( B ) Quantification ( % ) of explants presenting shoots after 21 days after incubation on SIM . Results were obtained from 3 independent experiments with at least 20 calli per condition . Bars marked with an asterisk ( * ) are statistically significant ( P < 0 . 05 ) according to the T - test .

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(B) Quantification (%) of explants presenting shoots after 21 days after incubation on SIM. Results were obtained from 3 independent experiments with at least 20 calli per condition. Bars marked with an asterisk (*) are statistically significant (P<0.05) according to the T -test.


( A ) Representative images of root explants from WT , atg2 - 2 and atg5 - 1 in CIM ( 21 days ) or CIM + SIM ( 21 + 21 days or 21 + 35 days ) . Scale bar : 2

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(A) Representative images of root explants from WT, atg2-2 and atg5-1 in CIM (21 days) or CIM + SIM (21+ 21 days or 21+ 35 days). Scale bar: 2 cm


( B and C ) Fresh weights of calli after incubation on CIM ( B ) or CIM + SIM ( 21 + 21 ) ( C ) . Box plots : centerlines show the medians ; box limits indicate the 25th and 75th percentiles ; whiskers extend to the minimum and maximum . Results were obtained from 3 independent experiments with at least 20 calli per condition , asteriscs mark statistical significance to WT according to the T - test ( * * P < 0 . 001 ; * * * P < 0 . 0001 ) .

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(B and C) Fresh weights of calli after incubation on CIM (B) or CIM + SIM (21+ 21) (C). Box plots: centerlines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the minimum and maximum. Results were obtained from 3 independent experiments with at least 20 calli per condition, asteriscs mark statistical significance to WT according to the T -test (** P<0.001; *** P<0.0001).


Labelling scheme and representative DNA fibers from U2OS cells transfected with siLuc or siChk1 .

Labelling scheme and representative DNA fibers from U2OS cells transfected with siLuc or siChk1.


Western Blot of Chk1 in U2OS cells , 48 h after transfection with siRNA . Actin was used as loading control .

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Western Blot of Chk1 in U2OS cells, 48 h after transfection with siRNA. Actin was used as loading control.


Control ( siLuc ) and Chk1 - depleted ( siChk1 ) U20S cells were labeled with CldU and IdU for 10 and 30 min , respectively . Mean ( + SEM ) track lengths ( top ) and CldU / IdU ratios ( bottom ) are shown . Data come from 6 independent experiments and a total of 579 ( siLuc ) and 590 ( siChk1 ) fibers were scored .

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Control (siLuc) and Chk1-depleted (siChk1) U20S cells were labeled with CldU and IdU for 10 and 30 min, respectively. Mean (+SEM) track lengths (top) and CldU/IdU ratios (bottom) are shown. Data come from 6 independent experiments and a total of 579 (siLuc) and 590 (siChk1) fibers were scored.


Western Blot of phospho - serine - 296 - Chk1 ( p - Chk1 ) in U2OS cells . Cells were treated 1 . 5 h with the Chk1 inhibitor Gö6976 ( Chk1i ) or DMSO , irradiated with 100J / m2 UV to induce high levels of p - Chk1 , and collected 1 . 5 h afterwards . Actin was used as a loading control .

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Western Blot of phospho-serine-296-Chk1 (p-Chk1) in U2OS cells. Cells were treated 1.5 h with the Chk1 inhibitor Gö6976 (Chk1i) or DMSO, irradiated with 100J/m2 UV to induce high levels of p-Chk1, and collected 1.5 h afterwards. Actin was used as a loading control.


U2OS cells treated with DMSO ( control ) or Chk1i were labeled with CldU and IdU for 10 and 30 min , respectively . Data come from 3 independent experiments and a total of 304 ( DMSO ) and 312 ( Chk1i ) fibers were scored .

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U2OS cells treated with DMSO (control) or Chk1i were labeled with CldU and IdU for 10 and 30 min, respectively. Data come from 3 independent experiments and a total of 304 (DMSO) and 312 (Chk1i) fibers were scored.


U2OS cells were labeled with CldU for 10 min , followed by increasing IdU labelling times and subjected to DNA stretching . ( A ) Labelling scheme and representative DNA fibers . Scale bars : 5 μm .

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U2OS cells were labeled with CldU for 10 min, followed by increasing IdU labelling times and subjected to DNA stretching. (A) Labelling scheme and representative DNA fibers. Scale bars: 5 μm.


U2OS cells were labeled with CldU for 10 min , followed by increasing IdU labelling times and subjected to DNA stretching . IdU track length after 10 - 80 min . Theoretical IdU track lengths are shown in red ; the theoretical 10 - min points were considered equal to the experimental values obtained for siLuc or siChk1 , and the following theoretical points were calculated proportionally . Data were calculated as the mean ( + SD ) of mean IdU track lengths ( B ) n = 4 for IdU times 20 - 60 min or n = 3 for IdU times 10 and 80 min ; for each experiment and condition 60 - 100 fibers were scored .

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U2OS cells were labeled with CldU for 10 min, followed by increasing IdU labelling times and subjected to DNA stretching. IdU track length after 10-80 min. Theoretical IdU track lengths are shown in red; the theoretical 10-min points were considered equal to the experimental values obtained for siLuc or siChk1, and the following theoretical points were calculated proportionally. Data were calculated as the mean (+SD) of mean IdU track lengths (B) n=4 for IdU times 20-60 min or n=3 for IdU times 10 and 80 min; for each experiment and condition 60-100 fibers were scored.


U2OS cells were labeled with CldU for 10 min , followed by increasing IdU labelling times and subjected to DNA stretching . Fork speed for each IdU labelling time . Data were calculated as the mean ( + SD ) of mean fork speeds ( C ) . n = 4 for IdU times 20 - 60 min or n = 3 for IdU times 10 and 80 min ; for each experiment and condition 60 - 100 fibers were scored .

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U2OS cells were labeled with CldU for 10 min, followed by increasing IdU labelling times and subjected to DNA stretching. Fork speed for each IdU labelling time. Data were calculated as the mean (+SD) of mean fork speeds (C). n=4 for IdU times 20-60 min or n=3 for IdU times 10 and 80 min; for each experiment and condition 60-100 fibers were scored.


DNA combing from HCT116 cells labeled with CldU and IdU for 20 min . The upper left panel shows the labelling scheme . The upper right panel shows representative sister replication forks that emanate from the same origin ( ORI = origin of replication ) ; a pair of sister forks is considered asymmetric if their lengths ( CldU + IdU ) differ in more than 33 % . The percentage of asymmetric forks ( lower left panel ; a Western Blot of Chk1 and Ku80 , loading control , performed in parallel to the combing experiments , is shown ) and the lengths of each pair of forks ( lower right panel ; points outside of the dotted lines correspond to asymmetric forks ) are shown . Data ( siLuc , n = 131 ; siChk1 , n = 117 ) come from 2 independent experiments ; the numbers indicate the mean ( + SD ) percentage of asymmetric forks .

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DNA combing from HCT116 cells labeled with CldU and IdU for 20 min. The upper left panel shows the labelling scheme. The upper right panel shows representative sister replication forks that emanate from the same origin (ORI=origin of replication); a pair of sister forks is considered asymmetric if their lengths (CldU+IdU) differ in more than 33%. The percentage of asymmetric forks (lower left panel; a Western Blot of Chk1 and Ku80, loading control, performed in parallel to the combing experiments, is shown) and the lengths of each pair of forks (lower right panel; points outside of the dotted lines correspond to asymmetric forks) are shown. Data (siLuc, n=131; siChk1, n=117) come from 2 independent experiments; the numbers indicate the mean (+SD) percentage of asymmetric forks.


Labelling protocol used here and in all figures that follow , except otherwise indicated . The lower panels show representative images and schemes of an ongoing fork and of origins that fired during the CldU ( 1st ) or IdU ( 2nd ) pulses . Gray dotted lines represent unlabeled DNA . Percentage of origins was calculated as : ( 1st + 2nd pulse origins ) / total number of fibers .

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Labelling protocol used here and in all figures that follow, except otherwise indicated. The lower panels show representative images and schemes of an ongoing fork and of origins that fired during the CldU (1st) or IdU (2nd) pulses. Gray dotted lines represent unlabeled DNA. Percentage of origins was calculated as: (1st+2nd pulse origins)/total number of fibers.


IdU track lengths from U2OS cells ( Rosc , roscovitine ) . 180 - 200 DNA fibers obtained from 2 independent experiments were measured for each condition . The upper panel shows a Western Blot of Polη , 48 h after transfection with siRNA . Actin was used as a loading control .

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IdU track lengths from U2OS cells (Rosc, roscovitine). 180-200 DNA fibers obtained from 2 independent experiments were measured for each condition. The upper panel shows a Western Blot of Polη, 48 h after transfection with siRNA. Actin was used as a loading control.


Percentage of origin firing ( mean + SD ) from DNA fibers in ( A ) ( n = 2 ) . The dotted line represents the mean percentage of origin firing in control samples .

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Percentage of origin firing (mean+SD) from DNA fibers in (A) (n=2). The dotted line represents the mean percentage of origin firing in control samples.


Representative images of siChk1 - depleted U2OS cells showing nuclear localization of GFP - Polη - WT / S687A / S687D . Numbers indicate percentage of cells showing nuclear GFP - Polη ( mean + SD , n = 2 ) . Scale bar : 100 μm .

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Representative images of siChk1-depleted U2OS cells showing nuclear localization of GFP-Polη-WT/S687A/S687D. Numbers indicate percentage of cells showing nuclear GFP-Polη (mean+SD, n=2). Scale bar: 100 μm.


Western Blot of GFP - Polη in U2OS cells , 48 / 24 h after transfection with siRNA / GFP ( - Polη ) . Actin was used as loading control .

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Western Blot of GFP-Polη in U2OS cells, 48/24 h after transfection with siRNA/GFP(-Polη). Actin was used as loading control.


IdU track lengths from U2OS cells transfected with GFP ( - ) or GFP - Polη - WT / S687A / S687D . > 180 fibers obtained from 2 independent experiments were measured for each condition .

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IdU track lengths from U2OS cells transfected with GFP (-) or GFP-Polη-WT/S687A/S687D. >180 fibers obtained from 2 independent experiments were measured for each condition.


Number of foci per cell after CSK extraction in siLuc and siChk1 - depleted U2OS cells transfected with GFP - Polη - WT / S687A / S687D . > 150 cells / sample were analyzed in 4 independent experiments . p < 0 . 05 was considered significant .

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Number of foci per cell after CSK extraction in siLuc and siChk1-depleted U2OS cells transfected with GFP-Polη-WT/S687A/S687D. >150 cells/sample were analyzed in 4 independent experiments. p<0.05 was considered significant.


Representative images of cells with GFP - Polη foci used for quantification in ( E ) . The number of foci per cell for each representative nucleus is indicated . Scale bar : 10 μm .

Representative images of cells with GFP-Polη foci used for quantification in (E). The number of foci per cell for each representative nucleus is indicated. Scale bar: 10 μm.


IdU track lengths and percentage of origin firing ( mean + SD ) from U2OS cells treated or not with a CDC7 inhibitor ( CDC7i ) . > 380 ( track length ) and > 600 ( origin frequency ) fibers obtained from 4 ( track length ) or 3 ( origin frequency ) independent experiments were measured for each condition .

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IdU track lengths and percentage of origin firing (mean+SD) from U2OS cells treated or not with a CDC7 inhibitor (CDC7i). >380 (track length) and >600 (origin frequency) fibers obtained from 4 (track length) or 3 (origin frequency) independent experiments were measured for each condition.


IdU track lengths and percentage of origin firing ( mean + SD ) from U2OS cells transfected or not with siCDT1 . Track lengths from > 290 fibers obtained from 3 independent experiments were measured for each condition . To calculate origin frequency > 700 DNA fibers from 3 independent experiments were scored .

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IdU track lengths and percentage of origin firing (mean+SD) from U2OS cells transfected or not with siCDT1. Track lengths from >290 fibers obtained from 3 independent experiments were measured for each condition. To calculate origin frequency >700 DNA fibers from 3 independent experiments were scored.


IdU track lengths and percentage of origin firing ( mean + SD ) from U2OS cells supplemented or not with nucleosides ( NS ) . > 195 ( track length ) and > 350 ( origin frequency ) fibers obtained from 2 independent experiments were measured for each condition .

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IdU track lengths and percentage of origin firing (mean+SD) from U2OS cells supplemented or not with nucleosides (NS). >195 (track length) and >350 (origin frequency) fibers obtained from 2 independent experiments were measured for each condition.


IdU track lengths and percentage of origin firing ( mean + SD ) from U2OS cells transfected or not with GFP - Polη . > 180 ( track length ) and > 400 ( origin frequency ) DNA fibers obtained from 2 independent experiments were measured for each condition .

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IdU track lengths and percentage of origin firing (mean+SD) from U2OS cells transfected or not with GFP-Polη. >180 (track length) and >400 (origin frequency) DNA fibers obtained from 2 independent experiments were measured for each condition.


Percentage of γH2AX - positive U2OS cells ( mean + SD ) after 5 h treatment with the Chk1 inhibitor Gö6976 ( Chk1i ) , in the presence of 5 μM NS , 1 , 25 μM CDC7i or 10 μM roscovitine . > 900 ( DMSO ) or > 2000 ( Chk1i ) cells / sample were analyzed in 2 independent experiments . Total γH2AX was used to perform the statistics . Representative images of data shown in ( B ) . Scale bar : 50 μm .

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Percentage of γH2AX-positive U2OS cells (mean+SD) after 5 h treatment with the Chk1 inhibitor Gö6976 (Chk1i), in the presence of 5 μM NS, 1,25 μM CDC7i or 10 μM roscovitine. >900 (DMSO) or >2000 (Chk1i) cells/sample were analyzed in 2 independent experiments. Total γH2AX was used to perform the statistics. Representative images of data shown in (B). Scale bar: 50 μm.


DSB accumulation measured by neutral comet assay after 8 h treatment with Chk1i , in the presence of 5 μM NS , siCDC7 or 10 μM roscovitine . 85 - 170 cells / sample were analyzed in 2 independent experiments . Representative images of data shown in ( D ) . Scale bar : 25 μm .

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DSB accumulation measured by neutral comet assay after 8 h treatment with Chk1i, in the presence of 5 μM NS, siCDC7 or 10 μM roscovitine. 85-170 cells/sample were analyzed in 2 independent experiments. Representative images of data shown in (D). Scale bar: 25 μm.


Percentage of parental ssDNA - positive U2OS cells ( mean + SD ) after 5 h treatment with Chk1i , in the presence of 5 μM NS , 1 , 25 μM CDC7i or 10 μM roscovitine . > 450 cells / sample were analyzed in 2 independent experiments . The induction of ssDNA on control samples was null . Representative images of data shown in ( F ) . Scale bar : 25 μm .

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Percentage of parental ssDNA-positive U2OS cells (mean+SD) after 5 h treatment with Chk1i, in the presence of 5 μM NS, 1,25 μM CDC7i or 10 μM roscovitine. >450 cells/sample were analyzed in 2 independent experiments. The induction of ssDNA on control samples was null. Representative images of data shown in (F). Scale bar: 25 μm.


Sensitivity of U2OS cells to Chk1 inhibition , roscovitine ( 5 μM ) and Polη depletion . Cell number was determined 4 days after a 24 h treatment ( 5 days in total ) with Chk1i ± Rosc . Data represent the mean ( + SD ) of 3 independent experiments .

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Sensitivity of U2OS cells to Chk1 inhibition, roscovitine (5 μM) and Polη depletion. Cell number was determined 4 days after a 24 h treatment (5 days in total) with Chk1i±Rosc. Data represent the mean (+SD) of 3 independent experiments.


Sensitivity of U2OS cells transduced with shRNA targeting Chk1 ( shChk1 ) or non - targeting shRNA ( shScr ) and Lenti - GFP - Polη or Lenti - GFP ( - ) . Cell number was determined 7 days after transduction ( 5 days after maximal downregulation ) . Data represent the mean ( + SD ) of 3 independent experiments .

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Sensitivity of U2OS cells transduced with shRNA targeting Chk1 (shChk1) or non-targeting shRNA (shScr) and Lenti-GFP-Polη or Lenti-GFP (-). Cell number was determined 7 days after transduction (5 days after maximal downregulation). Data represent the mean (+SD) of 3 independent experiments.


Western Blot of CDC45 and Chk1 in U2OS cells after an extraction with CSK buffer to separate the insoluble and soluble fractions . Roscovitine ( 25μM ) was added 2 h before harvesting . H2B was used as a loading control .

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Western Blot of CDC45 and Chk1 in U2OS cells after an extraction with CSK buffer to separate the insoluble and soluble fractions. Roscovitine (25μM) was added 2 h before harvesting. H2B was used as a loading control.


IdU track lengths from U2OS cells . 200 DNA fibers obtained from 2 independent experiments were measured for each condition .

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IdU track lengths from U2OS cells. 200 DNA fibers obtained from 2 independent experiments were measured for each condition.


IdU track lengths from U2OS cells . > 170 DNA fibers obtained from 2 independent experiments were measured for each condition . For comparison , the data showing that Polη is required for roscovitine - dependent rescue of fork elongation in Chk1 - depleted cells ( Fig 3B ) is shown on the right . The lower panel shows the corresponding Western Blot of Chk1 , Polη and CDC45 . Actin was used as a loading control .

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IdU track lengths from U2OS cells. >170 DNA fibers obtained from 2 independent experiments were measured for each condition. For comparison, the data showing that Polη is required for roscovitine-dependent rescue of fork elongation in Chk1-depleted cells (Fig 3B) is shown on the right. The lower panel shows the corresponding Western Blot of Chk1, Polη and CDC45. Actin was used as a loading control.


Percentage of origin firing ( mean + SD ) from DNA fibers in C ( n = 2 ) . > 490 fibers per condition were scored .

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Percentage of origin firing (mean+SD) from DNA fibers in C (n=2). >490 fibers per condition were scored.


Sensitivity of U2OS cells to Chk1 and CDC45 depletion . Cell number was determined 7 days after transfection ( 5 days after maximal downregulation ) . Data represent the mean ( + SD ) of 2 independent experiments . Representative image of data shown in ( E ) . Scale bar : 100 μm .

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Sensitivity of U2OS cells to Chk1 and CDC45 depletion. Cell number was determined 7 days after transfection (5 days after maximal downregulation). Data represent the mean (+SD) of 2 independent experiments. Representative image of data shown in (E). Scale bar: 100 μm.


Sensitivity of U2OS cells to Chk1 inhibition and roscovitine ( 5 μM ) . Cell number was determined 4 days after a 24 h treatment ( 5 days in total ) with Chk1i ± Rosc . Data represent the mean ( + SD ) of 2 independent experiments . Representative image of data shown in ( G ) . Scale bar : 100 μm .

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Sensitivity of U2OS cells to Chk1 inhibition and roscovitine (5 μM). Cell number was determined 4 days after a 24 h treatment (5 days in total) with Chk1i±Rosc. Data represent the mean (+SD) of 2 independent experiments. Representative image of data shown in (G). Scale bar: 100 μm.


HeLa and cyclin F Knock - Out ( CCNF K / O ) cells were treated with Kinase ChemoGenomic Set ( KSGS ) in 384 well format . After 72 Hours ( h ) viability was measured using rezasurin . Flowchart representation ( left ) . Robust Z - score difference is plotted for data acquired at 1 μM concentration ( right ) .

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HeLa and cyclin F Knock-Out (CCNF K/O) cells were treated with Kinase ChemoGenomic Set (KSGS) in 384 well format. After 72 Hours (h) viability was measured using rezasurin. Flowchart representation (left). Robust Z-score difference is plotted for data acquired at 1 μM concentration (right).


Cell survival measured using rezasurin and compared to controls treated with DMSO ( expressed as relative proliferation % ) . Cells were treated with the indicated inhibitors at 0 . 1 and 1 μM concentration .

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Cell survival measured using rezasurin and compared to controls treated with DMSO (expressed as relative proliferation %). Cells were treated with the indicated inhibitors at 0.1 and 1 μM concentration.


Cell survival measured using rezasurin and compared to controls ( 0 ) treated with DMSO ( expressed as relative proliferation % ) . Cells were treated with UCN - 01 at the indicated concentrations .

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Cell survival measured using rezasurin and compared to controls (0) treated with DMSO (expressed as relative proliferation %). Cells were treated with UCN-01 at the indicated concentrations.


Differences in viability of HeLa and CCNF K / O cells after treatment with specific Chk1i ( LY2603618 ) at the indicated concentrations plotted on a log10 scale to measure differences in IC50 .

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Differences in viability of HeLa and CCNF K/O cells after treatment with specific Chk1i (LY2603618) at the indicated concentrations plotted on a log10 scale to measure differences in IC50.


Number of cells was measured in HeLa and CCNF K / O cells left untreated or treated with Chk1i ( LY2603618 ) for 24h , 48h , or 72h as indicated . ( n . s . = non - significant ) .

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Number of cells was measured in HeLa and CCNF K/O cells left untreated or treated with Chk1i (LY2603618) for 24h, 48h, or 72h as indicated. (n.s. = non-significant).


Cell viability measurements using Propidium Iodide staining for HeLa and CCNF K / O cells after treatment with Chk1i ( LY2603618 ) .

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Cell viability measurements using Propidium Iodide staining for HeLa and CCNF K/O cells after treatment with Chk1i (LY2603618).


U - 2 - OS cells were seeded and transfected with non - targeting siRNA siNC ( Negative Control ) or siCyc F . 24 hours after transfection , cells were treated with UCN - 01 , or Chk1i ( LY2603618 ) at indicated concentrations for 3 days before viability was measured .

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U-2-OS cells were seeded and transfected with non-targeting siRNA siNC (Negative Control) or siCyc F. 24 hours after transfection, cells were treated with UCN-01, or Chk1i (LY2603618) at indicated concentrations for 3 days before viability was measured.


Cell viability measurements using Propidium Iodide staining in U - 2 - OS cells transfected with non - targeting siNC and siCyc F and treated with Chk1i ( LY2603618 ) as indicated .

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Cell viability measurements using Propidium Iodide staining in U-2-OS cells transfected with non-targeting siNC and siCyc F and treated with Chk1i (LY2603618) as indicated.


Cells treated with 1μM Chk1i ( LY2603618 ) for the indicated hours ( h ) were harvested and lysed using SDS . Indicated protein were resolved by SDS page and detected by Western blot ( WB ) . TFIIH was used as a loading control .

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Cells treated with 1μM Chk1i (LY2603618) for the indicated hours (h) were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by Western blot (WB). TFIIH was used as a loading control.


Percentage ( % ) of γ - H2AX positive cells in G1 cells after Chk1i ( LY2603618 ) treatment for the indicated hours ( h ) measured using Fluorescence Activated Cell Sorting ( FACS ) . G1 cells were considered having DAPI 2n staining and EdU negative . Percentage ( % ) of γ - H2AX positive cells in replicating ( EdU + ) cells after Chk1i ( LY2603618 ) treatment at the indicated time points . Percentage ( % ) of γ - H2AX positive cells in G2 / M cells after Chk1i ( LY2603618 ) treatment . G2 / M cells were considered having DAPI 4n staining and EdU negativity .

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Percentage (%) of γ-H2AX positive cells in G1 cells after Chk1i (LY2603618) treatment for the indicated hours (h) measured using Fluorescence Activated Cell Sorting (FACS). G1 cells were considered having DAPI 2n staining and EdU negative. Percentage (%) of γ-H2AX positive cells in replicating (EdU+) cells after Chk1i (LY2603618) treatment at the indicated time points. Percentage (%) of γ-H2AX positive cells in G2/M cells after Chk1i (LY2603618) treatment. G2/M cells were considered having DAPI 4n staining and EdU negativity.


Intensity of γ - H2AX measured as Relative Fluorescence Units ( RFU ) in early S phase cells treated with Chk1i . Mean intensity of γ - H2AX fluorescence was evaluated from EdU + cells with 2N DAPI content . Intensity of γ - H2AX measured as Relative Fluorescence Units ( RFU ) in replicating cells treated with Chk1i . Mean intensity of γ - H2AX fluorescence was evaluated from EdU + . Overall , cell cycle profile of HeLa and CCNF K / O cells after Chk1i treatment . Cells with 2n DAPI staining but EdU - were gated as G1 cells ; cells with > 2n but < 4n DAPI staining but EdU - were gated as EdU - S cells ; cells with > 2n but < 4n DAPI staining but EdU + were gated as EdU + S phase ; cells with 4n DAPI and pH3 S10 negative staining were gated as G2 phase and cells with 4n DAPI and pH3 S10 positive as M cells . Data from at least 3 independent replicates and plotted as Mean ± SD .

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Intensity of γ-H2AX measured as Relative Fluorescence Units (RFU) in early S phase cells treated with Chk1i. Mean intensity of γ-H2AX fluorescence was evaluated from EdU+ cells with 2N DAPI content. Intensity of γ-H2AX measured as Relative Fluorescence Units (RFU) in replicating cells treated with Chk1i. Mean intensity of γ-H2AX fluorescence was evaluated from EdU+. Overall, cell cycle profile of HeLa and CCNF K/O cells after Chk1i treatment. Cells with 2n DAPI staining but EdU- were gated as G1 cells; cells with >2n but <4n DAPI staining but EdU- were gated as EdU- S cells; cells with >2n but <4n DAPI staining but EdU+ were gated as EdU+ S phase; cells with 4n DAPI and pH3 S10 negative staining were gated as G2 phase and cells with 4n DAPI and pH3 S10 positive as M cells. Data from at least 3 independent replicates and plotted as Mean ± SD.


Representative plots of γ - H2AX vs RPA2 signal of HeLa and CCNF K / O cells after treatment with 1μM Chk1i ( LY2603618 ) at the indicated hours ( h ) . Blue labeled dots represent γ - H2AX positive cells . Relates to materials section for details on representation .

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Representative plots of γ-H2AX vs RPA2 signal of HeLa and CCNF K/O cells after treatment with 1μM Chk1i (LY2603618) at the indicated hours (h). Blue labeled dots represent γ-H2AX positive cells. Relates to materials section for details on representation.


Representative plots of γ - H2AX intensity vs DAPI signal of CCNF K / O cells transfected with empty GFP and GFP - CCNF untreated ( top panels ) and after treatment with 1μM Chk1i ( LY2603618 ) for 20h ( bottom panels ) . Normalised γ - H2AX intensity in CCNF K / O cells transfected with either GFP empty vector or GFP - Cyc F and treated with 1μM Chk1i for 20 hours . GFP positive cells were gated . Data from 3 independent experiments were plotted with Mean % ± SD .

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Representative plots of γ-H2AX intensity vs DAPI signal of CCNF K/O cells transfected with empty GFP and GFP-CCNF untreated (top panels) and after treatment with 1μM Chk1i (LY2603618) for 20h (bottom panels). Normalised γ-H2AX intensity in CCNF K/O cells transfected with either GFP empty vector or GFP-Cyc F and treated with 1μM Chk1i for 20 hours. GFP positive cells were gated. Data from 3 independent experiments were plotted with Mean % ± SD.


Representative images of neutral and alkaline comet assay from HeLa and CCNF K / O cells treated with Chk1i . Cells were treated with 1μM Chk1i for 24h before being harvested for neutral and alkaline comet assays as indicated . Quantification of comets in alkaline gels . At least 100 cells , across two slides , were analysed in each condition in two biological replicates . Data are shown as medians , with 25 / 75 % percentile range ( box ) and 10 - 90 % percentile range ( whiskers ) . Fold changes of median values are shown in bold . p - values were calculated using the Mann - Whitney test ( two - tailed ) . Olive tail moment = ( Tail . mean - Head . mean ) * Tail % DNA / 100 . Quantification of comets in neutral gels . At least 100 cells , across two slides , were analysed in each condition in two biological replicates . Data are shown as medians , with 25 / 75 % percentile range ( box ) and 10 - 90 % percentile range ( whiskers ) . Fold changes of median values are shown in bold . p - values were calculated using the Mann - Whitney test ( two - tailed ) . Olive tail moment = ( Tail . mean - Head . mean ) * Tail % DNA / 100 .

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

Representative images of neutral and alkaline comet assay from HeLa and CCNF K/O cells treated with Chk1i. Cells were treated with 1μM Chk1i for 24h before being harvested for neutral and alkaline comet assays as indicated. Quantification of comets in alkaline gels. At least 100 cells, across two slides, were analysed in each condition in two biological replicates. Data are shown as medians, with 25/75% percentile range (box) and 10-90% percentile range (whiskers). Fold changes of median values are shown in bold. p-values were calculated using the Mann-Whitney test (two-tailed). Olive tail moment = (Tail.mean - Head.mean)*Tail%DNA/100. Quantification of comets in neutral gels. At least 100 cells, across two slides, were analysed in each condition in two biological replicates. Data are shown as medians, with 25/75% percentile range (box) and 10-90% percentile range (whiskers). Fold changes of median values are shown in bold. p-values were calculated using the Mann-Whitney test (two-tailed). Olive tail moment = (Tail.mean - Head.mean)*Tail%DNA/100.


Percentage ( % ) of γ - H2AX positive cells in HeLa and CCNF K / O after transfection of the indicated siRNAs treated with Chk1i for 20 hours . Percent of γ - H2Ax positive cells were plotted as Mean % ± SD . Percentage ( % ) of hyper - RPA positive cells in HeLa and CCNF K / O after transfection of the indicated siRNAs treated with Chk1i for 20 hours . Percent of hyper RPA positive cells were plotted as Mean % ± SD .

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Percentage (%) of γ-H2AX positive cells in HeLa and CCNF K/O after transfection of the indicated siRNAs treated with Chk1i for 20 hours. Percent of γ-H2Ax positive cells were plotted as Mean % ± SD. Percentage (%) of hyper-RPA positive cells in HeLa and CCNF K/O after transfection of the indicated siRNAs treated with Chk1i for 20 hours. Percent of hyper RPA positive cells were plotted as Mean % ± SD.


Percentage ( % ) of γ - H2AX positive cells in S phase cells by DAPI content after Chk1i ( LY2603618 )

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Percentage (%) of γ-H2AX positive cells in S phase cells by DAPI content after Chk1i (LY2603618)


Relative γ - H2AX intensity in cells transfected with either Empty Vector ( EV ) or HA - E2F1 treated with Chk1i for 20 hours . γ - H2AX fluorescence in HA positive cells was normalized to the empty vector transfected cells and compared to cells not expressing HA .

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Relative γ-H2AX intensity in cells transfected with either Empty Vector (EV) or HA-E2F1 treated with Chk1i for 20 hours. γ-H2AX fluorescence in HA positive cells was normalized to the empty vector transfected cells and compared to cells not expressing HA.


HeLa and CCNF K / O cells transfected with the indicated siRNA and treated with 1μM Chk1i ( LY2603618 ) for 20 hours were harvested and lysed using SDS . Indicated protein were resolved by SDS page and detected by WB . TFIIH served as a loading control .

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HeLa and CCNF K/O cells transfected with the indicated siRNA and treated with 1μM Chk1i (LY2603618) for 20 hours were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by WB. TFIIH served as a loading control.


HeLa and CCNF K / O cells were harvested and lysed using SDS . Indicated protein were resolved by SDS page and detected by WB . Ponceau S staining was used as a loading control .

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HeLa and CCNF K/O cells were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by WB. Ponceau S staining was used as a loading control.


U - 2 - OS cells transfected with non - targeting siRNA ( siNC ) or with an siRNA targeting Cyc F were lysed using SDS . Whole cell lysates were analyzed by immunoblotting 48h after transfection . H3 was used as a loading control .

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U-2-OS cells transfected with non-targeting siRNA (siNC) or with an siRNA targeting Cyc F were lysed using SDS. Whole cell lysates were analyzed by immunoblotting 48h after transfection. H3 was used as a loading control.


Heatmap showing gene set variance analysis ( GSVA ) score for gene sets related to E2F targets ( PID _ E2F _ PATHWAY , ISHIDA _ E2F _ TARGETS and HALLMARK _ E2F _ TARGETS ) .

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Heatmap showing gene set variance analysis (GSVA) score for gene sets related to E2F targets (PID_E2F_PATHWAY, ISHIDA_E2F_TARGETS and HALLMARK_E2F_TARGETS).


HEK293T cells transfected with empty vector ( EV ) , FLAG - cyclin F WT , FLAG - cyclin F ( ∆ F ) and FLAG - cyclin F M309A were treated with MLN4924 for 4 hours and harvested for immunoprecipitation using FLAG antibodies . Immunoprecipitates were immunoblotted as indicated .

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HEK293T cells transfected with empty vector (EV), FLAG-cyclin F WT, FLAG-cyclin F (∆F) and FLAG-cyclin F M309A were treated with MLN4924 for 4 hours and harvested for immunoprecipitation using FLAG antibodies. Immunoprecipitates were immunoblotted as indicated.


HEK293T cells were cotransfected with MYC - tagged ubiquitin and HA - E2F1 in the presence of FLAG - cyclin F WT , FLAG - cyclin F ( ∆ F ) and FLAG - cyclin F M309A were indicated ( + ) . HA - E2F1 was immunoprecipitated with an anti - HA resin under denaturing conditions and immunoblotted .

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HEK293T cells were cotransfected with MYC-tagged ubiquitin and HA-E2F1 in the presence of FLAG-cyclin F WT, FLAG-cyclin F (∆F) and FLAG-cyclin F M309A were indicated (+). HA-E2F1 was immunoprecipitated with an anti-HA resin under denaturing conditions and immunoblotted.


HeLa and CCNF K / O cells treated with 10 μM Chk1i ( LY2603618 ) for the indicated time points ( h = hours ) were harvested and lysed using SDS . Indicated protein were resolved by SDS page and detected by WB . H2B served as a loading control .

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HeLa and CCNF K/O cells treated with 10 μM Chk1i (LY2603618) for the indicated time points (h=hours) were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by WB. H2B served as a loading control.


HEK293 cells transfected with empty vector ( EV ) or HA - E2F1 were treated with Chk1i for 20 hours and harvested for immunoprecipitation using HA antibodies . Immunoprecipitates were immunoblotted as indicated .

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HEK293 cells transfected with empty vector (EV) or HA-E2F1 were treated with Chk1i for 20 hours and harvested for immunoprecipitation using HA antibodies. Immunoprecipitates were immunoblotted as indicated.


Quantification of immunoprecipitation results of Figure 5G from three independent experiments . Data from 3 independent experiments were plotted with Mean % ± SD . * < 0 . 05 p - value and calculated by two - tailed paired t - test .

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Quantification of immunoprecipitation results of Figure 5G from three independent experiments. Data from 3 independent experiments were plotted with Mean % ± SD. *< 0.05 p-value and calculated by two-tailed paired t-test.


HEK293T cells were cotransfected with MYC - tagged ubiquitin and HA - E2F1 were treated with Chk1i for 20 hours and siRNA of cyclin F were indicated ( + ) . HA - E2F1 was immunoprecipitated with an anti - HA resin under denaturing conditions and immunoblotted .

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HEK293T cells were cotransfected with MYC-tagged ubiquitin and HA-E2F1 were treated with Chk1i for 20 hours and siRNA of cyclin F were indicated (+). HA-E2F1 was immunoprecipitated with an anti-HA resin under denaturing conditions and immunoblotted.


HEK293T cells transfected with HA - E2F1 Wild Type ( WT ) and HA - E2F1 lacking a CY motif ( ∆ RxL ) harvested for immunoprecipitation using HA antibodies . Immunoprecipitates were immunoblotted as indicated .

HEK293T cells transfected with HA-E2F1 Wild Type (WT) and HA-E2F1 lacking a CY motif (∆RxL) harvested for immunoprecipitation using HA antibodies. Immunoprecipitates were immunoblotted as indicated.


HEK293T cells were cotransfected with MYC - tagged ubiquitin and HA - E2F1 or HA - E2F1 ∆ RxL lacking the CY motif as indicated ( + ) . HA - E2F1 was immunoprecipitated with an anti - HA resin under denaturing conditions and immunoblotted .

HEK293T cells were cotransfected with MYC-tagged ubiquitin and HA-E2F1 or HA-E2F1 ∆RxL lacking the CY motif as indicated (+). HA-E2F1 was immunoprecipitated with an anti-HA resin under denaturing conditions and immunoblotted.


HeLa cells stably expressing HA - E2F1 WT and HA - E2F1 ∆ RxL under the control of a retroviral promoter were treated with Cycloheximide ( CHX ) at 50 μg / ml for the indicated minutes ( min ) Quantification of E2F1 half -

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HeLa cells stably expressing HA-E2F1 WT and HA-E2F1 ∆RxL under the control of a retroviral promoter were treated with Cycloheximide (CHX) at 50 μg/ml for the indicated minutes (min) Quantification of E2F1 half-life


HeLa cells stably expressing an Empty Vector ( EV ) , HA - E2F1 WT and HA - E2F1 ∆ RxL under the control of a retroviral promoter were treated with Chk1i for the indicated hours ( h ) . Indicated protein were resolved by SDS page and detected by WB . H3 was used as a loading control .

HeLa cells stably expressing an Empty Vector (EV), HA-E2F1 WT and HA-E2F1 ∆RxL under the control of a retroviral promoter were treated with Chk1i for the indicated hours (h). Indicated protein were resolved by SDS page and detected by WB. H3 was used as a loading control.


Relative survival ( % ) of HeLa cells stably expressing an Empty Vector ( EV ) , HA - E2F1 WT and HA - E2F1 ∆ RxL under the control of a retroviral promoter measured using rezasurin and compared to EV treated with Chk1i at the indicated concentrations .

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Relative survival (%) of HeLa cells stably expressing an Empty Vector (EV), HA-E2F1 WT and HA-E2F1 ∆RxL under the control of a retroviral promoter measured using rezasurin and compared to EV treated with Chk1i at the indicated concentrations.


A . - E . Staining for β - galactosidase in 3 - months old VEGF - Ahyper ( lacZ / wt ) mouseeyes . A . Low magnification image shows β - gal + cells ( in blue ) in the RPE ( white arrow ) , retina ( blue arrow ) , ciliary body ( black arrow ) , and lens ( orange arrow ) . Images of Figures 1B to 1E are higher magnification images of Figure 1A . Scale bar 200 μm . B . Strong expression of VEGF with β - gal + cells are observed in the RPE ( short white arrow ) and the inner nuclear layer ( INL ) ( long white arrow ) , while less staining is observed in the ganglion cell layer ( black arrow ) of the retina . Scale bar 100 μm . C . Ciliary body epithelial cells are β - gal + , similarly as RPE cells ( white arrow ) . Scale bar 100 μm . D . VEGF - A is strongly expressed in secondary nuclearfiber cells in the adult lens ( white arrow ) . Scale bar 50 μm . E . No β - gal + cells are observed in the corneas of adult VEGF - Ahyper ( lacZ / wt ) mouseeyes . White arrow shows the stroma of the cornea and the black arrow shows the corneal epithelium . Scale bar 100 μm .

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A.-E. Staining for β-galactosidase in 3-months old VEGF-Ahyper(lacZ/wt)mouseeyes.A. Low magnification image shows β-gal+ cells (in blue) in the RPE (white arrow), retina (blue arrow), ciliary body (black arrow), and lens (orange arrow). Images of Figures 1B to 1E are higher magnification images of Figure 1A. Scale bar 200 μm.B. Strong expression of VEGF with β-gal+ cells are observed in the RPE (short white arrow) and the inner nuclear layer (INL) (long white arrow), while less staining is observed in the ganglion cell layer (black arrow) of the retina. Scale bar 100 μm.C. Ciliary body epithelial cells are β-gal+, similarly as RPE cells (white arrow). Scale bar 100 μm.D. VEGF-A is strongly expressed in secondary nuclearfiber cells in the adult lens (white arrow). Scale bar 50 μm.E. No β-gal+ cells are observed in the corneas of adult VEGF-Ahyper(lacZ/wt)mouseeyes. White arrow shows the stroma of the cornea and the black arrow shows the corneal epithelium. Scale bar 100 μm.


G . RPE cells maintain strong expression of VEGF - A ( β - gal + cells [ white arrow ] ) in the retina with progressive age . Immunofluorescence for β - gal in the retina of a 24 - months old VEGF - Ahypermouse is shown . Ocular macrophages ( F4 / 80 + cells [ yellow arrow ] ) show no strong β - galactosidase expression , while some immunolabeling for β - galactosidase is detected in retinal cells of the inner nuclear layer ( orange arrow ) of the retina . RPE : retinal pigment epithelium ; ONL : outer nuclear layer ; INL : inner nuclear layer . Scale bar 50 μm .

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G. RPE cells maintain strong expression of VEGF-A (β-gal+ cells [white arrow]) in the retina with progressive age. Immunofluorescence for β-gal in the retina of a 24-months old VEGF-Ahypermouse is shown. Ocular macrophages (F4/80+ cells [yellow arrow]) show no strong β-galactosidase expression, while some immunolabeling for β-galactosidase is detected in retinal cells of the inner nuclear layer (orange arrow) of the retina. RPE: retinal pigment epithelium; ONL: outer nuclear layer; INL: inner nuclear layer. Scale bar 50 μm.


H . VEGF - A continues to be highly expressed in secondary nuclear fiber cells in the aged lens ( shown is a lens of a 24 - months old VEGF - Ahyper mouse ; nuclear β - galactosidasestaining ( arrow ) reflects cellular VEGF - A expression ) . Cor : lenscortex ; Cap : lens capsule . DAPI labels nuclei ( blue ) . Scale bar 25 μm .

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H. VEGF-A continues to be highly expressed in secondary nuclear fiber cells in the aged lens (shown is a lens of a 24-months old VEGF-Ahyper mouse; nuclear β-galactosidasestaining (arrow) reflects cellular VEGF-A expression). Cor: lenscortex; Cap: lens capsule. DAPI labels nuclei (blue). Scale bar 25 μm.


A . Left graph : VEGF - Aprotein levels in lenses of VEGF - Ahypermice ( KI ) are increased ~ 3 - fold throughout life ( measured at 2 - and at 8 . 5 - months of age ; n = 7 mice / group , 2 independent experiments ) . Graph shows mean ± SD . Right graph : VEGF - AmRNA levels are similarly increased in lenses of adult VEGF - Ahypermice ( 9 - months old ; n = 7 mice / group , 3 independent experiments ) . Graph shows mean ± SEM . * * P - value : 0 . 0095 .

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A. Left graph: VEGF-Aprotein levels in lenses of VEGF-Ahypermice (KI) are increased ~3-fold throughout life (measured at 2- and at 8.5-months of age; n=7 mice/group, 2 independent experiments). Graph shows mean ± SD. Right graph: VEGF-AmRNA levels are similarly increased in lenses of adult VEGF-Ahypermice (9-months old; n=7 mice/group, 3 independent experiments). Graph shows mean ± SEM. **P-value: 0.0095.


B . VEGF - A isoforms ( mainly VEGF - A120 and VEGF - A164 ) and the VEGF - A receptor Flk1 are expressed in the adult lens . Flt1 expression was not detected in the lens . N = 3 mice / group .

B. VEGF-A isoforms (mainly VEGF-A120 and VEGF-A164) and the VEGF-A receptor Flk1 are expressed in the adult lens. Flt1 expression was not detected in the lens. N=3 mice/group.


C . Increased serum levels of the lipid peroxidation marker 4 - HNE ( a marker of increased oxidative damage ) in aged , but not in young VEGF - Ahyper mice . * P - value : 0 . 0325 . N = 7 mice / group ( 2 independent experiments ) . Graph shows mean ± SEM .

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C. Increased serum levels of the lipid peroxidation marker 4-HNE (a marker of increased oxidative damage) in aged, but not in young VEGF-Ahyper mice. *P-value: 0.0325. N=7 mice/group (2 independent experiments). Graph shows mean ± SEM.


A . Increased ERK1 / 2 phosphorylation is observed in lenses from VEGF - Ahyper mice ( KI ) with increased VEGF - A levels already prior to cataract formation ( 11 - months old mice with no cataracts ( 0 cataract ) ) , which is maintained in lenses with mature cataracts ( lens of a 26 - months old VEGF - Ahyper mouse with + 3 cataract ) . Quantification of ERK1 / 2 phosphorylation in 11 - months old lenses ( n = 3 / group ) . Graph shows mean ± SEM . * * * P - value : 0 . 0005 .

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A. Increased ERK1/2 phosphorylation is observed in lenses from VEGF-Ahyper mice (KI) with increased VEGF-A levels already prior to cataract formation (11-months old mice with no cataracts (0 cataract)), which is maintained in lenses with mature cataracts (lens of a 26-months old VEGF-Ahyper mouse with +3 cataract). Quantification of ERK1/2 phosphorylation in 11-months old lenses (n=3/group). Graph shows mean ± SEM. ***P-value: 0.0005.


B . - C . Cataracts with opacification of the lens ( B . , black arrow ) and extruded nuclei ( C . , white arrow ) are observed with progressive age in VEGF - Ahyper mice ( here a representative 30 - months old lens with a cataract is shown ) .

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B.-C. Cataracts with opacification of the lens (B., black arrow) and extruded nuclei (C., white arrow) are observed with progressive age in VEGF-Ahyper mice (here a representative 30-months old lens with a cataract is shown).


D . - E . Histology of lens from b . - c . shows extrusion of sclerotic nucleus ( white arrows in C . and D . ) and massive degenerative changes of the lens cortex with vacuolization ( black arrows in D . and E . ) . Scale bar 500 μm in ( D . ) and 100 μm in ( E . ) .

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D.-E. Histology of lens from b.-c. shows extrusion of sclerotic nucleus (white arrows in C. and D.) and massive degenerative changes of the lens cortex with vacuolization (black arrows in D. and E.). Scale bar 500 μm in (D.) and 100 μm in (E.).


F . - G . Representative histological images of 21 - months old eyes from WT and VEGF - Ahyper mice . The sclerotic nucleus ( red arrow in G . ) was extruded and the lenscortex and capsule are separated from the nucleus ( white arrow in G . ) . The retina in aged VEGF - Ahyper mice is atrophic and shows degenerative changes ( yellow arrow in G . ) , while the retina ( * ) and lens ( * * ) appears normal in age - matched WT littermate mice . Scale bars 200 μm . DAPI ( blue , nuclei ) ; phalloidin ( green ) .

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F.-G. Representative histological images of 21-months old eyes from WT and VEGF-Ahyper mice. The sclerotic nucleus (red arrow in G.) was extruded and the lenscortex and capsule are separated from the nucleus (white arrow in G.). The retina in aged VEGF-Ahyper mice is atrophic and shows degenerative changes (yellow arrow in G.), while the retina (*) and lens (**) appears normal in age-matched WT littermate mice. Scale bars 200 μm. DAPI (blue, nuclei); phalloidin (green).


A . lens progress with age in VEGF - Ahypermice . lens were graded from + 1 ( mild ) , + 2 ( moderate ) , to + 3 ( mature cataract with fully opacified lens ) . While only a small subset of > 18 months - old WT mice have mature ( + 3 ) cataracts , the majority of VEGF - Ahypermice of this age group has mature cataracts . Percentile ( % ) of mice with graded cataracts is indicated in each age group . 0 = no cataract . Absolute mouse numbers of each group are shown in Appendix Figure S1 . B . Targeting NLRP3 inhibits normal age - dependent cataract formation , and delays ( but does not prevent ) VEGF - A - induced cataract formation in VEGF - Ahypermice . Percentile ( % ) of mice with graded cataracts is indicated in each age group . 0 = no cataract . Absolute mouse numbers of each group are shown in Appendix Figure S1 .

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A. lens progress with age in VEGF-Ahypermice. lens were graded from +1 (mild), +2 (moderate), to +3 (mature cataract with fully opacified lens). While only a small subset of >18 months-old WT mice have mature (+3) cataracts, the majority of VEGF-Ahypermice of this age group has mature cataracts. Percentile (%) of mice with graded cataracts is indicated in each age group. 0 = no cataract. Absolute mouse numbers of each group are shown in Appendix Figure S1.B. Targeting NLRP3 inhibits normal age-dependent cataract formation, and delays (but does not prevent) VEGF-A-induced cataract formation in VEGF-Ahypermice. Percentile (%) of mice with graded cataracts is indicated in each age group. 0 = no cataract. Absolute mouse numbers of each group are shown in Appendix Figure S1.


C . Expression of IL - 1β in lenses of 21 - months old VEGF - Ahyper mice with + 3 cataracts is increased compared to lenses from age - matched WT mice with + 3 cataracts , but not in lenses from 11 - months old mouse groups with no cataracts . * P - value : 0 . 0259 . N = 7 / group ( 3 independent experiments ) .

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C. Expression of IL-1β in lenses of 21-months old VEGF-Ahyper mice with +3 cataracts is increased compared to lenses from age-matched WT mice with +3 cataracts, but not in lenses from 11-months old mouse groups with no cataracts. *P-value: 0.0259. N=7/group (3 independent experiments).


D . VEGF - Ahyper mice have decreased levels of the major antioxidant in the lens , reduced glutathione ( μmol / gm lens weight ) , prior to cataract formation . Lenses with 0 to + 1 cataracts from 11 - months old mice were used for measurements . * * P - value : 0 . 0097 . N = 7 / group ( 2 independent experiments ) .

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D. VEGF-Ahyper mice have decreased levels of the major antioxidant in the lens, reduced glutathione (μmol/gm lens weight), prior to cataract formation. Lenses with 0 to +1 cataracts from 11-months old mice were used for measurements. ** P-value: 0.0097. N=7/group (2 independent experiments).


E - F . Overexpression of NADPH oxidase gp91phox [ * p - value : 0 . 0435 ] ( E . ) and of the antioxidant enzymes SOD1 [ * p - value : 0 . 0330 ] , catalase ( CAT ) [ * p - value : 0 . 0448 ] and GPx - 1 [ * p - value : 0 . 0443 ] ( F . ) in 21 - months old lenses of VEGF - Ahyper mice with cataracts ( + 3 ) , but not in 11 - months old lenses with no cataracts . N = 7 / group ( 3 independent experiments ) .

E-F. Overexpression of NADPH oxidase gp91phox [*p-value: 0.0435] (E.) and of the antioxidant enzymes SOD1 [*p-value: 0.0330], catalase (CAT) [*p-value: 0.0448] and GPx-1 [*p-value: 0.0443] (F.) in 21-months old lenses of VEGF-Ahyper mice with cataracts (+3), but not in 11-months old lenses with no cataracts. N=7/group (3 independent experiments).


A - B . Choroidalflat mount staining of a white VEGF - Ahypermouse shows choroidalvessels ( green [ fluorescein - conjugated isolectin B4 ] ) from which neovessels ( strong red [ CD31 ] ) originate and that are covered by a RPEcell layer ( white [ phalloidin ] ) , which separates the CNV lesion from the photoreceptors . White arrows demarcate CNV lesion . Yellow arrows show round autofluorescent deposits . Scale bars 100 μm .

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A-B. Choroidalflat mount staining of a white VEGF-Ahypermouse shows choroidalvessels (green [fluorescein-conjugated isolectin B4]) from which neovessels (strong red [CD31]) originate and that are covered by a RPEcell layer (white [phalloidin]), which separates the CNV lesion from the photoreceptors. White arrows demarcate CNV lesion. Yellow arrows show round autofluorescent deposits. Scale bars 100 μm.


A - B . Choroidalflat mount staining of a white VEGF - A hypermouse shows choroidal vessels ( green [ fluorescein - conjugated isolectin B4 ] ) from which neovessels ( strong red [ CD31 ] ) originate and that are covered by a RPEcell layer ( white [ phalloidin ] ) , which separates the CNV lesion from the photoreceptors . White arrows demarcate CNV lesion . Yellow arrows show round autofluorescent deposits . Scale bars 100 μm .

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A-B. Choroidalflat mount staining of a white VEGF-A hypermouse shows choroidal vessels (green [fluorescein-conjugated isolectin B4]) from which neovessels (strong red [CD31]) originate and that are covered by a RPEcell layer (white [phalloidin]), which separates the CNV lesion from the photoreceptors. White arrows demarcate CNV lesion. Yellow arrows show round autofluorescent deposits. Scale bars 100 μm.


C . Section through a CNV lesion in a VEGF - Ahyper mouse . Bruch ' s membrane location is indictaed by yellow arrows . Choroidal neovessels ( white arrows ) are covered by a layer of RPE cells ( black arrows ) . Chor : choroid ; ONL : outer nuclear layer ; PR : photoreceptors . Scale bar 20 μm . A . - C . 21 - months old VEGF - Ahyper mice .

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C. Section through a CNV lesion in a VEGF-Ahyper mouse. Bruch's membrane location is indictaed by yellow arrows. Choroidal neovessels (white arrows) are covered by a layer of RPE cells (black arrows). Chor: choroid; ONL: outer nuclear layer; PR: photoreceptors. Scale bar 20 μm. A.-C. 21-months old VEGF-Ahyper mice.


RPE - specific overexpression of VEGF - A in VMD2Cre + / WTROSA - STOPfl / fl - VEGF - A164mice ( VEGF - ARPEmice ) results in CNV at sites of VEGF - A overexpression ( Cre + patches ( white nuclei ) ; red arrow ) , while Cre - areas with normal VEGF - A expression show no CNV ( white arrow ) . Neovessels are CD31 + ( red ) . VEGF - A - induced CNV lesions and RPE barrier breakdown in VMD2Cre + / WT ROSA - STOPfl / fl - VEGF - A164mice resemble those seen in VEGF - Ahypermice ( B . - C . , arrows ; phalloidin staining in green ) . Scale bars 100 μm .

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RPE-specific overexpression of VEGF-A in VMD2Cre+/WTROSA-STOPfl/fl-VEGF-A164mice (VEGF-ARPEmice) results in CNV at sites of VEGF-A overexpression (Cre+ patches (white nuclei); red arrow), while Cre- areas with normal VEGF-A expression show no CNV (white arrow). Neovessels are CD31+ (red). VEGF-A-induced CNV lesions and RPE barrier breakdown in VMD2Cre+/WT ROSA-STOPfl/fl-VEGF-A164mice resemble those seen in VEGF-Ahypermice (B.-C., arrows; phalloidin staining in green). Scale bars 100 μm.


D . Genetic inactivation of Flk1 specifically in the RPE in VEGF - Ahypermice ( VMD2Cre + / WTFlk1fl / flVEGF - Ahypermice ) prevents RPE barrier breakdown and CNV lesion formation . Choroidalflat mounts of 5 months old VEGF - Ahypermice and VMD2Cre + / WTFlk1fl / flVEGF - Ahypermice are shown . β - gal shows uniform VEGF - A expression in these mice ( green ) . Crestaining ( red ) shows co - localization of β - gal and Crestaining in the nuclei of RPE cells in VMD2Cre + / WTFlk1fl / flVEGF - Ahypermice ( red areas of staining occur in CNV lesions of VEGF - Ahypermice due to labeling of anti - mouseIgG and not Cre , as the anti - mouseIgG secondary antibodies were used to detect the mouse anti - Cre antibody ) . Phalloidinstaining ( white ) reveals extensive RPE barrier breakdown and CNV lesions ( arrow ) in VEGF - Ahypermice , which is not observed in VMD2Cre + / WTFlk1fl / flVEGF - Ahypermice . Scale bar 100 μm .

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D. Genetic inactivation of Flk1 specifically in the RPE in VEGF-Ahypermice (VMD2Cre+/WTFlk1fl/flVEGF-Ahypermice) prevents RPE barrier breakdown and CNV lesion formation. Choroidalflat mounts of 5 months old VEGF-Ahypermice and VMD2Cre+/WTFlk1fl/flVEGF-Ahypermice are shown. β-gal shows uniform VEGF-A expression in these mice (green). Crestaining (red) shows co-localization of β-gal and Crestaining in the nuclei of RPE cells in VMD2Cre+/WTFlk1fl/flVEGF-Ahypermice (red areas of staining occur in CNV lesions of VEGF-Ahypermice due to labeling of anti-mouseIgG and not Cre, as the anti-mouseIgG secondary antibodies were used to detect the mouse anti-Cre antibody). Phalloidinstaining (white) reveals extensive RPE barrier breakdown and CNV lesions (arrow) in VEGF-Ahypermice, which is not observed in VMD2Cre+/WTFlk1fl/flVEGF-Ahypermice. Scale bar 100 μm.


A . NLRP3 is expressed in RPE cells at sites of CNV lesions ( arrow ; white ) . B . Lack of NLRP3 staining in VEGF - Ahyper / NLRP3 - / - mice ( control ) . Scale bars 100 μm .

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A. NLRP3 is expressed in RPE cells at sites of CNV lesions (arrow; white).B. Lack of NLRP3 staining in VEGF-Ahyper/NLRP3-/- mice (control). Scale bars 100 μm.


C . Expression of NLRP3 [ * p - value : 0 . 0478 ] and IL - 1β [ * p - value : 0 . 0398 ] , but not of IL - 18 [ p - value : 0 . 3529 ] is increased in RPE / choroid lysates from VEGF - Ahyper mice when compared to matched control littermate mice . N = 7 mice / group ( 3 independent experiments ) . Graphs show mean ± SEM .

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C. Expression of NLRP3 [*p-value: 0.0478] and IL-1β [*p-value: 0.0398], but not of IL-18 [p-value: 0.3529] is increased in RPE/choroid lysates from VEGF-Ahyper mice when compared to matched control littermate mice. N=7 mice/group (3 independent experiments). Graphs show mean ± SEM.


D . - G . Complement pathway activation ( immunolabeling for the product of complement pathway activation C5b - 9 , an inducer of the NLRP3 inflammasome ) is observed in CNV lesions of VEGF - Ahypermice ( white arrow in E . ) but not in age - matched control mice ( D . ) Co - localization of C5b - 9 in the same CNV lesion is observed with complement C1qA ( the initiator of the classical complement pathway and an inducer of the NLRP3 inflammasome ) ( F . ) , and with TLR2 ( G . ) ( white arrows ) . Autofluorescence in red in D . and E . CD31 + vessels in red in I . and J . Yellow arrows show site of choroid . Scale bars 50 μm .

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D.-G. Complement pathway activation (immunolabeling for the product of complement pathway activation C5b-9, an inducer of the NLRP3 inflammasome) is observed in CNV lesions of VEGF-Ahypermice (white arrow in E.) but not in age-matched control mice (D.) Co-localization of C5b-9 in the same CNV lesion is observed with complement C1qA (the initiator of the classical complement pathway and an inducer of the NLRP3 inflammasome) (F.), and with TLR2 (G.) (white arrows). Autofluorescence in red in D. and E. CD31+vessels in red in I. and J. Yellow arrows show site of choroid. Scale bars 50 μm.


A . CNV lesion numbers per eye in 6 - week old VEGF - Ahypermice compared with VEGF - Ahypermice lacking CASP1 / CASP11 , NLRP3 , IL1R1 , IL - 18 , TLR2 , or those heterozygous for beclin - 1 or homozygous for hypomorphic ATG16L1 . CASP1 / CASP11 , NLRP3 or IL - 1R1 deficiency results in significantly fewer CNV lesions . Each value represents the total CNV lesion number / eye for an individual mouse . Mean ± SEM is shown . P - values are based on comparison with the VEGF - Ahypermouse group . Absolute numbers of mice per group are indicated in ( ) . P - values were calculated with a two - tailed unpaired Student ' s t - test . Separately , p - values are shown between VEGF - Ahyper / CASP1 - / - / CASP11 - / - and VEGF - Ahyper / NLRP3 - / - or VEGF - Ahyper / IL1R1 - / - mouse groups . WT mice developed no CNV lesions .

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A. CNV lesion numbers per eye in 6-week old VEGF-Ahypermice compared with VEGF-Ahypermice lacking CASP1/CASP11, NLRP3, IL1R1, IL-18, TLR2, or those heterozygous for beclin-1 or homozygous for hypomorphic ATG16L1. CASP1/CASP11, NLRP3 or IL-1R1 deficiency results in significantly fewer CNV lesions. Each value represents the total CNV lesion number/eye for an individual mouse. Mean ± SEM is shown. P-values are based on comparison with the VEGF-Ahypermouse group. Absolute numbers of mice per group are indicated in ( ). P-values were calculated with a two-tailed unpaired Student's t-test. Separately, p-values are shown between VEGF-Ahyper/CASP1-/-/CASP11-/- and VEGF-Ahyper/NLRP3-/- or VEGF-Ahyper/IL1R1-/-mouse groups. WT mice developed no CNV lesions.


B . Average CNV lesion area ( in μm2 / lesion ) in 6 - week old VEGF - Ahypermice compared with VEGF - Ahypermice lacking CASP1 / CASP11 , NLRP3 , IL1R1 , IL - 18 , TLR2 , or those heterozygous for beclin - 1 or homozygous for hypomorphic ATG16L1 . Lack of TLR2 or CASP1 / CASP11 significantly reduces CNV lesion size , while reduced autophagy moderately increases CNV size . Each value represents the average CNV lesion size / eye for an individual mouse . Mean ± SEM is shown . P - values are based on comparison with the VEGF - Ahypermouse group . Absolute numbers of mice per group are indicated in ( ) . P - values were calculated with a two - tailed unpaired Student ' s t - test . Separately , p - values are shown between VEGF - Ahyper / CASP1 - / - / CASP11 - / - and VEGF - Ahyper / NLRP3 - / - or VEGF - Ahyper / IL1R1 - / - mouse groups .

B. Average CNV lesion area (in μm2/lesion) in 6-week old VEGF-Ahypermice compared with VEGF-Ahypermice lacking CASP1/CASP11, NLRP3, IL1R1, IL-18, TLR2, or those heterozygous for beclin-1 or homozygous for hypomorphic ATG16L1. Lack of TLR2 or CASP1/CASP11 significantly reduces CNV lesion size, while reduced autophagy moderately increases CNV size. Each value represents the average CNV lesion size/eye for an individual mouse. Mean ± SEM is shown. P-values are based on comparison with the VEGF-Ahypermouse group. Absolute numbers of mice per group are indicated in ( ). P-values were calculated with a two-tailed unpaired Student's t-test. Separately, p-values are shown between VEGF-Ahyper/CASP1-/-/CASP11-/- and VEGF-Ahyper/NLRP3-/- or VEGF-Ahyper/IL1R1-/-mouse groups.


C . - E . Representative choroidal flat mount images show multifocal CNV lesions in VEGF - Ahyper mice and a significant reduction in CNV numbers in those VEGF - Ahyper mice that lack either CASP1 / CASP11 or IL1R1 ( and therefore IL - 1β - signaling ) . Staining for phalloidin in green and for CD31 in red . Scale bars 200 μm .

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C.-E. Representative choroidal flat mount images show multifocal CNV lesions in VEGF-Ahyper mice and a significant reduction in CNV numbers in those VEGF-Ahyper mice that lack either CASP1/CASP11 or IL1R1 (and therefore IL-1β-signaling). Staining for phalloidin in green and for CD31 in red. Scale bars 200 μm.


. Laser - induced CNV experiments demonstrate that treatment with a caspase - 1 inhibitor ( compared to DMSO treated controls ) or a neutralizing TLR2 antibody ( compared to isotype matched IgG treated controls ) inhibits CNV lesion growth . P - values are shown . N = 15 mice / group . Representative CNV lesions in choroidal flat mounts are shown . Scale bars 100 μm .

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. Laser-induced CNV experiments demonstrate that treatment with a caspase-1 inhibitor (compared to DMSO treated controls) or a neutralizing TLR2 antibody (compared to isotype matched IgG treated controls) inhibits CNV lesion growth. P-values are shown. N=15 mice/group. Representative CNV lesions in choroidal flat mounts are shown. Scale bars 100 μm.


Figure 9 : VEGF - A - induced non - exudative AMD - like pathologies can be prevented by targeting NLRP3 , IL1R1 or caspase - 1 / caspase - 11 . A . No significant abnormalities of the choroid , RPE or retina are seen in control wild - type littermate mice . Representative image of a 28 - months old control eye is shown . White arrow indicates normal Bruch ' s membrane . Chor : choroid ; RPE ; PR : photoreceptor ; ONL : outer nuclear layer . B . Aged VEGF - Ahypermice develop a progressive age - dependent degeneration of the RPE ( red arrow ) and the photoreceptors ( yellow arrow ) with accumulation of thick sub - RPE basal laminar deposits ( black arrow ) . These non - exudative AMD - like pathologies occur also at sites devoid of CNV lesions . C . - E . Genetic inactivation of CASP1 / CASP11 , NLRP3 , or IL1R1 strongly inhibits the manifestation of RPE and photoreceptor degeneration in aged VEGF - Ahypermice . F . - G . VEGF - Ahyper / IL18 - / - mice show significant RPE and photoreceptor degeneration , with shortened and irregular photoreceptor outer segments ( white arrows ) . Thick sub - RPE deposits that protrude into the RPE plane are observed as well ( black arrows ) . H . VEGF - Ahyper / Becn1 + / - mice show significant RPE and photoreceptor degeneration . I . In contrast , age - matched VEGF - Ahypomice that are hypomorphic for VEGF - A and express β - galactosidase from the endogenous VEGF - A locus ( as do VEGF - Ahypermice ) do not show these eye pathologies . B - I . 21 - months old mice . Scale bars 20 μm .

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Figure 9: VEGF-A-induced non-exudative AMD-like pathologies can be prevented by targeting NLRP3, IL1R1 or caspase-1/caspase-11.A. No significant abnormalities of the choroid, RPE or retina are seen in control wild-type littermate mice. Representative image of a 28-months old control eye is shown. White arrow indicates normal Bruch's membrane. Chor: choroid; RPE; PR: photoreceptor; ONL: outer nuclear layer.B. Aged VEGF-Ahypermice develop a progressive age-dependent degeneration of the RPE (red arrow) and the photoreceptors (yellow arrow) with accumulation of thick sub-RPE basal laminar deposits (black arrow). These non-exudative AMD-like pathologies occur also at sites devoid of CNV lesions.C.-E. Genetic inactivation of CASP1/CASP11, NLRP3, or IL1R1 strongly inhibits the manifestation of RPE and photoreceptor degeneration in aged VEGF-Ahypermice.F.-G. VEGF-Ahyper/IL18-/-mice show significant RPE and photoreceptor degeneration, with shortened and irregular photoreceptor outer segments (white arrows). Thick sub-RPE deposits that protrude into the RPE plane are observed as well (black arrows).H. VEGF-Ahyper/Becn1+/-mice show significant RPE and photoreceptor degeneration.I. In contrast, age-matched VEGF-Ahypomice that are hypomorphic for VEGF-A and express β-galactosidase from the endogenous VEGF-A locus (as do VEGF-Ahypermice) do not show these eye pathologies.B-I. 21-months old mice. Scale bars 20 μm.


A . Graphs show means of a - and b - waves of dark - adapted and light - adapted ERG responses ( ERG intensities in μV ) for both the left and the right eyes from 21 - months old VEGF - Ahypermice , VEGF - Ahyper / CASP1 - / - / CASP11 - / - mice , CASP1 - / - / CASP11 - / - mice , VEGF - Ahyper / IL1R1 - / - mice , IL1R1 - / - mice , VEGF - Ahyper / IL18 - / - mice , and IL18 - / - mice . None of these mice had cataracts . Graphs show mean ± SEM .

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A. Graphs show means of a- and b-waves of dark-adapted and light-adapted ERG responses (ERG intensities in μV) for both the left and the right eyes from 21-months old VEGF-Ahypermice, VEGF-Ahyper/CASP1-/-/CASP11-/-mice, CASP1-/-/CASP11-/-mice, VEGF-Ahyper/IL1R1-/-mice, IL1R1-/-mice, VEGF-Ahyper/IL18-/-mice, and IL18-/-mice. None of these mice had cataracts. Graphs show mean ± SEM.


B . Representative ERG tracings are shown for VEGF - Ahyper mice and for VEGF - Ahyper / CASP1 - / - / CASP11 - / - mice . X - axes show milliseconds ( ms ) . Y - axes show ERG intensities ( in μV ) .

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B. Representative ERG tracings are shown for VEGF-Ahyper mice and for VEGF-Ahyper/CASP1-/-/CASP11-/- mice. X-axes show milliseconds (ms). Y-axes show ERG intensities (in μV).


Figure 11 : Lack of caspase - 1 / caspase - 11 inhibits CNV lesion formation , retinal degeneration , fundus abnormalities and fundus autofluorescence in aged VEGF - Ahyper mice . A . OCTs in aged VEGF - Ahypermice show retinal degeneration with a thinning of the retina and CNV lesions extending from the choroid into the subretinal space ( arrows ) . OCTs appear normal in age - matched VEGF - Ahyper / CASP1 - / - / CASP11 - / - mice and CASP1 - / - / CASP11 - / - mice . B . Fundus autofluorescence imaging shows areas of autofluorescence in the fundus of aged VEGF - Ahypermice , but not in age - matched VEGF - Ahyper / CASP1 - / - / CASP11 - / - mice and CASP1 - / - / CASP11 - / - mice . C . Abnormal pigment loss and degenerative changes are observed in the fundus of aged VEGF - Ahypermice , while the fundus of age - matched VEGF - Ahyper / CASP1 - / - / CASP11 - / - mice and CASP1 - / - / CASP11 - / - mice does not show these abnormalities . Representative images of 21 - months old mice are shown .

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Figure 11: Lack of caspase-1/caspase-11 inhibits CNV lesion formation, retinal degeneration, fundus abnormalities and fundus autofluorescence in aged VEGF-Ahyper mice.A. OCTs in aged VEGF-Ahypermice show retinal degeneration with a thinning of the retina and CNV lesions extending from the choroid into the subretinal space (arrows). OCTs appear normal in age-matched VEGF-Ahyper/CASP1-/-/CASP11-/-mice and CASP1-/-/CASP11-/-mice.B. Fundus autofluorescence imaging shows areas of autofluorescence in the fundus of aged VEGF-Ahypermice, but not in age-matched VEGF-Ahyper/CASP1-/-/CASP11-/-mice and CASP1-/-/CASP11-/-mice.C. Abnormal pigment loss and degenerative changes are observed in the fundus of aged VEGF-Ahypermice, while the fundus of age-matched VEGF-Ahyper/CASP1-/-/CASP11-/-mice and CASP1-/-/CASP11-/-mice does not show these abnormalities.Representative images of 21-months old mice are shown.


( A ) HeLa cells expressing HA - Parkin were treated with CCCP or DMSO ( control ) and then immunostained with the indicated antibodies .

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(A) HeLa cells expressing HA-Parkin were treated with CCCP or DMSO (control) and then immunostained with the indicated antibodies.


( B ) HeLa cells stably expressing HA - Parkin or intact SH - SY5Y cells were treated with CCCP or DMSO and subjected to fractionation experiments . I , C , and M indicate input , cytosol - rich supernatant , and mitochondria - rich membrane pellet , respectively .

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(B) HeLa cells stably expressing HA-Parkin or intact SH-SY5Y cells were treated with CCCP or DMSO and subjected to fractionation experiments. I, C, and M indicate input, cytosol-rich supernatant, and mitochondria-rich membrane pellet, respectively.


( D ) Polarized mitochondria stained with MitoTracker red ( red arrowheads ) were not labeled by Parkin . In contrast , damaged mitochondria marked by Parkin ( green arrowheads ) were not stained with MitoTracker red .

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(D) Polarized mitochondria stained with MitoTracker red (red arrowheads) were not labeled by Parkin. In contrast, damaged mitochondria marked by Parkin (green arrowheads) were not stained with MitoTracker red.


( E ) HeLa cells expressing HA - Parkin with various pathogenic mutations were treated with CCCP , followed by immunocytochemistry . ( A , D , and E ) Higher magnification views of the boxed areas are shown in the insets . ( F ) Parkin colocalization with mitochondria was analyzed in > 100 cells per mutation . Example figures indicative of robust colocalization ( counted as 1 ) , weak colocalization ( counted as 0 . 5 ) , and no colocalization ( counted as 0 ) are shown . Error bars represent the mean ± SD values of at least three experiments .

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(E) HeLa cells expressing HA-Parkin with various pathogenic mutations were treated with CCCP, followed by immunocytochemistry. (A, D, and E) Higher magnification views of the boxed areas are shown in the insets. (F) Parkin colocalization with mitochondria was analyzed in >100 cells per mutation. Example figures indicative of robust colocalization (counted as 1), weak colocalization (counted as 0.5), and no colocalization (counted as 0) are shown. Error bars represent the mean ± SD values of at least three experiments.


( A ) HeLa cells expressing wild - type Parkin or E3 - inactivating mutations were treated with CCCP and then immunostained with the indicated antibodies . When E3 - inactivating mutations were introduced into Parkin , the mitochondrial ubiquitylation signal disappeared .

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(A) HeLa cells expressing wild-type Parkin or E3-inactivating mutations were treated with CCCP and then immunostained with the indicated antibodies. When E3-inactivating mutations were introduced into Parkin, the mitochondrial ubiquitylation signal disappeared.


( B and C ) HeLa cells expressing HA - Parkin ( B ) or expressing both Mt - GFP and HA - Parkin ( C ) were treated with CCCP or DMSO ( control ) and then immunostained with the indicated antibodies .

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(B and C) HeLa cells expressing HA-Parkin (B) or expressing both Mt-GFP and HA-Parkin (C) were treated with CCCP or DMSO (control) and then immunostained with the indicated antibodies.


( D ) Localization of GFP - Parkin to the mitochondria after CCCP treatment . ( A - D ) Higher magnification views of the boxed areas are shown in the insets .

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(D) Localization of GFP-Parkin to the mitochondria after CCCP treatment. (A-D) Higher magnification views of the boxed areas are shown in the insets.


( E ) HeLa cell lysates expressing GFP - Parkin were immunoprecipitated by anti - GFP antibody , followed by immunoblotting with the indicated antibodies .

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(E) HeLa cell lysates expressing GFP-Parkin were immunoprecipitated by anti-GFP antibody, followed by immunoblotting with the indicated antibodies.


( F ) Straight immunoblotting of HA - and GFP - Parkin in the absence or presence of CCCP . Note the slower migrating ladders derived from ubiquitylation ( Ub ) in only the GFP - Parkin with CCCP lane .

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(F) Straight immunoblotting of HA- and GFP-Parkin in the absence or presence of CCCP. Note the slower migrating ladders derived from ubiquitylation (Ub) in only the GFP-Parkin with CCCP lane.


( G ) GFP - Parkin - expressing HeLa cells with various pathogenic mutations ( Fig . 1 C ) were treated with CCCP and subjected to immunoblotting . Asterisks show ubiquitylation of GFP - Parkin . Vertical black lines indicate that intervening lanes have been spliced out . IB , immunoblot ; IP , immunoprecipitation .

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(G) GFP-Parkin-expressing HeLa cells with various pathogenic mutations (Fig. 1 C) were treated with CCCP and subjected to immunoblotting. Asterisks show ubiquitylation of GFP-Parkin. Vertical black lines indicate that intervening lanes have been spliced out. IB, immunoblot; IP, immunoprecipitation.


( A ) The number of HeLa cells with N - terminal - or C - terminal - tagged PINK1 localized to the mitochondria was counted in > 100 cells .

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(A) The number of HeLa cells with N-terminal- or C-terminal-tagged PINK1 localized to the mitochondria was counted in >100 cells.


( B and C ) Exogenous nontagged PINK1 ( B ) or endogenous PINK1 ( C ) in HeLa cells was immunostained with the indicated antibodies . ( D ) The number of HeLa cells with endogenous PINK1 localized to the mitochondria was counted as in A . ( A and D ) Error bars represent the mean ± SD values of least three experiments .

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(B and C) Exogenous nontagged PINK1 (B) or endogenous PINK1 (C) in HeLa cells was immunostained with the indicated antibodies. (D) The number of HeLa cells with endogenous PINK1 localized to the mitochondria was counted as in A. (A and D) Error bars represent the mean ± SD values of least three experiments.


( E ) Endogenous PINK1 gradually accumulated after CCCP treatment . The first through the fifth lanes show endogenous PINK1 , and the sixth lane shows overexpressed untagged PINK1 . Note that the asterisk indicates a cross - reacting band because it was not affected by overproduction of untagged PINK1 .

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(E) Endogenous PINK1 gradually accumulated after CCCP treatment. The first through the fifth lanes show endogenous PINK1, and the sixth lane shows overexpressed untagged PINK1. Note that the asterisk indicates a cross-reacting band because it was not affected by overproduction of untagged PINK1.


( F ) Subcellular fractionation of endogenous PINK1 . Intact SH - SY5Y cells were treated with CCCP or DMSO and subjected to fractionation experiments ( same sample as Fig . 1 B ) . I , C , and M indicate input , cytosol - rich supernatant , and the mitochondria - rich membrane pellet , respectively .

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(F) Subcellular fractionation of endogenous PINK1. Intact SH-SY5Y cells were treated with CCCP or DMSO and subjected to fractionation experiments (same sample as Fig. 1 B). I, C, and M indicate input, cytosol-rich supernatant, and the mitochondria-rich membrane pellet, respectively.


( G ) HeLa cells were treated with CCCP and cycloheximide as depicted , followed by immunoblotting with the indicated antibodies . LDH , lactate dehydrogenase . ( F and G ) Asterisks indicate a cross - reacting band .

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(G) HeLa cells were treated with CCCP and cycloheximide as depicted, followed by immunoblotting with the indicated antibodies. LDH, lactate dehydrogenase. (F and G) Asterisks indicate a cross-reacting band.


( H ) N - terminal 34 aa of PINK1 recruited GFP to the mitochondria both in the absence and presence of CCCP . The top panel shows control HeLa cells expressing only GFP . ( B , C , and H ) Higher magnification views of the boxed areas are shown in the insets . Bars , 10 µm .

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(H) N-terminal 34 aa of PINK1 recruited GFP to the mitochondria both in the absence and presence of CCCP. The top panel shows control HeLa cells expressing only GFP. (B, C, and H) Higher magnification views of the boxed areas are shown in the insets. Bars, 10 µm.


( A ) PINK1 knockout ( PINK1 − / − ) or control ( PINK1 + / + ) MEFs were transfected with HA - Parkin , treated with CCCP , and subjected to immunocytochemistry with the indicated antibodies . Higher magnification views of the boxed areas are shown in the insets . ( B ) The number of MEFs with Parkin localized to the mitochondria was counted as in Fig . 3 A .

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(A) PINK1 knockout (PINK1−/−) or control (PINK1+/+) MEFs were transfected with HA-Parkin, treated with CCCP, and subjected to immunocytochemistry with the indicated antibodies. Higher magnification views of the boxed areas are shown in the insets. (B) The number of MEFs with Parkin localized to the mitochondria was counted as in Fig. 3 A.


( C and D ) Neither activation of Parkin nor mitochondrial degradation was observed in PINK1 − / − MEFs . MEFs stably expressing GFP - Parkin were treated with CCCP for 4 h and subjected to immunoblotting ( C )

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(C and D) Neither activation of Parkin nor mitochondrial degradation was observed in PINK1−/− MEFs. MEFs stably expressing GFP-Parkin were treated with CCCP for 4 h and subjected to immunoblotting (C)


( C and D ) Neither activation of Parkin nor mitochondrial degradation was observed in PINK1 − / − MEFs . MEFs stably expressing GFP - Parkin were treated with CCCP for 24 h , followed by immunocytochemistry ( D ) . IB , immunoblot ; Ub , ubiquitylation .

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(C and D) Neither activation of Parkin nor mitochondrial degradation was observed in PINK1−/− MEFs. MEFs stably expressing GFP-Parkin were treated with CCCP for 24 h, followed by immunocytochemistry (D). IB, immunoblot; Ub, ubiquitylation.


( E ) The number of MEFs without anti - Tom20 antibody - detectable mitochondria was counted as in Fig . 3 A . In the example figure ( left ) , blue arrowheads indicate cells without anti - Tom20 antibody - detectable mitochondria , and red arrowheads indicate cells harboring anti - Tom20 antibody - detectable mitochondria .

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(E) The number of MEFs without anti-Tom20 antibody-detectable mitochondria was counted as in Fig. 3 A. In the example figure (left), blue arrowheads indicate cells without anti-Tom20 antibody-detectable mitochondria, and red arrowheads indicate cells harboring anti-Tom20 antibody-detectable mitochondria.


( A ) Schematic depiction of pathogenic and deletion mutants of PINK1 used in this study . MTS , mitochondria - targeting sequence ; TMD , transmembrane domain . ( B ) Subcellular localization of Parkin in PINK1 − / − cells complemented by various pathogenic and deletion mutants of PINK1 - Flag . Cells were treated with CCCP . Higher magnification views of the boxed areas are shown in the insets . ( C ) The number of cells with Parkin - positive mitochondria was counted as in Fig . 3 A . Error bars represent the mean ± SD values of least three experiments .

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(A) Schematic depiction of pathogenic and deletion mutants of PINK1 used in this study. MTS, mitochondria-targeting sequence; TMD, transmembrane domain. (B) Subcellular localization of Parkin in PINK1−/− cells complemented by various pathogenic and deletion mutants of PINK1-Flag. Cells were treated with CCCP. Higher magnification views of the boxed areas are shown in the insets. (C) The number of cells with Parkin-positive mitochondria was counted as in Fig. 3 A. Error bars represent the mean ± SD values of least three experiments.


( D ) PINK1 − / − MEFs complemented by various PINK1 mutants were treated with CCCP and subjected to immunoblotting using anti - Parkin or anti - Flag ( tag of PINK1 ) antibodies . IB , immunoblot ; Ub , ubiquitylation .

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(D) PINK1−/− MEFs complemented by various PINK1 mutants were treated with CCCP and subjected to immunoblotting using anti-Parkin or anti-Flag (tag of PINK1) antibodies. IB, immunoblot; Ub, ubiquitylation.


A , B . Coronal CT image of the chest at baseline with pleural fluid and intralobar septal thickening ( arrow ) ( A ) , and 3 months after starting trametinib ( B ) .

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A, B. Coronal CT image of the chest at baseline with pleural fluid and intralobar septal thickening (arrow) (A), and 3 months after starting trametinib (B).


C , D . MR T2 weighted coronal slice of the chest at baseline demonstrating bilateral pleural effusions ( arrow heads ) and pulmonary interstitial edema ( arrows ) ( C ) , and 3 months on trametinib therapy ( D ) .

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C, D. MR T2 weighted coronal slice of the chest at baseline demonstrating bilateral pleural effusions (arrow heads) and pulmonary interstitial edema (arrows) (C), and 3 months on trametinib therapy (D).


IN - DCMRL coronal MIP of the chest at baseline demonstrating dilated and tortuous TD ( arrow ) with dilated central lymphatic networks and extensive bilateral pulmonary perfusion ( arrowheads ) ( E ) , and 6 months after trametinib therapy demonstrating dilated and tortuous TD ( arrow ) with reduction in the extent of the dilated central lymphatic networks and resolution of bilateral pulmonary perfusion

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IN-DCMRL coronal MIP of the chest at baseline demonstrating dilated and tortuous TD (arrow) with dilated central lymphatic networks and extensive bilateral pulmonary perfusion (arrowheads) (E), and 6 months after trametinib therapy demonstrating dilated and tortuous TD (arrow) with reduction in the extent of the dilated central lymphatic networks and resolution of bilateral pulmonary perfusion


IN - DCMRL coronal MIP of the chest at baseline demonstrating dilated and tortuous TD ( arrow ) with dilated central lymphatic networks and extensive bilateral pulmonary perfusion ( arrowheads ) ( E ) , and 6 months after trametinib therapy demonstrating dilated and tortuous TD ( arrow ) with reduction in the extent of the dilated central lymphatic networks and resolution of bilateral pulmonary

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