B . MITOL prevents caspase - dependent in mitophagy . WT or MITOL KO HeLa stably expressing HA - Parkin HeLa were treated with DMSO or CCCP ( 10 µM ) with or without Z - VAD - FMK ( 10 μM ) for 24 h . were stained with propidium iodide ( PI ) for counting microscopy of of dead . Data represent mean ± SD of three independent experiments ( > 100 were counted ) . For statistical , a one - way ANOVA with Tukey post - was performed , * * P < 0 . 01 . C . in mitophagy caused by MITOL deficiency depends on calcium leak . WT MITOL KO HeLa stably expressing HA - Parkin or HeLa were treated with DMSO or CCCP ( 10 µM ) with or without BAPTA - AM ( 10 μM ) for 24 h . were stained with propidium iodide ( PI ) for counting microscopy of of dead . Data represent mean ± SD of three independent experiments ( > 100 were counted ) . For statistical , a one - way ANOVA with Tukey post - was performed , * * P < 0 . 01 .

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B. MITOL prevents caspase-dependent cell death in mitophagy. WT or MITOL KO HeLa cells stably expressing HA-Parkin and HeLa cells were treated with DMSO or CCCP (10 µM) with or without Z-VAD-FMK (10 μM) for 24 h. Cells were stained with propidium iodide (PI) for counting fluorescence microscopy of the percentage of dead cells. Data represent the mean ± SD of three independent experiments (>100 individual cells were counted). For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01. C. Cell death in mitophagy caused by MITOL deficiency depends on calcium leak. WT and MITOL KO HeLa cells stably expressing HA-Parkin or HeLa cells were treated with DMSO or CCCP (10 µM) with or without BAPTA-AM (10 μM) for 24 h. Cells were stained with propidium iodide (PI) for counting fluorescence microscopy of the percentage of dead cells. Data represent the mean ± SD of three independent experiments (>100 individual cells were counted). For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01.


( C ) Expression of miRNAs miR - 146a miR - 505 correlated with overall survival in ovarian cancer . validation cohort ( n = 150 samples ) is shown . See Figure EV1 for training cohort .

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(C) Expression of miRNAs miR-146a and miR-505 correlated with overall survival in ovarian cancer patients. The validation cohort (n=150 samples) is shown. See Figure EV1 for the training cohort.


( A ) consequence of miR - 155 miR - 181b mimics on viability , 5 days post transfection , relative to a negative control miRNA mimic is shown across a panel of normal ovarian cancer lines as indicated . Each data point is mean of n = 3 / . In screen , miR - 155 was found to reduce viability ( Z - score < - 2 ) in one , TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors . miR - 181 was found to reduce viability ( Z - score < - 2 ) in 2 lines , TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors . N , normal lines ( IHH , HOSE , HBEC3 , HBEC13 , HBEC30 , HBEC34 ) , EOC , epithelial ovarian cancer lines . miR - 155 data points for each are shown in black while miR - 181b data points are displayed in red .

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(A) The consequence of miR-155 and miR-181b mimics on cell viability, 5 days post transfection, relative to a negative control miRNA mimic is shown across a panel of normal and ovarian cancer cell lines as indicated. Each data point is the mean of n=3/cell line. In the screen, miR-155 was found to reduce cell viability (Z-score < -2) in one cell line, and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors. miR-181 was found to reduce viability (Z-score < -2) in 2 cell lines, and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors. N, normal cell lines (IHH, HOSE, HBEC3, HBEC13, HBEC30, and HBEC34), EOC, epithelial ovarian cancer cell lines. miR-155 data points for each cell line are shown in black while miR-181b data points are displayed in red.


( B ) Immunoblots indicate suppression of serum - induced AKT phosphorylation at S473 T308 in response to miR - 155 suppression of T308 phosphorylation in response to miR - 181b .

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(B) Immunoblots indicate suppression of serum-induced AKT phosphorylation at S473 and T308 in response to miR-155 and suppression of T308 phosphorylation in response to miR-181b.


( C ) miR - 155 miR - 181b reduced viability in GDC0941 - sensitive breast cancer lines . Error bars indicate mean ± SD ( n = 3 ) .

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(C) miR-155 and miR-181b reduced cell viability in GDC0941-sensitive breast cancer cell lines. Error bars indicate mean ± SD (n=3).


( D ) Differential localization of E - cadherin in PEO1 PEO4 48 hours post transfection with indicated oligos . were counterstained with phalloidin DAPI .

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(D) Differential localization of E-cadherin in PEO1 and PEO4 cells 48 hours post transfection with the indicated oligos. Cells were counterstained with phalloidin and DAPI.


( E ) Stimulation of PEO4 with 10 ng / mL TGFβ1 significantly sensitized to AKT inhibition with AKT Inhibitor X . Each point represents mean of 3 experiments ± SD * denotes p - value < 0 . 05 by Student ' s T - .

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(E) Stimulation of PEO4 cells with 10 ng/mL TGFβ1 significantly sensitized the cells to AKT inhibition with AKT Inhibitor X. Each point represents the mean of 3 experiments ± SD and * denotes p-value < 0.05 by Student's T-test.


( F ) viability upon expression of miR - 155 or miR - 181a mimics in 41 NSCLC lines 5 human bronchial epithelial lines . Each data point is mean of n = 3 / . R2 from Pearson correlation .

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(F) Cell viability upon expression of miR-155 or miR-181a mimics in 41 NSCLC cell lines and 5 human bronchial epithelial cell lines. Each data point is the mean of n=3/cell line. R2 from Pearson correlation.


( A ) Consequence of miR - 517a on EOC viability 41 NSCLC lines ( as in 2A ) . N , normal lines ( IHH , HOSE , HBEC3 , HBEC13 , HBEC30 , HBEC34 ) . miR - 517a was found to significantly reduce viability ( Z - score < - 2 ) in 5 lines in screen , TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors .

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(A) Consequence of miR-517a on EOC cell viability and 41 NSCLC cell lines (as in 2A). N, normal cell lines (IHH, HOSE, HBEC3, HBEC13, HBEC30, and HBEC34). miR-517a was found to significantly reduce cell viability (Z-score < -2) in 5 cell lines in the screen, and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors.


( B , C ) In vivo delivery of neutral liposome incorporated miR - 517a mimic reduced burden by ( B ) nodule number ( C ) in an orthotopic xenograft model using SKOV3 . Box - - whisker plot from n = 8 . p - value from Student ' s T - . LD , low - dose ( 200 μg / kg ) ; HD , high - dose ( 400 μg / kg ) .

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(B, C) In vivo delivery of neutral liposome incorporated miR-517a mimic reduced tumor burden by weight (B) and nodule number (C) in an orthotopic xenograft model using SKOV3 cells. Box-and-whisker plot from n=8. p-value from Student's T-Test. LD, low-dose (200 μg/kg); HD, high-dose (400 μg/kg).


( D ) Correlation of consequence of ARCN1 depletion miR - 517a sensitivity in 12 NSCLC lines ( plot as in 2D ) .

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(D) Correlation of the consequence of ARCN1 depletion and miR-517a sensitivity in 12 NSCLC cell lines (plot as in 2D).


( E ) Immunoblots indicate miR - 517a - induced depletion of ARCN1 USP1 in indicated lines .

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(E) Immunoblots indicate miR-517a-induced depletion of ARCN1 and USP1 in the indicated cell lines.


( F ) Consequence of indicated siRNAs miR - 517a on viability of indicated lines .

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(F) Consequence of the indicated siRNAs and miR-517a on the viability of the indicated cell lines.


( G ) Immunoblots indicate that miR - 517a suppresses expression of ARCN1 in multiple miR - 517a sensitive lines .

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(G) Immunoblots indicate that miR-517a suppresses expression of ARCN1 in multiple miR-517a sensitive cell lines.


( A ) Consequence of miR - 124 on EOC viability ( as in 2A ) . N , normal lines ( IHH HOSE ) . In screen , miR - 124 was found to significantly reduce viability ( Z - score < - 2 ) in 6 lines , TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors .

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(A) Consequence of miR-124 on EOC cell viability (as in 2A). N, normal cell lines (IHH and HOSE). In the screen, miR-124 was found to significantly reduce cell viability (Z-score < -2) in 6 cell lines, and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors.


( B ) miR - 124 induced expression of neuronal marker Tuj1 ( TUBB3 ) MAP2 in ES2 48 hours post transfection . were counterstained with DAPI phalloidin .

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(B) miR-124 induced expression of neuronal marker proteins Tuj1 (TUBB3) and MAP2 in ES2 cells 48 hours post transfection. Cells were counterstained with DAPI and phalloidin.


( C ) miR - 124 responsive genes in PEO1 were enriched for TargetScan predicted targets . p - value from hypergeometric distribution .

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(C) miR-124 responsive genes in PEO1 cells were enriched for TargetScan predicted targets. p-value from hypergeometric distribution.


( A ) Immunoblots indicate miR - 124 - induced SIX4 depletion . siSIX4 is shown as an antibody control .

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(A) Immunoblots indicate miR-124-induced SIX4 depletion. siSIX4 is shown as an antibody control.


( B ) Consequence of SIX4 depletion on EOCcell viability ( as in 2A , N , normal lines : HOSE , HBEC30 ) .

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(B) Consequence of SIX4 depletion on EOCcell viability (as in 2A, N, normal cell lines: HOSE, HBEC30).


( C ) in vivo knockdown of SIX4 using neutral liposome incorporated siRNA reduced burden ascites volume in an orthotopic xenograft mouse model . Box - - whisker plot from n = 8 . p - value from Student ' s T - .

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(C) in vivo knockdown of SIX4 using neutral liposome incorporated siRNA reduced tumor burden and ascites volume in an orthotopic xenograft mouse model. Box-and-whisker plot from n=8. p-value from Student's T-Test.


( D ) Consequence of SIX4 depletion in PEO1 on E - cadherinplasma accumulation 48 hours after transfection . were counterstained with phalloidin DAPI .

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(D) Consequence of SIX4 depletion in PEO1 cells on E-cadherinplasma membrane accumulation 48 hours after transfection. Cells were counterstained with phalloidin and DAPI.


( E ) Immunoblots indicate consequence of SIX4 depletion on LKB1 accumulation AMPK pathway activation .

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(E) Immunoblots indicate consequence of SIX4 depletion on LKB1 accumulation and AMPK pathway activation.


ER Proliferation under UPR - Inducing Conditions ( A ) Determination of ER abundance in control UPR - induced . Representative are shown . UPR was induced in wild - type by addition of DTT . Ultrastructure of control UPR - induced was analyzed using ImageJ . lower show traces of cortical ER ( represented in magenta ) envelope ( NE , in blue ) . Vacuoles , nuclei , mitochondria are indicated as V , N , M , respectively . ( B ) of ER proliferation during UPR . UPR was induced were collected for EM at indicated time points . of ER ( as traced in [ A ] ) was measured divided by of section . Data are plotted relative to time 0 . Measurements for each time point correspond to mean of 25 independent . ( C ) Expression of HAC1i was induced by addition of 100 μM DOC for 3 h . ER was quantified as described above in ( B ) .

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ER Proliferation under UPR-Inducing Conditions(A) Determination of ER abundance in control and UPR-induced cells. Representative cells are shown. The UPR was induced in wild-type cells by addition of DTT. Ultrastructure of control cells and UPR-induced cells was analyzed using ImageJ. The lower images show traces of cortical ER (represented in magenta) and the nuclear envelope (NE, in blue). Vacuoles, nuclei, and mitochondria are indicated as V, N, and M, respectively.(B) Quantification of the ER proliferation during the UPR. UPR was induced and cells were collected for EM at the indicated time points. Length of the ER (as traced in [A]) was measured and divided by the area of the section. Data are plotted relative to time 0. Measurements for each time point correspond to the mean of 25 independent cell images.(C) Expression of HAC1i was induced by addition of 100 μM DOC for 3 h. ER was quantified as described above in (B).


ER Morphologically Changes during UPR ( A ) Control UPR - induced were used to analyze follow ER within a single using EM . Boxes indicate areas magnified in ( B ) . shown here correspond to full section of labeled " + 140 nm " in ( B ) . ( B ) Serial section of control UPR - induced . Sections are separated by 70 nm on z - axis . ER is represented in magenta NE in blue . ( C ) Electro from control UPR - induced showing that between ER membranes increases during UPR . For a better preservation of ultrastructure , samples for this experiment were prepared using high - pressure freezing / freeze substitution techniques ( see Material Methods ) .

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The ER Morphologically Changes during the UPR(A) Control cells and UPR-induced cells were used to analyze and follow the ER within a single cell using EM. Boxes indicate the areas magnified in (B). Cells shown here correspond to the full section of the images labeled "+140 nm" in (B).(B) Serial section of control and UPR-induced cells. Sections are separated by 70 nm on the z-axis. ER is represented in magenta and NE in blue.(C) Electro micrographs from control and UPR-induced cells showing that the distance between ER membranes increases during the UPR. For a better preservation of the ultrastructure, samples for this experiment were prepared using high-pressure freezing/freeze substitution techniques (see Material and Methods).


Characterization of ER - Containing Autophagosomes ( ERAs ) during UPR ( A ) of representative DTT - treated wild - type that contain ERAs . Nuclei cytoplasm are indicated as N C , respectively . ( B ) Enlargement of representative of ERAs from different . bottom right image is likely to show a section through a cup - shaped ERA . Note that there are no connections between stacked cisternae envelope . ( C ) High magnification of ERA double envelope . ( D ) Some ERAs are found attached to or are in close proximity to ER tubules / sheets ( indicated by arrow ) . Note that section in ( A ) includes two such junctions . ( E ) High - pressure freezing / freeze substitution image of an ERA linked to an ER tubule / sheet . osmium / lead used in this technique visualizes ribosomes demonstrates that outer ERA envelope , but not stacked internal cisternae , are tightly studded with ribosomes , indicating that they originate from ER membranes . ( F ) High - pressure freezing / freeze substitution image of an ER - ERA junction using an improved protocol to visualize membranes . ( G ) Using same technique as in ( F ) , we visualized internal content of an ERA . Note that both portions of internal membranes of sequestering double envelope contain bound ribosomes , hence are likely derived from ER .

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Characterization of ER-Containing Autophagosomes (ERAs) during the UPR(A) Images of representative DTT-treated wild-type cells that contain ERAs. Nuclei and cytoplasm are indicated as N and C, respectively.(B) Enlargement of representative images of ERAs from different cells. The bottom right image is likely to show a section through a cup-shaped ERA. Note that there are no connections between the stacked cisternae and the envelope.(C) High magnification of the ERA double membrane envelope.(D) Some ERAs are found attached to or are in close proximity to ER tubules/sheets (indicated by the arrow). Note that the section in (A) includes two such junctions.(E) High-pressure freezing/freeze substitution image of an ERA linked to an ER tubule/sheet. The osmium/lead staining used in this technique visualizes ribosomes and demonstrates that the outer ERA envelope membrane, but not the stacked internal cisternae, are tightly studded with ribosomes, indicating that they originate from ER membranes.(F) High-pressure freezing/freeze substitution image of an ER-ERA junction using an improved protocol to visualize membranes.(G) Using the same technique as in (F), we visualized the internal membrane content of an ERA. Note that both portions of the internal membranes and of the sequestering double membrane envelope contain bound ribosomes, and hence are likely derived from the ER.


Visualization of an ER Marker after UPR Induction ( A ) treated with UPR - inducing drug DTT ( + DTT ) or with no drug were visualized using a fusion between translocon component Sec61 red - " cherry . " Top panels show untreated , bottom panels show representative UPR - induced . ( B ) Representative showing UPR - induced that contain ERAs ( indicated by arrows ) .

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Fluorescence Visualization of an ER Marker after UPR Induction(A) Cells treated with the UPR-inducing drug DTT (+DTT) or with no drug were visualized using a fusion protein between the translocon component Sec61 and the red-fluorescent protein "cherry." Top panels show untreated cells, and bottom panels show representative UPR-induced cells.(B) Representative images showing UPR-induced cells that contain ERAs (indicated by arrows).


Immunogold Labeling of ERAs with an Antibody Directed against an ER Marker ( A ) Representative section of a immunolabeled against a myc - tagged Sec63 , an integral ER . As a antibody , we used a rabbit polyclonal anti - myc , as a secondary , we used 15 - nm gold particles - conjugated anti - rabbit antibody . Nucleus , envelope , ER , ERA are indicated as N , NE , ER , ERA , respectively . ( B ) High magnification of an electron micrograph of a section of ER . showed that there are 5 ± 2 gold particles per linear micrometer of ER . ( C ) High magnification of ERAs . To predict how many gold particles one should expect in a particular ERA , we first calculated averaged amount of ER ( expressed as in linear micrometers ) present in an ERA ( similar to ones shown in Figure 3B ) , normalized value for its . These calculations determined that there are 20 . 8 ± 3 . 3 μm of ER per μm2 inside ERAs . These values allowed us to predict how many gold particles would be expected over a section of an ERA if it were packed with ER membranes . Two representative ERAs are shown . ERA shown in middle picture should hold 2 . 4 μm of ER inside , therefore , should have 12 gold particles . We counted 12 gold particles . ERA on right could contain 2 . 7 μm of ER should contain14 gold particles ; we counted 16 gold particles . image on right shows a representative view of a nucleoplasmic region . ( D ) of gold - labeling density per . To assess signal - to - noise of our immunogold - labeling procedure , we assessed background labeling by counting number of gold particles over an areas of nucleoplasm ( N ) over ERAs , normalized to respective areas .

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Immunogold Labeling of ERAs with an Antibody Directed against an ER Membrane Marker(A) Representative section of a cell immunolabeled against a myc-tagged Sec63, an integral ER membrane protein. As a primary antibody, we used a rabbit polyclonal anti-myc and, as a secondary, we used 15-nm gold particles-conjugated anti-rabbit antibody. Nucleus, nuclear envelope, ER, and ERA are indicated as N, NE, ER, and ERA, respectively.(B) High magnification of an electron micrograph of a section of ER. Quantification showed that there are 5 ± 2 gold particles per linear micrometer of ER.(C) High magnification of ERAs. To predict how many gold particles one should expect in a particular ERA, we first calculated and averaged the amount of ER (expressed as length in linear micrometers) present in an ERA (similar to the ones shown in Figure 3B), and normalized the value for its area. These calculations determined that there are 20.8 ± 3.3 μm of ER per μm2 inside the ERAs. These values allowed us to predict how many gold particles would be expected over a section of an ERA if it were packed with ER membranes. Two representative ERAs are shown. The ERA shown in the middle picture should hold 2.4 μm of ER inside and, therefore, should have 12 gold particles. We counted 12 gold particles. The ERA on the right could contain 2.7 μm of ER and should contain14 gold particles; we counted 16 gold particles. The image on the right shows a representative view of a nucleoplasmic region.(D) Quantification of gold-labeling density per area. To assess the signal-to-noise ratio of our immunogold-labeling procedure, we assessed background labeling by counting the number of gold particles over an areas of nucleoplasm (N) and over ERAs, and normalized the counts to the respective areas.


( A ) Wild - type transformed with a plasmid containing GFP - Atg8 were grown for 4 h in synthetic media with no drug , with UPR - inducing conditions ( + DTT + TM ) , or under nitrogen starvation conditions ( N starv ) , then harvested for preparation . extracts were analyzed by Western blotting using antibodies against GFP ( top panel ) or Hac1 ( bottom panel ) . Total was measured by BCA . Same of was loaded in each lane , transfer efficiency was checked by Ponceau . identities of different bands are indicated .

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(A) Wild-type cells transformed with a plasmid containing GFP-Atg8 were grown for 4 h in synthetic media with no drug, with UPR-inducing conditions (+DTT and +TM), or under nitrogen starvation conditions (N starv), and then harvested for protein preparation. Protein extracts were analyzed by Western blotting using antibodies against GFP (top panel) or Hac1 (bottom panel). Total protein concentration was measured by BCA protein assay. Same concentration of protein was loaded in each lane, and transfer efficiency was checked by Ponceau staining. The identities of the different bands are indicated.


( B ) Wild - type expressing GFP - Atg8 grown under conditions described above were visualized by microscopy .

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(B) Wild-type cells expressing GFP-Atg8 grown under the conditions described above were visualized by fluorescence microscopy.


( C ) GFP - Atg8 was detected in extracts from untreated hac1Δ or expressing HAC1i ( + DOC ) by Western blotting using antibodies against GFP .

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(C) GFP-Atg8 was detected in extracts from untreated hac1Δ cells or cells expressing HAC1i (+DOC) by Western blotting using antibodies against GFP.


( D ) Western blot using antibodies against GFP of extracts from hac1Δ , ire1Δ , or vps4Δ pep4Δ expressing GFP - Atg8 . Mutant were grown under regular conditions , UPR - inducing conditions ( + DTT ) , or nitrogen starvation conditions ( N starv ) .

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(D) Western blot using antibodies against GFP of extracts from hac1Δ, ire1Δ, or vps4Δ pep4Δ cells expressing GFP-Atg8. Mutant cells were grown under regular conditions, UPR-inducing conditions (+DTT), or nitrogen starvation conditions (N starv).


Localization of GFP - Atg8 during UPR InductionSome of DTT - treated shown in Figure 4B expressing GFP - Atg8 Sec61 - cherry ( as an ER marker ) were visualized using microscopy . GFP - Atg8 localizes in close proximity to ERAs detected by ER marker .

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Localization of GFP-Atg8 during UPR InductionSome of the DTT-treated cells shown in Figure 4B expressing GFP-Atg8 and Sec61-cherry (as an ER marker) were visualized using fluorescence microscopy. GFP-Atg8 localizes in close proximity to the ERAs detected by the ER marker.


Atg8 Other ATG Genes Are Necessary during UPR InductionSerial dilutions for wild - type , hac1Δ , atg1Δ , atg8Δ , atg9Δ , atg16Δ , atg20Δ deletion vps4Δ pep4Δ double deletion were grown on rich - media plates with no drug ( YPD ) or with different concentrations of tunicamycin ( TM ; 0 . 2 or 1 . 0 μg / ml ) . atg19Δ gave an identical result to other autophagy genes shown here ( unpublished data ) .

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Atg8 and Other ATG Genes Are Necessary during UPR InductionSerial dilutions for wild-type, hac1Δ, atg1Δ, atg8Δ, atg9Δ, atg16Δ, and atg20Δ deletion cells and vps4Δ pep4Δ double deletion cells were grown on rich-media plates with no drug (YPD) or with different concentrations of tunicamycin (TM; 0.2 or 1.0 μg/ml). atg19Δ gave an identical result to the other autophagy genes shown here (unpublished data).


A Left : Overlay image of a confocal recording of an apoptotic U2OS surrounded by non - apoptotic labeled with Bax ( green ) Tom22 ( red ) . Bax translocates to mitochondria in apoptotic . Right : display of two color channels as shown in overlay image . asterisk denotes apoptotic . Scale bars : 10 µm .

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A Left: Overlay image of a confocal recording of an apoptotic U2OS cell surrounded by non-apoptotic cells labeled with Bax (green) and Tom22 (red). Bax translocates to the mitochondria in apoptotic cells. Right: Individual display of the two color channels as shown in the overlay image. The asterisk denotes the apoptotic cell. Scale bars: 10 µm.


B , C Dual color image of an apoptotic U2OS decorated with antibodies against Bax ( green ) Tom20 ( red ) . Bax signal was recorded in STED mode Tom20 signal in diffraction - limited confocal mode . ( B ) ( C ) display same data set . small are magnifications of areas indicated by rectangles . In ( B ) color table was adjusted to brightest Bax signal in image resulting in a clear display of clusters . In ( C ) color table was adjusted such that dimmer Bax - rings are visible clusters are saturated . arrows point to Bax - rings . Scale bars : 1 µm .

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B, C Dual color image of an apoptotic U2OS cell decorated with antibodies against Bax (green) and Tom20 (red). The Bax signal was recorded in the STED mode and the Tom20 signal in the diffraction-limited confocal mode. (B) and (C) display the same data set. The small images are magnifications of the areas indicated by the rectangles. In (B) the color table was adjusted to the brightest Bax signal in the image resulting in a clear display of the clusters. In (C) the color table was adjusted such that the dimmer Bax-rings are visible and the clusters are saturated. The arrows point to Bax-rings. Scale bars: 1 µm.


D Optical sections of an apoptotic mitochondrion labelled for Bax ( green ) Tom22 ( red ) . Shown are three optical sections a maximum projection of entire image stack . Scale bar : 0 . 5µm .

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D Optical sections of an apoptotic mitochondrion labelled for Bax (green) and Tom22 (red). Shown are three optical sections and a maximum intensity projection of the entire image stack. Scale bar: 0.5µm.


E Bax - rings on apoptotic mitochondria of various mammalian types . Shown are apoptotic mitochondria labelled for Bax ( green ) Tom22 ( red ) in HT1080 , SH - SY5Y , HeLa CV - 1 , as indicated . Scale bars : 0 . 5 µm .

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E Bax-rings on apoptotic mitochondria of various mammalian cell types. Shown are apoptotic mitochondria labelled for Bax (green) and Tom22 (red) in HT1080, SH-SY5Y, HeLa and CV-1 cells, as indicated. Scale bars: 0.5 µm.


F of diameter of Bax - rings in apoptotic U2OS . Only long ring - axis was measured . Bax - rings smaller than 200 nm exist , but were not considered .

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F Quantification of the diameter of Bax-rings in apoptotic U2OS cells. Only the long ring-axis was measured. Bax-rings smaller than 200 nm exist, but were not considered.


Apoptotic mitochondria were decorated with antibodies against Bax ( green ) Tom22 ( red ) . Bax signal was recorded in STED mode . Shown are color channels as well as an overlay ( from top to bottom ) of several apoptotic mitochondria . We used Lagrange interpolation to double number of pixels . Note that throughout this manuscript except for contrast stretching no image manipulation was applied . Scale bar : 0 . 5 µm .

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Apoptotic mitochondria were decorated with antibodies against Bax (green) and Tom22 (red). The Bax signal was recorded in the STED mode. Shown are the individual color channels as well as an overlay (from top to bottom) of several apoptotic mitochondria. We used Lagrange interpolation to double the number of pixels. Note that throughout this manuscript except for contrast stretching no image manipulation was applied. Scale bar: 0.5 µm.


A , B Three - color of mitochondria of unchallenged ( A ) apoptotic ( B ) U2OS . From left to right : Tom22 , Bax ( recorded in STED - mode ) , cytochrome c an overlay of three channels . arrows denote Bax - rings . Scale bars : 2 µm .

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A, B Three-color images of mitochondria of unchallenged (A) and apoptotic (B) U2OS cells. From left to right: Tom22, Bax (recorded in the STED-mode), cytochrome c and an overlay of all three channels. The arrows denote Bax-rings. Scale bars: 2 µm.


A In apoptotic Drp1 knockdown U2OS , cytochrome c release is delayed . Confocal overview image of an apoptotic Drp1 knockdown labelled with antibodies against Tom20 ( red ) , cytochrome c ( blue ) Bax ( green ) . Large image : Overlay of three channels . Small : Magnifications of indicated by rectangle in large image . Shown are channels , as indicated . arrows point to mitochondria that had not released cytochrome c ; asterisks denote mitochondria that had released cytochrome c . Scale bars : 10 µm ( overview ) 2 µm ( magnifications ) .

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A In apoptotic Drp1 knockdown U2OS cells, cytochrome c release is delayed. Confocal overview image of an apoptotic Drp1 knockdown cell labelled with antibodies against Tom20 (red), cytochrome c (blue) and Bax (green). Large image: Overlay of all three channels. Small images: Magnifications of the area indicated by the rectangle in the large image. Shown are the individual channels, as indicated. The arrows point to mitochondria that had not released cytochrome c; the asterisks denote mitochondria that had released cytochrome c. Scale bars: 10 µm (overview) and 2 µm (magnifications).


B In Drp1 knockdown Bax - ring formation cytochrome c release is not correlated . Three - color of apoptotic mitochondria of Drp1 knockdown . From left to right : Tom22 , Bax ( recorded in STED - mode ) , cytochrome c an overlay of three channels . arrows point to Bax - rings . Scale bar : 1 µm .

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B In Drp1 knockdown cells Bax-ring formation and cytochrome c release is not correlated. Three-color images of apoptotic mitochondria of Drp1 knockdown cells. From left to right: Tom22, Bax (recorded in the STED-mode), cytochrome c and an overlay of all three channels. The arrows point to Bax-rings. Scale bar: 1 µm.


Drp1 knockdown were treated with DMSO ( control ) or with actinomycin D for 14 h to induce apoptosis were decorated with antisera against Bax , cytochrome c , Mic27 or Mic60 ( see Appendix Figure S4 ) . Three - color were taken , whereby Mic27 or Mic60 were recorded in STED mode . Relying on Bax cytochrome c signals , we discriminated between healthy apoptotic mitochondria before after cytochrome c release . distributions of Mic27 or Mic60 were analyzed by determining normalized variance of , which is a sensitive measure for distribution of labelled . numbers within columns represent numbers of analyzed error bars represent standard error of mean .

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Drp1 knockdown cells were treated with DMSO (control) or with actinomycin D for 14 h to induce apoptosis and were decorated with antisera against Bax, cytochrome c, and Mic27 or Mic60 (see Appendix Figure S4). Three-color images were taken, whereby Mic27 or Mic60 were recorded in the STED mode. Relying on the Bax and the cytochrome c signals, we discriminated between healthy and apoptotic mitochondria before and after cytochrome c release. The distributions of Mic27 or Mic60 were analyzed by determining the normalized variance of the fluorescence intensity, which is a sensitive measure for the distribution of the labelled protein. The numbers within the columns represent the numbers of images analyzed and the error bars represent the standard error of the mean.


Shown are seven optical sections through a mitochondrion of an apoptotic U2OS . Top middle row : Tom22 Bax ( recorded in STED ) channel , respectively . Bottom row : overlay ( Tom22 : red ; Bax : green ) . Note absence of Tom22 signal in enclosed by Bax - ring . Displayed are raw data . Scale bar : 0 . 5 µm .

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Shown are seven optical sections through a mitochondrion of an apoptotic U2OS cell. Top and middle row: Tom22 and Bax (recorded in STED) channel, respectively. Bottom row: overlay (Tom22: red; Bax: green). Note the absence of Tom22 signal in the area enclosed by the Bax-ring. Displayed are raw data. Scale bar: 0.5 µm.


principle : RNAi was performed with luciferase - expressing that were challenged with or without CTLs bi - specific antibody . Before readout , supernatant was removed remaining intact were lysed to measure residual - - associated luciferase . To identify immune - checkpoint regulators , difference between normalized luciferase measurements for conditions with CTLs without CTLs was calculated . siRNAs enhancing CTL cytotoxicity would only reduce normalized luciferase under conditions with CTLs ; hence , difference between luciferase measurements will be > 0 .

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Assay principle: RNAi was performed with luciferase-expressing cells that were challenged with or without CTLs and bi-specific antibody. Before readout, cell supernatant was removed and the remaining intact cells were lysed to measure the residual-cell-associated luciferase. To identify immune-checkpoint regulators, the difference between normalized luciferase measurements for conditions with CTLs and without CTLs was calculated. siRNAs enhancing CTL cytotoxicity would only reduce normalized luciferase levels under conditions with CTLs; hence, the difference between luciferase measurements will be > 0.


Luc - CTL performed at different T to MCF7 with PBMC - derived CD8 + T anti - EpCAM x CD3 bi - specific antibody ( ○ ) . Anti - CD19 × CD3 bi - specific Ab ( ▪ ) was used as a specificity control since CD19 is a B - lymphocyte - specific antigen therefore this bsAb fails to cross - link to T . Lower luciferase indicates higher lysis . Error bars denote ± .

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Luc-CTL assay performed at different T cell to MCF7 cell ratio with PBMC-derived CD8+ T cells and anti-EpCAM x CD3 bi-specific antibody (○). Anti-CD19 × CD3 bi-specific Ab (▪) was used as a specificity control since CD19 is a B-lymphocyte-specific antigen and therefore this bsAb fails to cross-link tumor to T cells. Lower luciferase intensity indicates higher lysis. Error bars denote ± SEM.


Luc - CTL was performed with MCF7 transfected with control or PD - L1 - specific siRNAs cocultured with or without CTLs bsAb . For each condition , luciferase activity of PD - L1 - siRNA - treated was normalized to that of control siRNA ; n = 8 . Error bars denote ± .

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Luc-CTL assay was performed with MCF7 cells transfected with control or PD-L1-specific siRNAs and cocultured with or without CTLs and bsAb. For each condition, the luciferase activity of PD-L1-siRNA-treated cells was normalized to that of the control siRNA; n = 8. Error bars denote ± SEM.


Comparison between Luc - CTL ( ▪ ) classical chromium ( Cr ) - release ( ○ ) with MCF7 as target survivin - specific T as effector at varying effector to target ( E : T ) ratios . Error bars denote ± .

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Comparison between the Luc-CTL assay (▪) and the classical chromium (Cr)-release assay (○) with MCF7 as target cells and survivin-specific T cells as effector cells at varying effector to target (E:T) ratios. Error bars denote ± SEM.


B Correlation between replicates for screen 2 for both viability toxicity sets , as determined by Pearson rank correlation .

B Correlation between replicates for screen 2 for both viability and toxicity sets, as determined by the Pearson rank correlation test.


C - E Graphical summary of gene function related to modification of T - mediated lysis cellviability for screens 1 , 2 , 3 , respectively . Positive score = reduced cancer cellviability ; negative score = increased viability . X - axis : influence on cellviability without addition of T . Y - axis : influence on cellviability with addition of T . Appropriate immune - suppressive ( PD - L1 , CEACAM - 6 , GAL - 3 ) lethality ( UBC , PLK - 1 ) controls , along with few positive ( GRM4 , GRK5 ) negative ( CCR9 , IL8 ) candidate immune - modulatory hits are highlighted herein .

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C-E Graphical summary of gene function related to modification of T cell-mediated tumor lysis and cellviability for screens 1, 2, and 3, respectively. Positive score = reduced cancer cellviability; negative score = increased viability. X-axis: influence on cellviability without addition of T cells. Y-axis: influence on cellviability with addition of T cells. Appropriate immune-suppressive (PD-L1, CEACAM-6, GAL-3) and lethality (UBC, PLK-1) controls, along with few positive (GRM4, GRK5) and negative (CCR9, IL8) candidate immune-modulatory hits are highlighted herein.


A MCF7 were transfected with described siRNA sequences harvested after 72 h for mRNA estimation using RT - PCR ( upper ) immunoblot ( lower ) , respectively . GAPDH beta - actin were used as controls for normalization , respectively .

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A MCF7 cells were transfected with the described siRNA sequences and harvested after 72 h for mRNA and protein estimation using RT-PCR (upper) and immunoblot (lower) analysis, respectively. GAPDH and beta-actin were used as controls for RNA and protein normalization, respectively.


B Luc - CTL cytotoxicity with PBMC - derived CTLs bi - specific Ab as effector population MCF7 as target , which were transfected with ( s1 - s4 ) or pooled CCR9 siRNA sequences . PD - L1 non - specific control siRNAs were used as positive negative controls , respectively , for CTL - mediated cytotoxicity .

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B Luc-CTL cytotoxicity assay with PBMC-derived CTLs and bi-specific Ab as effector population and MCF7 as target cells, which were transfected with individual (s1-s4) or pooled CCR9 siRNA sequences. PD-L1 and non-specific control siRNAs were used as positive and negative controls, respectively, for CTL-mediated cytotoxicity.


C , D Cr - release showing % specific lysis of MCF7 by survivin - specific T at different ratios upon CCR9 knockdown ( C ) or overexpression ( D ) . MCF7 were transfected with either CCR9 siRNA s1 ( Δ ) , pooled siRNA sequences ( ○ ) , positive control PD - L1 ( □ ) , non - specific control siRNA ( ▪ ) ( C ) or with pCMV6 - AC - His control vector ( ▪ ) pCMV6 - AC - His - CCR9 expression construct ( ○ ) ( D ) 72 h prior to .

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C, D Cr-release assay showing % specific lysis of MCF7 cells by survivin-specific T cells at different ratios upon CCR9 knockdown (C) or overexpression (D). MCF7 cells were transfected with either CCR9 siRNA s1 (Δ), pooled siRNA sequences (○), positive control PD-L1 (□), and non-specific control siRNA (▪) (C) or with pCMV6-AC-His control vector (▪) and pCMV6-AC-His-CCR9 expression construct (○) (D) 72 h prior to the assay.


E Cr - release showing % specific lysis of MDA - MB - 231 breast by survivin - specific T at different ratios upon CCR9 knockdown ( ○ ) in comparison to control knockdown ( ▪ ) .

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E Cr-release assay showing % specific lysis of MDA-MB-231 breast tumor cell line by survivin-specific T cells at different ratios upon CCR9 knockdown (○) in comparison to the control knockdown (▪).


F , G Cr - release showing lysis of - derived melanoma ( M579 - A2 ) by - infiltrating lymphocytes ( TIL 412 ) ( F ) or lysis of PANC - 1 pancreatic adenocarcinoma by - derived pancreatic TIL 53 ( G ) at different E : T ratios upon CCR9 ( ○ ) or control ( ▪ ) knockdown .

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F, G Cr-release assay showing lysis of patient-derived melanoma cells (M579-A2) by tumor-infiltrating lymphocytes (TIL 412) (F) or lysis of PANC-1 pancreatic adenocarcinoma cells by patient-derived pancreatic TIL 53 (G) at different E:T ratios upon CCR9 (○) or control (▪) knockdown.


A , B ELISpot showing IFN - γ ( A ) granzyme B ( B ) secretion by survivin - specific T , as spot numbers , upon CCR9 knockdown ( black bars ) in MCF7 compared to control knockdown ( bars ) . T ( TC ) alone ( grey bars ) were used as control for background spot numbers .

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A, B ELISpot assay showing IFN-γ (A) and granzyme B (B) secretion by survivin-specific T cells, as spot numbers, upon CCR9 knockdown (black bars) in MCF7 cells compared to the control knockdown (white bars). T cells (TC) alone (grey bars) were used as control for background spot numbers.


C Luminex showing cytokine in supernatant from coculture of survivin - specific TC either CCR9hi MCF7 ( transfected with CCR9 - specific siRNA ) or CCR9lo MCF7 ( transfected with control siRNA ) .

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C Luminex assay showing cytokine levels in the supernatant from the coculture of survivin-specific TC and either CCR9hi MCF7 (transfected with CCR9-specific siRNA) or CCR9lo MCF7 (transfected with control siRNA) cells.


D Phospho - plex showing phospho - STAT in survivin - specific TC upon encountering CCR9hi or CCR9lo MCF7 . Log2 of mean ( MFI ) of respective analytes to unstimulated TC is plotted herein .

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D Phospho-plex analysis showing the phospho-STAT levels in survivin-specific TC upon encountering CCR9hi or CCR9lo MCF7 cells. Log2 ratio of mean fluorescent intensity (MFI) of the respective analytes to the unstimulated TC is plotted herein.


E Immunoblot showing phospho - STAT1 in CCR9hi - treated , CCR9lo - treated or unstimulated TC using phospho - specific STAT1 ( pTyr701 ) antibody . Beta - actin was used as loading control .

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E Immunoblot analysis showing the phospho-STAT1 levels in the CCR9hi-treated, CCR9lo-treated or unstimulated TC using the phospho-specific STAT1 (pTyr701) antibody. Beta-actin was used as the loading control.


A ELISA showing CCL25 in lysates from indicated tumorcell lines . CCR9 knockdown ( k . d . ) in MCF7 was achieved using specific shRNA ( see Materials Methods ) .

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A ELISA showing CCL25 levels in cell lysates from indicated tumorcell lines. CCR9 knockdown (k.d.) in MCF7 cells was achieved using specific shRNA (see Materials and Methods).


B Cr - release showing % specific lysis of MCF7 by survivin TC upon CCL25 ( □ ) or CCR9 ( ○ ) inhibition using specific siRNAs in comparison to control siRNA ( ▪ ) . Mean ± are depicted herein .

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B Cr-release assay showing % specific lysis of MCF7 cells by survivin TC upon CCL25 (□) or CCR9 (○) inhibition using specific siRNAs in comparison to the control siRNA (▪). Mean ± SEM are depicted herein.


C MCF7 were transfected with control or CCR9 - specific siRNAs , 48 h later , supernatants ( CCR9lo or CCR9hi SSN , respectively ) were used to culture survivin TCs overnight . Supernatant - treated TCs were then used as effector against CCR9lo or CCR9hi MCF7 in Cr - release along with wild - type MCF7 . Mean ± are depicted herein .

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C MCF7 cells were transfected with control or CCR9-specific siRNAs, and 48 h later, the supernatants (CCR9lo or CCR9hi SSN, respectively) were used to culture survivin TCs overnight. Supernatant-treated TCs were then used as effector cells against CCR9lo or CCR9hi MCF7 tumor cells in the Cr-release assay along with wild-type MCF7 cells. Mean ± SEM are depicted herein.


D Cr - release showing % specific lysis of MCF7 that were pre - treated with or without pertussis toxin ( PTX ) , or knocked down for CCR9 using specific siRNA . Mean ± are depicted herein .

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D Cr-release assay showing % specific lysis of MCF7 cells that were pre-treated with or without pertussis toxin (PTX), or knocked down for CCR9 using specific siRNA. Mean ± SEM are depicted herein.


E , F MCF7 transfected with control siRNA ( CCR9hi ) or CCR9 siRNA ( CCR9lo ) were cocultured with survivin TCs for 12 h . Gene microarray was performed with total extracted from purified T after coculture . Volcano plot ( E ) illustrating fold change ( FC ; log2 ) in gene expression intensities compared with P - value ( − log2 ) between CCR9hi - CCR9lo - treated TCs . Horizontal bar at y = 4 . 32 represents a statistical significance of P = 0 . 05 ( genes in gray below this did not reach significance ) . LogFC cutoff at ± 0 . 5 is represented by vertical lines . representation of top upregulated ( LogFC > 0 . 5 ) downregulated ( LogFC < − 0 . 85 ) genes ( F ) with P ≤ 0 . 05 . replicates per sample group are shown herein .

O O O O O O O O O B-CONTROLLED_VAR 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 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 O O O O 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 MCF7 cells transfected with control siRNA (CCR9hi) or CCR9 siRNA (CCR9lo) were cocultured with survivin TCs for 12 h. Gene microarray was performed with the total RNA extracted from purified T cells after the coculture. Volcano plot (E) illustrating fold change (FC; log2) in gene expression intensities compared with P-value (−log2) between CCR9hi- and CCR9lo-treated TCs. Horizontal bar at y = 4.32 represents a statistical significance of P = 0.05 (genes in gray below this line did not reach significance). LogFC cutoff at ± 0.5 is represented by the vertical lines. Heatmap representation of the top upregulated (LogFC > 0.5) and downregulated (LogFC < −0.85) genes (F) with P ≤ 0.05. Individual replicates per sample group are shown herein.


A Cr - release showing TIL 209 - mediated lysis of CCR9 + M579 - A2 ( transduced with control shRNA ) or CCR9 − M579 - A2 ( transduced with CCR9 - specific shRNA ) . Curves represent mean ± .

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A Cr-release assay showing TIL 209-mediated lysis of CCR9+ M579-A2 (transduced with control shRNA) or CCR9− M579-A2 cells (transduced with CCR9-specific shRNA). Curves represent mean ± SEM.


C , D Tumorgrowth curves showing mean ± SEMtumor volume of CCR9 + or CCR9 − M579 - A2tumors in TIL - treated mice ( C ) or PBS alone group ( D ) . Statistical difference was calculated using unpaired one - sided Mann - Whitney U - .

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C, D Tumorgrowth curves showing mean ± SEMtumor volume of CCR9+ or CCR9−M579-A2tumors in TIL-treated mice (C) or the PBS alone group (D). Statistical difference was calculated using the unpaired one-sided Mann-Whitney U-test.


A ) SH - SY5Y neuroblastoma were either cultivated in RPMI + 10 % FCS or starved in HBSS with or without addition of LY294002 or rapamycin . After 16 h PINK1 mRNA expression was determined by RT - qPCR PINK1 mRNA content of kept in RPMI + 10 % FCS was set as 1 . Induction of autophagy by starvation or rapamycin resulted in an induction of PINK1 expression , comparable to positive control LY294002 ; n = 4 ; * expression changes compared to PINK1 expression in RPMI + 10 % FCS : LY294002 : p < 0 . 01 ; rapamycin : p < 1 × 10 − 4 ; HBSS : p < 0 . 005 ; HBSS + LY294002 : p < 5 × 10 − 6 ; HBSS + rapamycin : p < 0 . 0005 ; # expression changes compared to PINK1 expression in RPMI + 10 % FCS + LY294002 : p < 0 . 05 ; + expression changes compared to PINK1 expression in RPMI + 10 % FCS + rapamycin : p < 0 . 0005 .

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

A) SH-SY5Y neuroblastoma cells were either cultivated in RPMI+10% FCS or starved in HBSS with or without addition of LY294002 or rapamycin. After 16 h PINK1 mRNA expression was determined by RT-qPCR and the PINK1 mRNA content of cells kept in RPMI+10% FCS was set as 1. Induction of autophagy by starvation or rapamycin resulted in an induction of PINK1 expression, comparable to the positive control LY294002; n = 4; * expression changes compared to the PINK1 expression in RPMI+10% FCS: LY294002: p<0.01; rapamycin: p<1×10−4; HBSS: p<0.005; HBSS+LY294002: p<5×10−6; HBSS+ rapamycin: p<0.0005; # expression changes compared to the PINK1 expression in RPMI+10% FCS+LY294002: p<0.05; + expression changes compared to the PINK1 expression in RPMI+10% FCS+rapamycin: p<0.0005.


B ) SH - SY5Y were either stably transduced with a control ( nt ) shRNA or a shRNA directed against PINK1 cultivated in RPMI medium containing 5 % or 10 % FCS . Their PINK1 mRNA content was determined by RT - qPCR PINK1 mRNA content of nt kept in medium with 10 % FCS was set as 1 . Serum reduction increased PINK1 mRNA in nt in accordance with data shown in Fig . 1A , while stable PINK1 knockdown ( kd ) reduced PINK1 content under both conditions ; n = 3 ; * expression changes compared to PINK1 expression in RPMI + 10 % FCS : PINK1 kd 10 % FCS p < 0 . 0005 ; PINK1 induction by 5 % FCS : p < 0 . 05 ; # expression changes compared to PINK1 expression in RPMI + 5 % FCS : PINK1 kd 5 % FCS : p < 0 . 005 .

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

B) SH-SY5Y cells were either stably transduced with a control (nt) shRNA or a shRNA directed against PINK1 and cultivated in RPMI medium containing 5% or 10% FCS. Their PINK1 mRNA content was determined by RT-qPCR and PINK1 mRNA content of nt cells kept in medium with 10% FCS was set as 1. Serum reduction increased PINK1 mRNA in nt cells in accordance with the data shown in Fig. 1A, while stable PINK1 knockdown (kd) reduced PINK1 content under both conditions; n = 3; * expression changes compared to the PINK1 expression in RPMI+10% FCS: PINK1 kd 10% FCS p<0.0005; PINK1 induction by 5% FCS: p<0.05; # expression changes compared to the PINK1 expression in RPMI+5% FCS: PINK1 kd 5% FCS: p<0.005.


C ) SH - SY5Y without ( nt ) or with stable PINK1 knockdown ( kd ) were kept in medium with 5 % FCS either untreated or treated for 2 h with CCCP to stabilize PINK1 . Afterwards PINK1 63 kDa ( arrowhead ) actin were determined by western blotting ( see representative gel on right ) . revealed a reduction of PINK1 under both conditions in PINK1 kd .

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

C) SH-SY5Y cells without (nt) or with stable PINK1 knockdown (kd) were kept in medium with 5% FCS and either untreated or treated for 2 h with CCCP to stabilize PINK1. Afterwards the PINK1 63 kDa protein (arrowhead) and actin protein levels were determined by western blotting (see representative gel on the right). The quantification revealed a reduction of PINK1 protein under both conditions in PINK1 kd cells.


2A ) SH - SY5Y were starved for 2 h in HBSS with Bafilomycin ( + Baf ) or left untreated in RPMI + 5 % FCS ( - Baf ) . LC3 - II actin content were determined by western blotting . A representative blot is shown on right , showing only LC3 - II bands in Bafilomycin - treated samples . LC3 - II bands of Bafilomycin treated samples were normalized to actin relative LC3 - II content of nt was set as 1 . with stable PINK1 knockdown exhibited a reduced LC3 - II / actin compared to control ( nt ) ; n = 4 , p < 0 . 005 .

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

2A) SH-SY5Y cells were starved for 2 h in HBSS with Bafilomycin (+Baf) or left untreated in RPMI+5% FCS (- Baf). The LC3-II and actin content were determined by western blotting. A representative blot is shown on the right, showing only LC3-II bands in the Bafilomycin-treated samples. The LC3-II bands of Bafilomycin treated samples were normalized to actin and the relative LC3-II content of nt cells was set as 1. Cells with stable PINK1 knockdown exhibited a reduced LC3-II/actin ratio compared to the control (nt) cells; n = 4, p<0.005.


2B ) Cortical neurons from 3 different isolations ( 10 - 20 DIV ) of WT PINK1 KO mice were either non - starved or starved for 2 h in HBSS LC3 - II actin content was determined by western blotting . A representative blot is shown on right . relative LC3 - II content of WT PINK1 KO mice , respectively , was set as 1 . neurons showed a strong upregulation of autophagy in reaction to starvation but derived from PINK1 KO mice exhibited a reduced LC3 - II / actin compared to WT ; n = 5 , p < 0 . 001 .

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

2B) Cortical neurons from 3 different isolations (10-20 DIV) of WT and PINK1 KO mice were either non-starved or starved for 2 h in HBSS and the LC3-II and actin content was determined by western blotting. A representative blot is shown on the right. The relative LC3-II content of WT and PINK1 KO mice, respectively, was set as 1. Primary neurons showed a strong upregulation of autophagy in reaction to starvation but cells derived from PINK1 KO mice exhibited a reduced LC3-II/actin ratio compared to the WT cells; n = 5, p<0.001.


2C ) HeLa were transiently transfected with scrambled siRNA or PINK1 siRNA . After 48 h LC3 - II actin content was determined by western blotting . A representative blot is shown on right . relative LC3 - II content of transfected with scrambled siRNA was set as 1 . Transient PINK1 knockdown resulted in a reduced LC3 - II / actin compared to transfected with scrambled siRNA ; n = 6 ; p < 0 . 005 .

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

2C) HeLa cells were transiently transfected with scrambled siRNA or PINK1 siRNA. After 48 h the LC3-II and actin content was determined by western blotting. A representative blot is shown on the right. The relative LC3-II content of cells transfected with scrambled siRNA was set as 1. Transient PINK1 knockdown resulted in a reduced LC3-II/actin ratio compared to the cells transfected with scrambled siRNA; n = 6; p<0.005.


2D ) HeLa were transiently transfected with PINK1 - GFP , PINK1G309D - GFP or GFP . After 24 h were starved for 2 h in HBSS . Afterwards LC3 - II actin content were determined by western blotting . A representative blot is shown on right . relative LC3 - II content of transfected with GFP was set as 1 . Transient PINK1 or PINK1G309D overexpression resulted in an increased LC3 - II / actin compared to transfected with GFP ; n = 3 , PINK1 : p < 0 . 01 ; PINK1G309D : p < 0 . 05 .

O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 B-CONTROLLED_VAR O O O O O O O

2D) HeLa cells were transiently transfected with PINK1-GFP, PINK1G309D-GFP or GFP. After 24 h cells were starved for 2 h in HBSS. Afterwards the LC3-II and actin content were determined by western blotting. A representative blot is shown on the right. The relative LC3-II content of cells transfected with GFP was set as 1. Transient PINK1 or PINK1G309D overexpression resulted in an increased LC3-II/actin ratio compared to cells transfected with GFP; n = 3, PINK1: p<0.01; PINK1G309D: p<0.05.


3A ) Regular , non - transduced SH - SY5Y were cultivated either in RPMI + 10 % FCS or starved in HBSS medium for 24 40 h . LAMP - 2 actin content were determined by western blotting relative LAMP - 2 content of untreated was set as 1 . Starvation enhanced LAMP - 2protein ; n = 3 , p < 0 . 05 .

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

3A) Regular, non-transduced SH-SY5Y cells were cultivated either in RPMI+10% FCS or starved in HBSS medium for 24 and 40 h. The LAMP-2 and actin content were determined by western blotting and the relative LAMP-2 content of untreated cells was set as 1. Starvation enhanced LAMP-2protein levels; n = 3, p<0.05.


3B ) Transduced nt PINK1 knockdown ( kd ) SH - SY5Y were cultivated either in RPMI + 5 % FCS or starved for 24 h 40 h in HBSS . LAMP - 2 actin content were determined by western blotting . A representative blot after 40 h starvation in HBSS is shown on right . relative LAMP - 2 content of untreated was set as 1 . with stable PINK1 knockdown exhibited after 40 h a reduced LAMP - 2 / actin compared to nt ; n = 4 , p < 0 . 05 .

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

3B) Transduced nt and PINK1 knockdown (kd) SH-SY5Y cells were cultivated either in RPMI+5% FCS or starved for 24 h and 40 h in HBSS. The LAMP-2 and actin content were determined by western blotting. A representative blot after 40 h starvation in HBSS is shown on the right. The relative LAMP-2 content of untreated cells was set as 1. Cells with stable PINK1 knockdown exhibited after 40 h a reduced LAMP-2/actin ratio compared to the nt cells; n = 4, p<0.05.


3C ) nt PINK1 knockdown ( kd ) SH - SY5Y were starved for indicated times with HBSS afterwards stained for Lamp - 2 . were taken with constant microscopical settings . LAMP - 2 positive signals in each were quantified with ImageJ as described in Material Methods . Knockdown of PINK1 resulted in a decreased amount of LAMP - 2 positive stainings / after starvation ; n = 2 , at least 8 fields of view / condition , 89 - 124 / condition ; 24 h : p < 0 . 001 ; 40 h : p < 0 . 05 .

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

3C) nt and PINK1 knockdown (kd) SH-SY5Y cells were starved for the indicated times with HBSS and afterwards stained for Lamp-2. Micrographs were taken with constant microscopical settings. LAMP-2 positive signals in each cell were quantified with ImageJ as described in Material and Methods. Knockdown of PINK1 resulted in a decreased amount of LAMP-2 positive stainings/cell after starvation; n = 2, at least 8 fields of view/condition, 89-124 cells/condition; 24 h: p<0.001; 40 h: p<0.05.


3D ) nt PINK1 knockdown ( kd ) SH - SY5Y were stained with photo - reactive dye MTR either irradiated for 45 min or left untreated . 24 h 48 h after irradiation LAMP - 2 actin content were determined by western blotting . A representative blot 48 h after irradiation is shown on right . relative LAMP - 2 content of untreated was set as 1 . with stable PINK1 knockdown exhibited after 48 h a reduced Lamp - 2 / actin compared to control ( nt ) ; n = 5 , p < 0 . 001 .

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

3D) nt and PINK1 knockdown (kd) SH-SY5Y cells were stained with the photo-reactive dye MTR and either irradiated for 45 min or left untreated. 24 h and 48 h after irradiation the LAMP-2 and actin content were determined by western blotting. A representative blot 48 h after irradiation is shown on the right. The relative LAMP-2 content of untreated cells was set as 1. Cells with stable PINK1 knockdown exhibited after 48 h a reduced Lamp-2/actin ratio compared to the control (nt) cells; n = 5, p<0.001.


3E ) Acid phosphatase activity as parameter for lysosomal activity was quantified in nt PINK1 knockdown ( kd ) SH - SY5Y cultivated in RPMI medium with 5 % FCS . phosphatase activity was measured normalized to content . phosphatase activity of nt was set as 1 . PINK1 knockdown mediated a reduction of lysosomal activity ; n = 3 ; p < 0 . 05 .

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3E) Acid phosphatase activity as parameter for lysosomal activity was quantified in nt and PINK1 knockdown (kd) SH-SY5Y cells cultivated in RPMI medium with 5% FCS. The phosphatase activity was measured and normalized to the protein content. The phosphatase activity of nt cells was set as 1. PINK1 knockdown mediated a reduction of lysosomal activity; n = 3; p<0.05.


4A ) Oxygen consumption rate ( OCR ) was measured in nt PINK1 knockdown ( kd ) SH - SY5Y without ( nt ) or with PINK1 knockdown ( kd ) cultivated in RPMI medium with 5 % FCS . Although oxygen consumption appeared to be slightly impaired in with PINK1 kd , no significant changes were observed ; n = 1 ( quadruplicates ) .

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

4A) Oxygen consumption rate (OCR) was measured in nt and PINK1 knockdown (kd) SH-SY5Y cells without (nt) or with PINK1 knockdown (kd) cultivated in RPMI medium with 5% FCS. Although oxygen consumption appeared to be slightly impaired in cells with PINK1 kd, no significant changes were observed; n = 1 (quadruplicates).


4B ) Energy charge ( EC ) of nt PINK1 knockdown ( kd ) SH - SY5Y grown in RPMI + 5 % FCS was determined as detailed in Material Methods . PINK1 knockdown resulted in a lower EC compared to nt ; n = 5 , p0 . 05 .

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4B) Energy charge (EC) of nt and PINK1 knockdown (kd) SH-SY5Y cells grown in RPMI+5% FCS was determined as detailed in Material Methods. PINK1 knockdown resulted in a lower EC compared to nt cells; n = 5, p0.05.


4C ) nt PINK1 knockdown ( kd ) SH - SY5Y were grown for 2 months in RPMI + 5 % FCS their population doublings were determined . Starting from day 12 with PINK1 kd exhibited impaired population growth ; n = 1 ( triplicates ) ; * p < 0 . 05 ( day 12 ) - p < 1 × 10 − 6 ( day 61 ) .

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 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

4C) nt and PINK1 knockdown (kd) SH-SY5Y cells were grown for 2 months in RPMI+5% FCS and their population doublings were determined. Starting from day 12 cells with PINK1 kd exhibited impaired cell population growth; n = 1 (triplicates); * p<0.05 (day 12) - p<1×10−6 (day 61).


4D ) SH - SY5Y were grown in RPMI + 5 % FCS amount of nt PINK1 knockdown ( kd ) in S - phase was determined by BrdU incorporation . No differences in proliferation were visible between without ( nt ) with PINK1 kd ; n = 3 .

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 B-CONTROLLED_VAR O O O O O O O O

4D) SH-SY5Y cells were grown in RPMI+5% FCS and the amount of nt and PINK1 knockdown (kd) cells in S-phase was determined by BrdU incorporation. No differences in cell proliferation were visible between cells without (nt) and with PINK1 kd; n = 3.


5A ) nt PINK1 knockdown ( kd ) SH - SY5Y were either kept in RPMI medium with 5 % FCS or starved with HBSS for 12 h . DEVD cleavage as parameter for caspase - 3 activity was analyzed normalized to content . with PINK1 knockdown demonstrated a significantly increased caspase - 3 activity under both conditions ; n = 4 ; RPMI + 5 % FCS : p < 0 . 05 ; HBSS : p < 0 . 01 .

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

5A) nt and PINK1 knockdown (kd) SH-SY5Y cells were either kept in RPMI medium with 5% FCS or starved with HBSS for 12 h. DEVD cleavage as parameter for caspase-3 activity was analyzed and normalized to the protein content. Cells with PINK1 knockdown demonstrated a significantly increased caspase-3 activity under both conditions; n = 4; RPMI+5% FCS: p<0.05; HBSS: p<0.01.


5B ) nt PINK1 knockdown ( kd ) SH - SY5Y were either kept in RPMI medium with 5 % FCS for 48 h or starved with HBSS for indicated time points . In representative western blot appearance of PARP 89 kDa product is depicted as well as GAPDH for normalizing .

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5B) nt and PINK1 knockdown (kd) SH-SY5Y cells were either kept in RPMI medium with 5% FCS for 48 h or starved with HBSS for the indicated time points. In the representative western blot the appearance of the PARP 89 kDa cleaved product is depicted as well as GAPDH for normalizing.


5C ) nt PINK1 knockdown ( kd ) SH - SY5Y were either kept in RPMI medium with 5 % FCS for 48 h or starved with HBSS for indicated time points . For PARP 89 kDa product was normalized to full 113 kDa PARP resulting value normalized to GAPDH . with PINK1 knockdown demonstrated increased PARP cleavage that was significant after 24 h starvation ; n = 4 ; 24 h : p < 0 . 005 .

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5C) nt and PINK1 knockdown (kd) SH-SY5Y cells were either kept in RPMI medium with 5% FCS for 48 h or starved with HBSS for the indicated time points. For quantification the cleaved PARP 89 kDa product was normalized to the full length 113 kDa PARP and the resulting value normalized to GAPDH. Cells with PINK1 knockdown demonstrated increased PARP cleavage that was significant after 24 h starvation; n = 4; 24 h: p<0.005.


5D ) HeLa were transfected with scrambled siRNA or PINK1 siRNA GFP or GFP - Parkin or PINK1 - GFP or PINK1G309D - GFP . After transfection were starved for additional 24 h with HBSS afterwards amount of adherent was determined . number of adherent transfected with scrambled siRNA indicated plasmid was set as 1 . Starvation - mediated increased loss after PINK1 knockdown could be rescued by PINK1 or PINK1G309D - GFP but not by GFP or Parkin ; GFP : n = 5 , p < 0 . 005 ; Parkin : n = 3 , p < 0 . 05 ; PINK1 : n = 5 , ns ; PINK1G309D - GFP : n = 3 , ns .

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5D) HeLa cells were transfected with scrambled siRNA or PINK1 siRNA and GFP or GFP-Parkin or PINK1-GFP or PINK1G309D-GFP. After transfection cells were starved for additional 24 h with HBSS and afterwards the amount of adherent cells was determined. The number of adherent cells transfected with scrambled siRNA and the indicated plasmid was set as 1. Starvation-mediated increased cell loss after PINK1 knockdown could be rescued by PINK1 or PINK1G309D-GFP but not by GFP or Parkin; GFP: n = 5, p<0.005; Parkin: n = 3, p<0.05; PINK1: n = 5, ns; PINK1G309D-GFP: n = 3, ns.


5E ) HeLa HeLa stably overexpressing LC3 were transfected with scrambled siRNA or PINK1 siRNA . After 48 h were starved in HBSS for additional 24 h afterwards DEVD cleavage as parameter for caspase - 3 activity was analyzed normalized to 1 , 000 , 000 . PINK1 knockdown increased caspase - 3 activity significantly , which was prevented by LC3 overexpression ; n = 3 ; p < 0 . 05 .

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5E) HeLa cells and HeLa cells stably overexpressing LC3 were transfected with scrambled siRNA or PINK1 siRNA. After 48 h cells were starved in HBSS for additional 24 h and afterwards DEVD cleavage as parameter for caspase-3 activity was analyzed and normalized to 1,000,000 cells. PINK1 knockdown increased caspase-3 activity significantly, which was prevented by LC3 overexpression; n = 3; p<0.05.


5F ) HeLa HeLa stably expressing LC3 were transfected with scrambled or PINK1 siRNA . 48 h post transfection were either left untreated ( + medium ) or starved ( + HBSS ) for additional 24 h afterwards amount of adherent was determined . amount of non - starved was set as 1 . PINK1 knockdown resulted in elevated loss , which was prevented by LC3 overexpression ; HeLa : n = 6 , p < 0 . 05 ; HeLa LC3 : n = 5 .

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5F) HeLa cells and HeLa cells stably expressing LC3 were transfected with scrambled or PINK1 siRNA. 48 h post transfection cells were either left untreated (+ medium) or starved (+HBSS) for additional 24 h and afterwards the amount of adherent cells was determined. The amount of non-starved cells was set as 1. PINK1 knockdown resulted in elevated cell loss, which was prevented by LC3 overexpression; HeLa: n = 6, p<0.05; HeLa LC3: n = 5.


fragments in senescent . ( A ) fragments ( CCFs ) in senescent are strongly positive for histone H3 . Yellow arrow marks CCF . ( B ) Increased proportion of with CCFs in RS or OIS . Mean ± , n = 3 ; P < 0 . 01 ( OIS ) , P < 0 . 002 ( RS ) . Assays were performed 10 d after induction with tamoxifen ( OIS ) or 4 wk after last passage > 90 % SA β - gal + ( RS ) .

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Cytoplasmic chromatin fragments in senescent cells. (A) Cytoplasmic chromatin fragments (CCFs) in senescent cells are strongly positive for histone H3. Yellow arrow marks CCF. (B) Increased proportion of cells with CCFs in RS or OIS. Mean ± SEM, n = 3; P < 0.01 (OIS), P < 0.002 (RS). Assays were performed 10 d after induction with tamoxifen (OIS) or 4 wk after the last passage and >90% SA β-gal+ (RS).


( C ) CCFs in OIS are lamin A / C negative . ( D ) from C were scored for lamin A / C + lamin A / C − CCFs . Note that majority of micronuclei in control are lamin A / C positive . Mean ± , n = 3 ; P < 0 . 0006 for lamin A / C negative CCF con vs . OIS ; P < 0 . 6 for lamin A / C positive CCF con vs . OIS .

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(C) CCFs in OIS cells are lamin A/C negative. (D) Cells from C were scored for lamin A/C+ and lamin A/C− CCFs. Note that the majority of micronuclei in control cells are lamin A/C positive. Mean ± SEM, n = 3; P < 0.0006 for lamin A/C negative CCF con vs. OIS; P < 0.6 for lamin A/C positive CCF con vs. OIS.


( E ) CCFs in OIS are strongly positive for heterochromatic histone mark H3K27me3 but not for euchromatic mark H3K9ac . ( F ) from E were scored for indicated histone modifications in CCFs . A single representative experiment is shown at least 100 were scored per slide . Note majority of micronuclei in control are positive for both marks .

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(E) CCFs in OIS are strongly positive for heterochromatic histone mark H3K27me3 but not for euchromatic mark H3K9ac. (F) Cells from E were scored for indicated histone modifications in CCFs. A single representative experiment is shown and at least 100 cells were scored per slide. Note the majority of micronuclei in control cells are positive for both marks.


( G ) CCFs in RS contain γ - H2AX but not 53BP1 ( top ) . Intranuclear γ - H2AX foci colocalize with 53BP1 ( bottom ) . ( H ) CCF + CCF − from G were scored for colocalizing intranuclear γ - H2AX 53BP1 foci . Mean ± , n = 3 ; P < 0 . 019 . Bars ( in IF panels ) , 10 µm .

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(G) CCFs in RS contain γ-H2AX but not 53BP1 (top). Intranuclear γ-H2AX foci colocalize with 53BP1 (bottom). (H) CCF+ and CCF− cells from G were scored for colocalizing intranuclear γ-H2AX and 53BP1 foci. Mean ± SEM, n = 3; P < 0.019. Bars (in IF panels), 10 µm.


- to - cytoplasm blebbing in senescent associated with depletion of lamin B1 . ( A ) Extrusion of GFP - H2B - positive fragment from nonmitotic RS nucleus . Confocal time - lapse microscopy of transiently transfected GFP - H2B - expressing , 60 × original magnification , 150 nm optical section , time in mins . Bars : 10 µm ; ( insert ) 2 µm .

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Nuclear-to-cytoplasm chromatin blebbing in senescent cells associated with depletion of lamin B1. (A) Extrusion of GFP-H2B-positive chromatin fragment from nonmitotic RS nucleus. Confocal time-lapse microscopy of transiently transfected GFP-H2B-expressing cell, 60× original magnification, 150 nm optical section, time in mins. Bars: 10 µm; (insert) 2 µm.


( B ) fragments blebbing out of nucleus are γ - H2AX positive . Bars : ( top ) 10 µm ; ( bottom ) 5 µm .

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(B) Chromatin fragments blebbing out of nucleus are γ-H2AX positive. Bars: (top) 10 µm; (bottom) 5 µm.


( C ) Down - regulation of lamin B1 lamin B receptor ( LBR ) in RS , assayed by Western blot . See Materials methods for time - course details .

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(C) Down-regulation of lamin B1 and lamin B receptor (LBR) in RS cells, assayed by Western blot. See Materials and methods for time-course details.


( D ) Depletion of lamin B1 from OIS , assayed by immunofluorescence . ( E ) A - scan of lamin B1 along arrows in D . Data shown are from a single representative experiment out of three repeats .

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(D) Depletion of lamin B1 from OIS cells, assayed by immunofluorescence. (E) A line-scan of lamin B1 fluorescence intensity along the arrows in D. Data shown are from a single representative experiment out of three repeats.


. ( F ) Depletion of lamin B1 in BRAFV600E - induced OIS melanocytes in vitro . Bars , 10 µm . ( G ) Quantitative immunofluorescence of lamin B1 in melanocytes from F . A representative experiment out of two repeats is shown . At least 150 randomly selected were assessed .

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. (F) Depletion of lamin B1 in BRAFV600E-induced OIS melanocytes in vitro. Bars, 10 µm. (G) Quantitative immunofluorescence of lamin B1 in melanocytes from F. A representative experiment out of two repeats is shown. At least 150 randomly selected cells were assessed.


( H ) Senescent nevusmelanocytes ( n ) stain less intensely for lamin B1 as compared with nonsenescent melanocytes in epidermis ( e ) . dotted shows basement between dermis epidermis . Bottom panels ( Epi Nevus ) are expanded from boxed areas on top . Confocal microscopy for DAPI ( blue ) , MelanA ( red ) , lamin B1 ( green ) stained tissue . Bars , 20 µm . ( I ) Quantitation of results from H for three different nevi . Epifluorescence of lamin B1 - MelanA - stained nevi were obtained epidermal or nevusMelanA - positive were scored for presence ( positive ) of a characteristic lamin B1 " ring . " At least 50 MelanA - expressing epidermalmelanocytes at least 100 nevusmelanocytes were scored . Average of three different nevi ± ; P < 0 . 0005 .

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(H) Senescent nevusmelanocytes (n) stain less intensely for lamin B1 as compared with nonsenescent melanocytes in epidermis (e). White dotted line shows the basement membrane between the dermis and epidermis. Bottom panels (Epi and Nevus) are expanded from white boxed areas on top. Confocal microscopy for DAPI (blue), MelanA (red), and lamin B1 (green) stained tissue. Bars, 20 µm. (I) Quantitation of results from H for three different nevi. Epifluorescence images of lamin B1- and MelanA-stained nevi were obtained and epidermal or nevusMelanA-positive cells were scored for the presence (positive) of a characteristic lamin B1 "ring." At least 50 MelanA-expressing epidermalmelanocytes and at least 100 nevusmelanocytes were scored. Average of three different nevi ± SEM; P < 0.0005.


Blebbing of from nucleus to cytoplasm in senescent is associated with loss of envelope integrity . ( A ) Herniation of H3K27me3 - positive into cytoplasm of OIS . Yellow boxed is magnified in bottom panels . Bars : ( top panels ) 10 µm ; ( bottom panels ) 5 µm . ( B ) Quantitation of lamin B1 gaps or herniations in OIS . Mean ± , n = 2 ; P < 0 . 01 .

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Blebbing of chromatin from nucleus to cytoplasm in senescent cells is associated with loss of nuclear envelope integrity. (A) Herniation of H3K27me3-positive chromatin into the cytoplasm of OIS cells. Yellow boxed area is magnified in bottom panels. Bars: (top panels) 10 µm; (bottom panels) 5 µm. (B) Quantitation of lamin B1 gaps or herniations in OIS. Mean ± SEM, n = 2; P < 0.01.


( C ) Isolated nuclei of senescent are permeable to both 70 - 500 - kD dextrans , indicating impairment of barrier function of envelope . nuclei exclude dextran . Bars , 10 µm . ( D ) Quantitation of results from C . Mean ± SD , n = 4 ; P < 0 . 000001 for both 70 - 500 - kD dextrans .

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(C) Isolated nuclei of senescent cells are permeable to both 70- and 500-kD dextrans, indicating impairment of the barrier function of nuclear envelope. Dark nuclei exclude fluorescent dextran. Bars, 10 µm. (D) Quantitation of results from C. Mean ± SD, n = 4; P < 0.000001 for both 70- and 500-kD dextrans.


( E ) Representative OIS SAHF + nucleus showing dextran permeability . Bars , 10 µm . ( F ) Quantitation of dextran 70 uptake in SAHF + SAHF − OIS . P < 1 . 9 × 10 − 5 . ( G ) In nuclei , extrusion dextran intrusion occurred in close physical proximity . Right - panels are expanded from yellow boxed areas on left . Bars : ( left ) 10 µm ; ( right ) 2 µm .

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(E) Representative OIS SAHF+ nucleus showing dextran permeability. Bars, 10 µm. (F) Quantitation of dextran 70 uptake in SAHF+ and SAHF− OIS cells. P < 1.9 × 10−5. (G) In individual nuclei, chromatin extrusion and dextran intrusion occurred in close physical proximity. Right-hand panels are expanded from yellow boxed areas on the left. Bars: (left) 10 µm; (right) 2 µm.


histone is processed by a lysosomal / autophagy pathway . ( A ) Close juxtaposition of senescence - associated CCF ( RS ) with p62 bodies . Bars : 10 µm ; ( inset ) 1 µm . ( B ) Quantitation of control OIS with CCF overlapping p62 ( p62 + ) or not ( p62 − ) . Mean ± , n = 3 .

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Cytoplasmic histone is processed by a lysosomal/autophagy pathway. (A) Close juxtaposition of senescence-associated CCF (RS) with p62 nuclear bodies. Bars: 10 µm; (inset) 1 µm. (B) Quantitation of control and OIS cells with CCF overlapping p62 (p62+) or not (p62−). Mean ± SEM, n = 3.


( C ) Quantitation of proliferating RS with CCF overlapping p62 ( p62 + ) or not ( p62 − ) ubiquitination ( FK2 + ) or not ( FK2 − ) . Mean ± , n = 3 ; P < 0 . 0008 for FK2 + / p62 + CCF , comparing proliferating RS . ( D ) Ubiquitinated appear to surface in CCF . Yellow boxed areas are magnified in right - panels . Bars : ( left panels ) 10 µm ; ( right panels ) 1 µm .

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(C) Quantitation of proliferating and RS cells with CCF overlapping p62 (p62+) or not (p62−) and protein ubiquitination (FK2+) or not (FK2−). Mean ± SEM, n = 3; P < 0.0008 for FK2+/p62+ CCF, comparing proliferating and RS cells. (D) Ubiquitinated proteins appear to line chromatin surface in CCF. Yellow boxed areas are magnified in right-hand panels. Bars: (left panels) 10 µm; (right panels) 1 µm.


( E ) Apparent activation of cathepsin L in RS IMR90 . In contrast to RS , majority of cathepsin L in proliferating control exists in its inactive pro - cathepsin L form . Loading was normalized by number lamin A / C was used as a loading control . See Materials methods for details of time course .

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(E) Apparent activation of cathepsin L in RS IMR90 cells. In contrast to RS cells, the majority of cathepsin L in proliferating control cells exists in its inactive pro-cathepsin L form. Loading was normalized by cell number and lamin A/C was used as a loading control. See Materials and methods for details of time course.


( F ) Accumulation of H3cs . 1 , a specific N - terminal cleavage product of H3 , in OIS . See Materials methods for details of time course . Western blotting was performed using same lysates as in Fig . S4 B , same lamin A / C panel is shown in both figures .

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(F) Accumulation of H3cs.1, a specific N-terminal cleavage product of H3, in OIS cells. See Materials and methods for details of time course. Western blotting was performed using the same cellular lysates as in Fig. S4 B, and the same lamin A/C panel is shown in both figures.


( G ) Accumulation of H3cs . 1 corresponding high mobility H3 in OIS .

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(G) Accumulation of H3cs.1 and corresponding high mobility H3 in OIS.


( H ) Predominant localization of H3cs . 1 in senescent . α - Tubulin lamin A / C were used as fractionation controls for fractions , respectively .

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(H) Predominant cytoplasmic localization of H3cs.1 in senescent cells. α-Tubulin and lamin A/C were used as fractionation controls for cytoplasmic and nuclear fractions, respectively.


Senescent in vitro in vivo contain reduced histone content . ( A ) Immunofluorescent confocal of histone H3 in control or OIS ( 11 d after activation of ER - RASG12V ) . Yellow arrows indicate with most pronounced SAHF . ( B ) Quantitative immunofluorescence of histone H3 in RS OIS . Representative of two independent experiments .

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Senescent cells in vitro and in vivo contain reduced histone content. (A) Immunofluorescent confocal images of histone H3 in control or OIS cells (11 d after activation of ER-RASG12V). Yellow arrows indicate cells with most pronounced SAHF. (B) Quantitative immunofluorescence analysis of histone H3 in RS and OIS cells. Representative of two independent experiments.