Figure 3C . Endogenous MITOL translocates to the ER in later phase of mitophagy . EGFP - MITOL knock in HeLa cells were transfected with HA - Parkin and treated with DMSO or CCCP ( 10 µM ) as indicated times . Cells were fixed , permeabilized and subjected to immunofluorescence analysis with the indicated antibodies . Colocalization was quantified by Manders ' s coefficient . Means ± SEM of more than 10 cells obtained from three independent experiments . For statistical analysis , a one - way ANOVA with Tukey ' s multiple comparisons test were performed , * * * * P < 0 . 0001 . Scale bar represents 1 μm . E . ER - localized MITOL is transported from the mitochondria , and is not newly synthesized protein . HeLa cells stably expressing HA - Parkin were co - transfected with vectors for MITOL - KikGR and FLAG - FKBP38 . After ultraviolet light ( 365 nm ) exposure to the whole plate , cells were incubated with or without CCCP ( 10 µM ) for indicated time , and monitored for red and green KikGR fluorescence . Scale bar , 10 μm . Higher magnification images of the boxed regions are shown in the small panel . Right panel is schematic experimental model for KikGR in this study .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3C. Endogenous MITOL translocates to the ER in later phase of mitophagy. EGFP-MITOL knock in HeLa cells were transfected with HA-Parkin and treated with DMSO or CCCP (10 µM) as indicated times. Cells were fixed, permeabilized and subjected to immunofluorescence analysis with the indicated antibodies. Colocalization was quantified by Manders's coefficient. Means ± SEM of more than 10 cells obtained from three independent experiments. For statistical analysis, a one-way ANOVA with Tukey's multiple comparisons test were performed, ****P < 0.0001. Scale bar represents 1 μm.E. ER-localized MITOL is transported from the mitochondria, and is not newly synthesized protein. HeLa cells stably expressing HA-Parkin were co-transfected with vectors for MITOL-KikGR and FLAG-FKBP38. After ultraviolet light (365 nm) exposure to the whole plate, cells were incubated with or without CCCP (10 µM) for indicated time, and monitored for red and green KikGR fluorescence. Scale bar, 10 μm. Higher magnification images of the boxed regions are shown in the small panel. Right panel is schematic experimental model for KikGR in this study.


Figure 4C . Phosphorylated Parkin accumulates in the ER fraction in the absence of MITOL . WT or MITOL KO HeLa cells stably expressing HA - Parkin were treated with DMSO or CCCP ( 10 µM ) for 48 h . Lysates of cells were fractionated into cytosolic , mitochondrial and ER fractions , and then subjected to an IB assay with the indicated antibodies . Phospho - Ser65 - Parkin is detected by using specific antibody . Right panels are the quantification of IB about Phospho - Ser65 - Parkin protein levels of the ER fraction . The data represent the mean ± SD for three independent experiments . For statistical analysis , a one - way ANOVA with Tukey post - test was performed , * * P < 0 . 01 . D . Parkin is detected at the ER in the absence of MITOL . HeLa cells were transfected with vectors for KikGR - Parkin . After ultraviolet light ( 365 nm ) exposure to the whole plate , cells were incubated with or without CCCP ( 10 µM ) for indicated time , and monitored for red and green KikGR fluorescence . Scale bar , 10 μm . Higher magnification images of the boxed regions are shown in the small panel .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4C. Phosphorylated Parkin accumulates in the ER fraction in the absence of MITOL. WT or MITOL KO HeLa cells stably expressing HA-Parkin were treated with DMSO or CCCP (10 µM) for 48 h. Lysates of cells were fractionated into cytosolic, mitochondrial and ER fractions, and then subjected to an IB assay with the indicated antibodies. Phospho-Ser65-Parkin is detected by using specific antibody. Right panels are the quantification of IB about Phospho-Ser65-Parkin protein levels of the ER fraction. The data represent the mean ± SD for three independent experiments. For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01.D. Parkin is detected at the ER in the absence of MITOL. HeLa cells were transfected with vectors for KikGR-Parkin. After ultraviolet light (365 nm) exposure to the whole plate, cells were incubated with or without CCCP (10 µM) for indicated time, and monitored for red and green KikGR fluorescence. Scale bar, 10 μm. Higher magnification images of the boxed regions are shown in the small panel.


Figure 5D . MITOL KO increases degradation of ER targeting FKBP38 . HeLa cells stably expressing HA - Parkin were transfected with mitochondrial targeting FLAG - FKBP38 ( FKBP38ActA ) and ER targeting FLAG - FKBP38 ( FKBP38IYFFT ) , and treated with DMSO or CCCP ( 10 µM ) for 24 h . Lysates of cells were subjected to an IB assay with FLAG antibodies for organelle targeting FKBP38 or indicated antibodies . Another cell lysates were subsequently separated on a Phos - tag gel , followed by IB with anti - Parkin antibodies ( Phos - tag ) . E - G . MITOL partly prevents CCCP - induced cell death by reducing Parkin - mediated degradation of FKBP38 . WT or MITOL KO HeLa cells stably expressing HA - Parkin and HeLa cells were transfected with the indicated vectors , and treated with DMSO or CCCP ( 10 µM ) for 24 h . Cells were stained with propidium iodide ( PI ) for counting the percentage of dead cells using fluorescence microscopy . Data represent the mean ± SD of five independent experiments ( > 100 individual cells were counted ) . For statistical analysis , a one - way ANOVA with Tukey post - test was performed , * * P < 0 . 01 . KR , FKBP38 K271 / 273R .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5D. MITOL KO increases degradation of ER targeting FKBP38. HeLa cells stably expressing HA-Parkin were transfected with mitochondrial targeting FLAG-FKBP38 (FKBP38ActA) and ER targeting FLAG-FKBP38 (FKBP38IYFFT), and treated with DMSO or CCCP (10 µM) for 24 h. Lysates of cells were subjected to an IB assay with FLAG antibodies for organelle targeting FKBP38 or indicated antibodies. Another cell lysates were subsequently separated on a Phos-tag gel, followed by IB with anti-Parkin antibodies (Phos-tag).E-G. MITOL partly prevents CCCP-induced cell death by reducing Parkin-mediated degradation of FKBP38. WT or MITOL KO HeLa cells stably expressing HA-Parkin and HeLa cells were transfected with the indicated vectors, and treated with DMSO or CCCP (10 µM) for 24 h. Cells were stained with propidium iodide (PI) for counting the percentage of dead cells using fluorescence microscopy. Data represent the mean ± SD of five independent experiments (>100 individual cells were counted). For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01. KR, FKBP38 K271/273R.


Figure 6A . Mislocalization of Bcl - 2 is observed in MITOL KO cells in mitophagy . WT or MITOL KO HeLa cells stably expressing HA - Parkin were transfected with FLAG - Bcl2 and treated with DMSO or CCCP ( 10 µM ) for the indicated times . Cells were immunostained with indicated antibody . Scale bar , 1 μm . 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 .

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Figure 6A. Mislocalization of Bcl-2 is observed in MITOL KO cells in mitophagy. WT or MITOL KO HeLa cells stably expressing HA-Parkin were transfected with FLAG-Bcl2 and treated with DMSO or CCCP (10 µM) for the indicated times. Cells were immunostained with indicated antibody. Scale bar, 1 μm.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.


Figure 1 ( 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 .

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Figure 1(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.


Figure 2 ( 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 and T308 in response to miR - 155 and suppression of T308 phosphorylation in response to miR - 181b . ( 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 and PEO4 cells 48 hours post transfection with the indicated oligos . Cells were counterstained with phalloidin and DAPI . ( 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 ) 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 .

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Figure 2(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 and T308 in response to miR-155 and suppression of T308 phosphorylation in response to miR-181b.(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 and PEO4 cells 48 hours post transfection with the indicated oligos. Cells were counterstained with phalloidin and DAPI.(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) 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.


Figure 3 ( 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 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 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 and USP1 in the indicated cell lines . ( 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 cell lines .

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Figure 3(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 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 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 and USP1 in the indicated cell lines.(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 cell lines.


Figure 4 ( 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 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 cells were enriched for TargetScan predicted targets . p - value from hypergeometric distribution .

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Figure 4(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 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 cells were enriched for TargetScan predicted targets. p-value from hypergeometric distribution.


Figure 5 ( 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 cell lines : HOSE , HBEC30 ) . ( 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 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 and AMPK pathway activation .

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Figure 5(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 cell lines: HOSE, HBEC30).(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 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 and AMPK pathway activation.


Figure 1ER 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 ) .

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Figure 1ER 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).


Figure 2The 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 ) .

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Figure 2The 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).


Figure 3Characterization 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 .

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Figure 3Characterization 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.


Figure 4Fluorescence 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 ) .

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Figure 4Fluorescence 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).


Figure 5Immunogold 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 .

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Figure 5Immunogold 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.


Figure 6 ( 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 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 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 cells expressing GFP - Atg8 . Mutant cells were grown under regular conditions , UPR - inducing conditions ( + DTT ) , or nitrogen starvation conditions ( N starv ) .

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Figure 6(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 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 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 cells expressing GFP-Atg8. Mutant cells were grown under regular conditions, UPR-inducing conditions (+DTT), or nitrogen starvation conditions (N starv).


Figure 7Localization 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 .

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Figure 7Localization 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.


Figure 8Atg8 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 ) .

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Figure 8Atg8 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).


Figure 1A 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 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 ) 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 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 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 .

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Figure 1A 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 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) 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 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 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.


Figure 2Apoptotic 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 .

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Figure 2Apoptotic 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.


Figure 3A , 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 .

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Figure 3A, 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.


Figure 4A 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 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 .

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Figure 4A 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 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.


Figure 5Drp1 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 .

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Figure 5Drp1 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.


Figure 6Shown 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 .

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Figure 6Shown 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.


Figure 1Assay 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 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 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 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 .

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Figure 1Assay 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 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 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 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.


Figure 2B 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 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B 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 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.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.


Figure 4A 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 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 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 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 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 .

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Figure 4A 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 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 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 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 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.


Figure 5A , 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A, 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 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 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 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.


Figure 6A 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 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A 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 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 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 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 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.


Figure 7A 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 the PBS alone group ( D ) . Statistical difference was calculated using the unpaired one - sided Mann - Whitney U - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A 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 the PBS alone group (D). Statistical difference was calculated using the unpaired one-sided Mann-Whitney U-test.


Figure 1A ) 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A) 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 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 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.


Figure 22A ) 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 22A) 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 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 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 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.


Figure 33A ) 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 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 33A) 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 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 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 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 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.


Figure 44A ) 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 44A) 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 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 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 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.


Figure 55A ) 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 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 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 55A) 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 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 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 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 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 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.


Figure 1 . 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 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 ) 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 + 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 .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1.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 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) 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+ 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.