7 . control cells ( left panel , clone 38 ) , MCF7 . beclin1 cells ( middle panel , clone 17 ) and MCF7 . beclin1stop cells ( right panel , clone 70 ) 48 h after seeding at similar densities .

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c, Photomicrographs of MCF7.control cells (left panel, clone 38), MCF7. beclin1 cells (middle panel, clone 17) and MCF7.beclin1stop cells (right panel, clone 70) 48 h after seeding at similar densities.


d , Proliferation of MCF7 . control cells ( clones 37 and 38 ; closed squares and circles , respectively , solid lines ) , MCF7 . beclin1 cells ( clones 1 , 6 , 17 ; open squares , circles , triangles , respectively , solid lines ) and MCF7 . beclin1stop cells ( clone 70 , closed triangles , dotted line ) . Results represent mean Absorbance ( A ) ( ± s . e . m . ) for six wells ; similar results were obtained in three independent experiments .

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d, Proliferation of MCF7.control cells (clones 37 and 38; closed squares and circles, respectively, solid lines), MCF7.beclin1 cells (clones 1, 6, 17; open squares, circles, triangles, respectively, solid lines) and MCF7.beclin1stop cells (clone 70, closed triangles, dotted line). Results represent mean Absorbance (A) (±s.e.m.) for six wells; similar results were obtained in three independent experiments.


e , Cell viability of MCF7 clones determined by trypan blue staining . Symbols for each clone are as in

e, Cell viability of MCF7 clones determined by trypan blue staining. Symbols for each clone are as in d


f , Clonigenicity in semisolid medium ( soft agar ) of MCF7 clones . Results are mean ( ± s . e . m . ) for pooled triplicate wells from 3 - 4 independent experiments .

f, Clonigenicity in semisolid medium (soft agar) of MCF7 clones. Results are mean (±s.e.m.) for pooled triplicate wells from 3-4 independent experiments.


g , Tumour formation in NCR nude mice injected subcutaneously with MCF7 clones . Numbers on top of bar indicate number of autopsy - confirmed tumours at 8 weeks per number of mice injected with each clone .

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g, Tumour formation in NCR nude mice injected subcutaneously with MCF7 clones. Numbers on top of bar indicate number of autopsy-confirmed tumours at 8 weeks per number of mice injected with each clone.


a , Western blot analysis of human breast carcinoma cell lines . Results are from 8 representative cell lines out of a total of 11 analysed .

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a, Western blot analysis of human breast carcinoma cell lines. Results are from 8 representative cell lines out of a total of 11 analysed.


b , Western blot analysis of matched normal breast and breast carcinoma tissue from patients with sporadic invasive breast carcinoma . Results are from 7 representative cases out of a total of 17 analysed . N , normal breast tissue ; T , tumour tissue ; Mr , relative molecular mass .

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b, Western blot analysis of matched normal breast and breast carcinoma tissue from patients with sporadic invasive breast carcinoma. Results are from 7 representative cases out of a total of 17 analysed. N, normal breast tissue; T, tumour tissue; Mr, relative molecular mass.


c , Immunohistochemical analysis of paraffin - embedded sections of breast tissue from patient 441 shown in b . Adjacent sections are stained with pre - immune 843 rabbit serum ( left column ) , 843 anti - Beclin 1serum ( middle column ) and anti - cytokeratin ( right column ) . Top row shows low power view of region of breast containing tumour ( T ) and normal mammary epithelial duct ( N ) . Middle row shows higher power view of normal mammary epithelial duct . Bottom row shows higher power view of tumour . Scale bars , 1 mm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Immunohistochemical analysis of paraffin-embedded sections of breast tissue from patient 441 shown in b. Adjacent sections are stained with pre-immune 843 rabbit serum (left column), 843 anti-Beclin 1serum (middle column) and anti-cytokeratin (right column). Top row shows low power view of region of breast containing tumour (T) and normal mammary epithelial duct (N). Middle row shows higher power view of normal mammary epithelial duct. Bottom row shows higher power view of tumour. Scale bars, 1 mm.


( A ) Western blot detection of p - CHK2 Thr68 , CHK2 , p62 and LC3 in H1299 cells in normal medium ( HBSS ' ' - ' ' ) or after 1 hr HBSS starvation ( HBSS ' ' + ' ' ) pretreated with or without NAC ( 3mM ) .

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

(A) Western blot detection of p-CHK2 Thr68, CHK2, p62 and LC3 in H1299 cells in normal medium (HBSS ''-'') or after 1 hr HBSS starvation (HBSS ''+'') pretreated with or without NAC (3mM).


( B ) Western blot detection of p62 and LC3 in H1299 cells transfected with the indicated shRNA in normal medium or after H2O2 ( 500μM ) treatment .

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

(B) Western blot detection of p62 and LC3 in H1299 cells transfected with the indicated shRNA in normal medium or after H2O2 (500μM) treatment.


( C ) Western blot detection of p62 and LC3 in H1299 cells cotransfected with the indicated shRNA and the indicated plasmid and cultured in normal medium or after H2O2 ( 500μM ) treatment . CHK2 NTm , a shRNA nontargetable mutant CHK2 rescue plasmid .

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(C) Western blot detection of p62 and LC3 in H1299 cells cotransfected with the indicated shRNA and the indicated plasmid and cultured in normal medium or after H2O2 (500μM) treatment. CHK2 NTm, a shRNA nontargetable mutant CHK2 rescue plasmid.


( D ) Autophagic flux is shown by representative confocal microscopic images of 293 cells stably - expressing GFP - mCherry - LC3 transfected with the indicated shRNA following HBSS starvation and H2O2 ( 500μM ) treatment for 3h . Scale bar , 10 μm .

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(D) Autophagic flux is shown by representative confocal microscopic images of 293 cells stably-expressing GFP-mCherry-LC3 transfected with the indicated shRNA following HBSS starvation and H2O2 (500μM) treatment for 3h. Scale bar, 10 μm.


( E ) Quantitation of autophagosomal ( yellow ) and autolysosomal ( red ) LC3 puncta following HBSS starvation and H2O2 ( 500μM ) treatment for 1h ( n = 30 ) . Data are presented as mean ± s . e . m . from 3 independent experiments ; * * P < 0 . 01 ( Student ' s t - test ) .

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(E) Quantitation of autophagosomal (yellow) and autolysosomal (red) LC3 puncta following HBSS starvation and H2O2 (500μM) treatment for 1h (n=30). Data are presented as mean±s.e.m. from 3 independent experiments; **P < 0.01 (Student's t-test).


( F ) Representative electron microscopic image of autophagic vesicles or autophagosomes in H1299 cells transfected with the indicated shRNA in normal medium or after 3 hr HBSS starvation or treated with H2O2 ( 500μM ) . Scale bars , 500 nm . The red arrows indicate double - membraned autophagic structures .

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(F) Representative electron microscopic image of autophagic vesicles or autophagosomes in H1299 cells transfected with the indicated shRNA in normal medium or after 3 hr HBSS starvation or treated with H2O2 (500μM). Scale bars, 500 nm. The red arrows indicate double-membraned autophagic structures.


( G ) Electron microscopic quantification of autophagy vacuole in H1299 cells transfected with the indicated shRNA in normal medium or after 3 hr HBSS starvation or treated with H2O2 ( 500μM ) . Data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 05 ; * * P < 0 . 01 ( Student ' s t - test ) .

O O O O O 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

(G) Electron microscopic quantification of autophagy vacuole in H1299 cells transfected with the indicated shRNA in normal medium or after 3 hr HBSS starvation or treated with H2O2 (500μM). Data are presented as mean±s.e.m. from 3 independent experiments; *P < 0.05; **P < 0.01 (Student's t-test).


( A ) Immunoprecipitation assays testing the physical interaction between CHK2 and proteins encoded by autophagy - related genes in HCT116 cells . Lysates were extracted for immunoprecipitation with CHK2 - specific antibody or control IgG , followed by probing with antibodies specific for Ulk1 , Atg5 , Beclin1 , Atg7 or LC3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoprecipitation assays testing the physical interaction between CHK2 and proteins encoded by autophagy-related genes in HCT116 cells. Lysates were extracted for immunoprecipitation with CHK2-specific antibody or control IgG, followed by probing with antibodies specific for Ulk1, Atg5, Beclin1, Atg7 or LC3.


( B ) Interaction between endogenous CHK2 and Beclin 1 in HCT116 cells under fed conditions ( t = 0 ) and after HBSS starvation in the cytoplasmic fractions .

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(B) Interaction between endogenous CHK2 and Beclin 1 in HCT116 cells under fed conditions (t=0) and after HBSS starvation in the cytoplasmic fractions.


( C ) Interaction between endogenous CHK2 and Beclin 1 in HCT116 cells under fed conditions ( t = 0 ) and after H2O2 ( 500μM ) stimulation in the cytoplasmic fractions .

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(C) Interaction between endogenous CHK2 and Beclin 1 in HCT116 cells under fed conditions (t=0) and after H2O2 (500μM) stimulation in the cytoplasmic fractions.


( D ) HCT116 cells were pretreated with NAC in RPMI 1640 complete medium for 4 h and then cultured for 1 hr in HBSS starvation . The cytoplasmic lysates were subjected to immunoprecipitation with anti - Beclin 1 antibody followed by immunoblotting with anti - CHK2 and anti - Beclin 1 antibody . The expression of p - CHK2 Thr68 , CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions . Tubulin was used as a loading control .

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

(D) HCT116 cells were pretreated with NAC in RPMI 1640 complete medium for 4 h and then cultured for 1 hr in HBSS starvation. The cytoplasmic lysates were subjected to immunoprecipitation with anti-Beclin 1 antibody followed by immunoblotting with anti-CHK2 and anti-Beclin 1 antibody. The expression of p-CHK2 Thr68, CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions. Tubulin was used as a loading control.


HCT116 cells were pretreated with CHK2 inhibitorⅡ in RPMI 1640 complete medium for 4 hr and then cultured for 1 hr in HBSS starvation ( E ) The cytoplasmic lysates were subjected to immunoprecipitation with anti - Beclin 1 antibody followed by immunoblotting with anti - CHK2 and anti - Beclin 1 antibody . The expression of p - CHK2 Thr68 , CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions . Tubulin was used as a loading control .

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HCT116 cells were pretreated with CHK2 inhibitorⅡ in RPMI 1640 complete medium for 4 hr and then cultured for 1 hr in HBSS starvation (E) The cytoplasmic lysates were subjected to immunoprecipitation with anti-Beclin 1 antibody followed by immunoblotting with anti-CHK2 and anti-Beclin 1 antibody. The expression of p-CHK2 Thr68, CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions. Tubulin was used as a loading control.


HCT116 cells were pretreated with CHK2 inhibitorⅡ in RPMI 1640 complete medium for 4 hr or H2O2 ( 500μM ) stimulation ( F ) . The cytoplasmic lysates were subjected to immunoprecipitation with anti - Beclin 1 antibody followed by immunoblotting with anti - CHK2 and anti - Beclin 1 antibody . The expression of p - CHK2 Thr68 , CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions . Tubulin was used as a loading control .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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

HCT116 cells were pretreated with CHK2 inhibitorⅡ in RPMI 1640 complete medium for 4 hr or H2O2 (500μM) stimulation (F). The cytoplasmic lysates were subjected to immunoprecipitation with anti-Beclin 1 antibody followed by immunoblotting with anti-CHK2 and anti-Beclin 1 antibody. The expression of p-CHK2 Thr68, CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions. Tubulin was used as a loading control.


HCT116 cells were transiently transfected with the expression plasmids as indicated . After 36 hr post - transfection , cells were untreated ( G ) for 1hr and then collected for immunoprecipitation and western blotting analysis . Immunoprecipitation was performed using anti - Beclin 1 antibody followed by immunoblotting with anti - Flag or anti - Beclin 1 antibody .

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HCT116 cells were transiently transfected with the expression plasmids as indicated. After 36 hr post-transfection, cells were untreated (G) for 1hr and then collected for immunoprecipitation and western blotting analysis. Immunoprecipitation was performed using anti-Beclin 1 antibody followed by immunoblotting with anti-Flag or anti-Beclin 1 antibody.


HCT116 cells were transiently transfected with the expression plasmids as indicated . After 36 hr post - transfection , cells were treated with HBSS starvation ( H ) for 1hr and then collected for immunoprecipitation and western blotting analysis . Immunoprecipitation was performed using anti - Beclin 1 antibody followed by immunoblotting with anti - Flag or anti - Beclin 1 antibody .

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

HCT116 cells were transiently transfected with the expression plasmids as indicated. After 36 hr post-transfection, cells were treated with HBSS starvation (H) for 1hr and then collected for immunoprecipitation and western blotting analysis. Immunoprecipitation was performed using anti-Beclin 1 antibody followed by immunoblotting with anti-Flag or anti-Beclin 1 antibody.


HCT116 cells were transiently transfected with the expression plasmids as indicated . After 36 hr post - transfection , cells were treated with H2O2 ( 500μM ) stimulation ( I ) for 1hr and then collected for immunoprecipitation and western blotting analysis . Immunoprecipitation was performed using anti - Beclin 1 antibody followed by immunoblotting with anti - Flag or anti - Beclin 1 antibody .

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

HCT116 cells were transiently transfected with the expression plasmids as indicated. After 36 hr post-transfection, cells were treated with H2O2 (500μM) stimulation (I) for 1hr and then collected for immunoprecipitation and western blotting analysis. Immunoprecipitation was performed using anti-Beclin 1 antibody followed by immunoblotting with anti-Flag or anti-Beclin 1 antibody.


( J ) Western blot detection of p62 and LC3 in H1299 cells transfected with indicated plasmid in normal medium or after H2O2 ( 500 μM ) cultured for 3 hr .

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(J) Western blot detection of p62 and LC3 in H1299 cells transfected with indicated plasmid in normal medium or after H2O2 (500 μM) cultured for 3 hr.


( K ) H1299 cells were pretreated with CHK2 inhibitorⅡ in complete medium for 4 h and then cultured for 3 hr in H2O2 ( 500μM ) stimulation . Western blot detection of p - CHK2 Thr68 , CHK2 , p62 and LC3 .

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(K) H1299 cells were pretreated with CHK2 inhibitorⅡ in complete medium for 4 h and then cultured for 3 hr in H2O2 (500μM) stimulation. Western blot detection of p-CHK2 Thr68, CHK2, p62 and LC3.


( A ) The indicated recombinant fragments of GST - Beclin1 were incubated with recombinant CHK2 in the presence of 32P - labeled ATP for in vitro kinase assays . MBP was used as a positive control . Ponceau staining indicates the expression of GST - Beclin 1 fragments for in vitro kinase assays . The asterisks in the blot indicate the domain of phosphorylated Beclin 1 .

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(A) The indicated recombinant fragments of GST-Beclin1 were incubated with recombinant CHK2 in the presence of 32P-labeled ATP for in vitro kinase assays. MBP was used as a positive control. Ponceau staining indicates the expression of GST-Beclin 1 fragments for in vitro kinase assays. The asterisks in the blot indicate the domain of phosphorylated Beclin 1.


( C ) In vitro kinase assays to test the ability of recombinant CHK2 to phosphorylate recombinant GST - Beclin 1 - WT , S9093A ( AA ) , S90A and S93A protein . Reactions were analyzed by SDS - PAGE followed by autoradiography . The asterisks in the blot indicate the phosphorylation of Beclin 1 mutants .

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(C) In vitro kinase assays to test the ability of recombinant CHK2 to phosphorylate recombinant GST-Beclin 1-WT, S9093A (AA), S90A and S93A protein. Reactions were analyzed by SDS-PAGE followed by autoradiography. The asterisks in the blot indicate the phosphorylation of Beclin 1 mutants.


Western blot detection of p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in MEFs indicated genotype in normal medium or after 1 hr HBSS starvation ( D )

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Western blot detection of p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in MEFs indicated genotype in normal medium or after 1 hr HBSS starvation (D)


Western blot detection of p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in MEFs indicated genotype in normal medium or H2O2 ( 500μM ) stimulation ( E ) .

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Western blot detection of p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in MEFs indicated genotype in normal medium or H2O2 (500μM) stimulation (E).


Western blot detection of ATM , p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with indicated shRNA in normal medium or after HBSS starvation ( F )

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

Western blot detection of ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with indicated shRNA in normal medium or after HBSS starvation (F)


Western blot detection of ATM , p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with indicated shRNA in normal medium or H2O2 ( 500μM ) stimulation ( G ) .

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

Western blot detection of ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with indicated shRNA in normal medium or H2O2 (500μM) stimulation (G).


( H ) Western blot detection of p - ATM Ser1981 , ATM , p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells pretreated with NAC for 4 h and then cultured for 1 hr in HBSS starvation .

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(H) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells pretreated with NAC for 4 h and then cultured for 1 hr in HBSS starvation.


( A ) The effect of Beclin 1 Ser90 / 93 phosphorylation on the interaction between Beclin 1 and Bcl - 2 . GST - Beclin1 WT , AA mutant and DD mutant ( bait ) were incubated with or without recombinant CHK2 in the presence of ATP and then incubated with flag - Bcl - 2 ( preys ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The effect of Beclin 1 Ser90/93 phosphorylation on the interaction between Beclin 1 and Bcl-2. GST-Beclin1 WT, AA mutant and DD mutant (bait) were incubated with or without recombinant CHK2 in the presence of ATP and then incubated with flag-Bcl-2 (preys).


( B ) Immunoprecipitation of Bcl - 2 with Flag - Beclin 1 WT , AA mutant and DD mutant in HCT116 cells in normal medium or after H2O2 ( 500μM ) treatment for 1h .

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(B) Immunoprecipitation of Bcl-2 with Flag-Beclin 1 WT, AA mutant and DD mutant in HCT116 cells in normal medium or after H2O2 (500μM) treatment for 1h.


( C - D ) Autophagy levels were determined in Beclin 1 - depleted H1299 cells with reconstituted expression of Beclin 1 WT , AA mutant or DD mutant in normal medium or after H2O2 ( 500μM ) treatment for 3h in the presence ( C ) or absence ( D ) of CHK2 .

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(C-D) Autophagy levels were determined in Beclin 1-depleted H1299 cells with reconstituted expression of Beclin 1 WT, AA mutant or DD mutant in normal medium or after H2O2 (500μM) treatment for 3h in the presence (C) or absence (D) of CHK2.


( E ) Representative electron microscopic image of an autophagosome ( arrow , left panel , AP ) and an autolysosome ( arrow , right panel , AL ) in H1299 Beclin 1 WT cells treated with H2O2 ( 500μM ) for 6 hr . Scale bars , 500 nm . Electron microscopic quantification of autophagosomes ( AP ) and autolysosomes ( AL ) in H1299 cells with the indicated plasmids cultured for 6 hr in normal or H2O2 ( 500μM ) treatment . All quantitative data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 001 compared to Beclin 1 WT ; # P < 0 . 001 compared to Beclin 1 WT treated with H2O2 ; & P < 0 . 001 compared to Beclin 1 WT treated with H2O2 in the absence of CHK2 .

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

(E) Representative electron microscopic image of an autophagosome (arrow, left panel, AP) and an autolysosome (arrow, right panel, AL) in H1299 Beclin 1 WT cells treated with H2O2 (500μM) for 6 hr. Scale bars, 500 nm. Electron microscopic quantification of autophagosomes (AP) and autolysosomes (AL) in H1299 cells with the indicated plasmids cultured for 6 hr in normal or H2O2 (500μM) treatment. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.001 compared to Beclin 1 WT; #P < 0.001 compared to Beclin 1 WT treated with H2O2; &P < 0.001 compared to Beclin 1 WT treated with H2O2 in the absence of CHK2.


( F ) Western blot detection of p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 , Beclin 1 , p62 and LC3 in H1299 cells transfected with the indicated shRNA in normal medium or after H2O2 ( 500μM ) treatment for 3h .

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(F) Western blot detection of p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93, Beclin 1, p62 and LC3 in H1299 cells transfected with the indicated shRNA in normal medium or after H2O2 (500μM) treatment for 3h.


( A ) Western blot detection of p - CHK2 Thr68 , CHK2 , p - AMPKα Thr172 , AMPKα , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after glucose starvation .

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(A) Western blot detection of p-CHK2 Thr68, CHK2, p-AMPKα Thr172, AMPKα, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after glucose starvation.


( B ) Western blot detection of p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after hypoxia .

O O O O O O O O O O O O O O O O O O O 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) Western blot detection of p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after hypoxia.


( C ) Western blot detection of ATM , p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after glucose starvation .

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

(C) Western blot detection of ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after glucose starvation.


( D ) Western blot detection of p - ATM Ser1981 , ATM , p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after hypoxia .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after hypoxia.


( E ) P - ATM Ser1981 , ATM , p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 were analyzed in H1299 cells , and the cells were subjected to hypoxia with or without pretreatment with NAC .

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

(E) P-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 were analyzed in H1299 cells, and the cells were subjected to hypoxia with or without pretreatment with NAC.


( F ) P - ATM Ser1981 , ATM , p - CHK2 Thr68 , CHK2 , p - AMPKα Thr172 , AMPKα , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 were analyzed in H1299 cells , and the cells were subjected to glucose starvation with or without pretreatment with NAC .

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

(F) P-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-AMPKα Thr172, AMPKα, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 were analyzed in H1299 cells, and the cells were subjected to glucose starvation with or without pretreatment with NAC.


( G ) Western blot detection of p - ATM Ser1981 , ATM , p - CHK2 Thr68 , CHK2 , p - AMPKα Thr172 , and AMPKα in H1299 cells in normal medium or after H2O2 ( 25μM ) stimulation .

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

(G) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-AMPKα Thr172, and AMPKα in H1299 cells in normal medium or after H2O2 (25μM) stimulation.


( H ) Western blot detection of p - ATM Ser1981 , ATM , p - CHK2 Thr68 , CHK2 , p - AMPKα Thr172 and AMPKα in H1299 cells in normal medium or after 2DG ( 5mM ) and oligomycin ( 2 . 5μM ) 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 B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O

(H) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-AMPKα Thr172 and AMPKα in H1299 cells in normal medium or after 2DG (5mM) and oligomycin (2.5μM) treatment.


Intracellular ROS levels detected in H1299 cells transfected with the indicated shRNA in normal medium or after 12hr glucose starvation ( Glu - )

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

Intracellular ROS levels detected in H1299 cells transfected with the indicated shRNA in normal medium or after 12hr glucose starvation (Glu-)


Intracellular ROS levels detected in H1299 cells transfected with the indicated shRNA in normal medium or after 36hr hypoxia ( B ) .

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

Intracellular ROS levels detected in H1299 cells transfected with the indicated shRNA in normal medium or after 36hr hypoxia (B).


( C ) Western blot detection of CHK2 , HSP60 , TOM20 and TIM23 in H1299 cells transfected with the indicated shRNA in normal medium or after12 hr glucose starvation ( Glu - ) or 36 hr hypoxia .

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

(C) Western blot detection of CHK2, HSP60, TOM20 and TIM23 in H1299 cells transfected with the indicated shRNA in normal medium or after12 hr glucose starvation (Glu-) or 36 hr hypoxia.


( D - E ) Transmission electron microscopy of indicated H1299 cells cultured in glucose starvation ( Glu - ) medium or hypoxia . The yellow asterisks mark mitochondria . The red arrows indicate autophagic structures . All quantitative data are presented as mean ± s . e . m . from 3 independent experiments ; * * * P < 0 . 001 compared to shNC treated with H2O2 or hypoxia ( Mann - Whitney test ) ; Scale bar , 1 μm .

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

(D-E) Transmission electron microscopy of indicated H1299 cells cultured in glucose starvation (Glu-) medium or hypoxia. The yellow asterisks mark mitochondria. The red arrows indicate autophagic structures. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; ***P < 0.001 compared to shNC treated with H2O2 or hypoxia (Mann-Whitney test); Scale bar, 1 μm.


( F ) Representative confocal images of the shift in the fluorescence emission of Keima from 458 to 561 nm . Scale bar , 10 μm .

O O O 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) Representative confocal images of the shift in the fluorescence emission of Keima from 458 to 561 nm. Scale bar, 10 μm.


The mitochondrial membrane potential analysis of 293 cells transfected with the indicated shRNA in normal medium or after 18 hr glucose starvation ( G ) All results are from three independent experiments . All quantitative data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 05 ( Mann - Whitney test ) .

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

The mitochondrial membrane potential analysis of 293 cells transfected with the indicated shRNA in normal medium or after 18 hr glucose starvation (G) All results are from three independent experiments. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 (Mann-Whitney test).


The mitochondrial membrane potential analysis of 293 cells transfected with the indicated shRNA in normal medium or 48 hr hypoxia ( H ) . All results are from three independent experiments . All quantitative data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 05 ( Mann - Whitney test ) .

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

The mitochondrial membrane potential analysis of 293 cells transfected with the indicated shRNA in normal medium or 48 hr hypoxia (H). All results are from three independent experiments. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 (Mann-Whitney test).


H1299 cells transfected with the indicated shRNA in normal medium or after 12h glucose starvation ( A ) Representative FACS analysis of apoptosis . Data are presented as mean ± s . e . m . from 3 independent experiments ; * p < 0 . 05 compared to NC glucose starvation ( A ) ( Student ' s t - test ) .

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

H1299 cells transfected with the indicated shRNA in normal medium or after 12h glucose starvation (A) Representative FACS analysis of apoptosis. Data are presented as mean ± s.e.m. from 3 independent experiments; *p < 0.05 compared to NC glucose starvation (A) (Student's t-test).


H1299 cells transfected with the indicated shRNA in normal medium or 48h hypoxia stimulation ( B ) . Representative FACS analysis of apoptosis . Data are presented as mean ± s . e . m . from 3 independent experiments * * p < 0 . 01 compared to NC hypoxia ( B ) ( Student ' s t - test ) .

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

H1299 cells transfected with the indicated shRNA in normal medium or 48h hypoxia stimulation (B). Representative FACS analysis of apoptosis. Data are presented as mean ± s.e.m. from 3 independent experiments **p < 0.01 compared to NC hypoxia (B) (Student's t-test).


H1299 cells transfected with the indicated plasmids in normal medium or after 12h glucose starvation ( C ) Representative FACS analysis of apoptosis . Results from three independent experiments are presented as a histogram . Data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 05 compared to Beclin 1 S9093A ( AA ) treated with H2O2 or hypoxia ( Mann - Whitney test ) .

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

H1299 cells transfected with the indicated plasmids in normal medium or after 12h glucose starvation (C) Representative FACS analysis of apoptosis. Results from three independent experiments are presented as a histogram. Data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 compared to Beclin 1 S9093A (AA) treated with H2O2 or hypoxia (Mann-Whitney test).


H1299 cells transfected with the indicated plasmids in normal medium or 48h hypoxia stimulation ( D ) . Representative FACS analysis of apoptosis . Results from three independent experiments are presented as a histogram . Data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 05 compared to Beclin 1 S9093A ( AA ) treated with H2O2 or hypoxia ( Mann - Whitney test ) .

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

H1299 cells transfected with the indicated plasmids in normal medium or 48h hypoxia stimulation (D). Representative FACS analysis of apoptosis. Results from three independent experiments are presented as a histogram. Data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 compared to Beclin 1 S9093A (AA) treated with H2O2 or hypoxia (Mann-Whitney test).


( E ) CHK2 + / + and CHK2 - / - mice were subjected to MCAO for 1 hr and reperfusion for 12 h . Contralateral ( C ) and ipsilateral ( I ) tissues of the mouse brain were coronally sectioned and stained with 2 % TTC .

(E) CHK2+/+ and CHK2-/- mice were subjected to MCAO for 1 hr and reperfusion for 12 h. Contralateral (C) and ipsilateral (I) tissues of the mouse brain were coronally sectioned and stained with 2 % TTC.


( F ) The infarct volume was determined by measuring infarct size relative to normal in the slice . ( n = 5 mice ) . Data are presented as mean ± s . e . m . ; * * P < 0 . 01 ( Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The infarct volume was determined by measuring infarct size relative to normal in the slice. (n = 5 mice). Data are presented as mean ± s.e.m.; **P < 0.01 (Student's t-test).


( G - J ) Immunoblots for p62 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in the cortical extracts from ischemia and reperfusion - treated CHK2 + / + and CHK2 - / - mice . Quantification of p62 , p - Beclin 1 Ser90 and p - Beclin 1 Ser93 protein levels ( n = 3 mice ) . Data are presented as mean ± s . e . m . ; * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J) Immunoblots for p62, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in the cortical extracts from ischemia and reperfusion-treated CHK2+/+ and CHK2-/- mice. Quantification of p62, p-Beclin 1 Ser90 and p-Beclin 1 Ser93 protein levels (n = 3 mice). Data are presented as mean ± s.e.m.; *P < 0.05, **P < 0.01 (Student's t-test).


Alveolar macrophages from different donors were purified from BAL fluid by allowing them to attach to the culture dish for 2 hours . Subsequently , they were pre - treated overnight with either type I or type III IFNs ( 1 ng / mL IFN - λ1 and 10 U / mL IFN - α2 , respectively ) before infection with Sendai virus ( SeV ) and influenza A virus ( IAV , strain PR8 ) for 6 hours as indicated . Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2 , IFNB1 , and IFNL1 . ISG response measured by ISG15 and RSAD2 . Each colour corresponds to a donor ; 9 different donors were analysed . The dashed line represents the detection limit . An ordinary one - way - ANOVA test was used for the statistical analysis : ns , not significant ; * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 .

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

Alveolar macrophages from different donors were purified from BAL fluid by allowing them to attach to the culture dish for 2 hours. Subsequently, they were pre-treated overnight with either type I or type III IFNs (1 ng/mL IFN-λ1 and 10 U/mL IFN-α2, respectively) before infection with Sendai virus (SeV) and influenza A virus (IAV, strain PR8) for 6 hours as indicated. Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2, IFNB1, and IFNL1. ISG response measured by ISG15 and RSAD2. Each colour corresponds to a donor; 9 different donors were analysed. The dashed line represents the detection limit. An ordinary one-way-ANOVA test was used for the statistical analysis: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.