Figure 1A , B . A fraction of OTULIN is localized at mitochondria where it suppresses M1 - linked ubiquitination . HEK293T cells were transfected with control or OTULIN siRNA . Cells were harvested 72 h ( A ) or 48 h ( B ) after transfection or 15 min after TNF treatment ( 25 ng / ml ) . Mitochondria were isolated by differential centrifugation and purified by ultracentrifugation using an OptiPrepTM density gradient . 38 % ( A ) or 20 % ( B ) of the mitochondrial fractions and 2 % ( A , B ) of whole cell lysates were analyzed by immunoblotting using the antibodies indicated . For the detection of M1 - linked ubiquitin chains , either the 1E3 ( A ) or the 1F11 / 3F5 / Y102L ( B ) antibody was used . C . TNF induces M1 - ubiquitination at mitochondria in various cell types . The indicated cell types were treated with TNF ( 25 ng / ml , 15 min ) and purified mitochondrial fractions analyzed as described in A . D . TNF - induced mitochondrial M1 - ubiquitination does not occur in HOIP - deficient cells . Wildtype ( WT ) and HOIP - KO HAP cells were treated with TNF ( 25 ng / ml , 15 min ) and analyzed as described in A . E . Mitochondria and M1 - linked ubiquitin co - localize after TNF treatment . SH - SY5Y cells were treated with TNF ( 25 ng / ml , 15 min ) , fixed , stained with antibodies against HSP60 ( green ) and M1 - ubiquitin ( 1E3 , red ) and analyzed by SR - SIM . F . TNF induces recruitment of LUBAC components to mitochondria . HEK293T cells were treated with TNF ( 25 ng / ml , 15 min ) and analyzed as described in A . G . TNF induces a fast and transient increase in M1 - and K63 - specific ubiquitination at mitochondria . HEK 293T cells were treated with TNF ( 25 ng / ml ) for the indicated time and the mitochondrial fractions were analyzed by immunoblotting using M1 - , K63 - , and K48 - specific ubiquitin antibodies .

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Figure 1A, B. A fraction of OTULIN is localized at mitochondria where it suppresses M1-linked ubiquitination. HEK293T cells were transfected with control or OTULIN siRNA. Cells were harvested 72 h (A) or 48 h (B) after transfection or 15 min after TNF treatment (25 ng/ml). Mitochondria were isolated by differential centrifugation and purified by ultracentrifugation using an OptiPrepTM density gradient. 38% (A) or 20% (B) of the mitochondrial fractions and 2% (A, B) of whole cell lysates were analyzed by immunoblotting using the antibodies indicated. For the detection of M1-linked ubiquitin chains, either the 1E3 (A) or the 1F11/3F5/Y102L (B) antibody was used.C. TNF induces M1-ubiquitination at mitochondria in various cell types. The indicated cell types were treated with TNF (25 ng/ml, 15 min) and purified mitochondrial fractions analyzed as described in A.D. TNF-induced mitochondrial M1-ubiquitination does not occur in HOIP-deficient cells. Wildtype (WT) and HOIP-KO HAP cells were treated with TNF (25 ng/ml, 15 min) and analyzed as described in A.E. Mitochondria and M1-linked ubiquitin co-localize after TNF treatment. SH-SY5Y cells were treated with TNF (25 ng/ml, 15 min), fixed, stained with antibodies against HSP60 (green) and M1-ubiquitin (1E3, red) and analyzed by SR-SIM.F. TNF induces recruitment of LUBAC components to mitochondria. HEK293T cells were treated with TNF (25 ng/ml, 15 min) and analyzed as described in A.G. TNF induces a fast and transient increase in M1- and K63-specific ubiquitination at mitochondria. HEK 293T cells were treated with TNF (25 ng/ml) for the indicated time and the mitochondrial fractions were analyzed by immunoblotting using M1-, K63-, and K48-specific ubiquitin antibodies.


Figure 2A , B . PINK1 is stabilized by TNF treatment . HEK293T cells were treated with TNF ( 25 ng / ml , 15 min ) or CCCP ( 10 µM , 90 min ) before harvesting or incubated with OTULIN - specific siRNA for 48 h ( A ) . Purified mitochondrial fractions were analyzed by immunoblotting using the indicated antibodies . Quantification of PINK1 - specific signals normalized to TIM23 is shown in the right panel . Data represent the mean values with standard deviations of 4 independent experiments . * p < 0 . 05 . A two - tailed non - parametric Mann - Whitney U - test was used to analyze statistical significance . In ( A ) , * unprocessed PINK1 ; * * processed PINK1 . C . TNF - induced PINK1 stabilization does not induce mitophagy . HeLa cells expressing mt - mKeima were treated with TNF ( 25 ng / ml ) for 30 min or 16 h . As a control , mt - mKeima - expressing Hela cells were treated with antimycin A and oligomycin ( A / O , 10 µM each ) for 1 h . The analysis was done by flow cytometry gating of lysosomal and neutral mt - mKeima . D . Catalytically active HOIP ubiquitinates overexpressed PINK1 . HEK293T cells were transfected with the plasmids indicated . After 24 h , the cells were harvested under denaturing conditions and PINK1 was immunoprecipitated via the V5 tag followed by immunoblotting against ubiquitin . E . Recombinant HOIP ubiquitinates PINK1 in vitro . V5 - tagged - PINK1 immunoprecipitated from transiently transfected HEK293T cells via the V5 tag was incubated with recombinant mouse Ube1 , UBE2L3 , C - terminal HOIP and ubiquitin for in vitro ubiquitination . The samples were then analyzed by immunoblotting using V5 antibodies . F . Endogenous PINK1 is modified with M1 ubiquitin chains after TNF treatment . HEK293T cells were treated with TNF ( 25 ng / ml , 15 min ) , then endogenous PINK1 was immunoprecipitated from mitochondrial fractions . Cells mildly overexpressing PINK1 - V5 were also included ( lanes 4 , 5 , 8 ) , to make sure that the immunoreactive bands seen for endogenous PINK1 indeed correspond to PINK1 . As a control for the presence of M1 - linked ubiquitin chains , immunoprecipitated PINK1 was treated with recombinant OTULIN . As controls for the specificity of the immunoprecipitation , beads only ( lanes 6 - 8 ) and beads plus IgG ( lane 9 ) were included . The samples were analyzed by immunoblotting using antibodies against PINK1 , M1 - ubiquitin , and phosphorylated ubiquitin . For immunoprecipitation of overexpressed PINK1 , only 50 % of cells were used in comparison to the immunoprecipitation of endogenous PINK1 .

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 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 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 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 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

Figure 2A, B. PINK1 is stabilized by TNF treatment. HEK293T cells were treated with TNF (25 ng/ml, 15 min) or CCCP (10 µM, 90 min) before harvesting or incubated with OTULIN-specific siRNA for 48 h (A). Purified mitochondrial fractions were analyzed by immunoblotting using the indicated antibodies. Quantification of PINK1-specific signals normalized to TIM23 is shown in the right panel. Data represent the mean values with standard deviations of 4 independent experiments. *p < 0.05. A two-tailed non-parametric Mann-Whitney U-test was used to analyze statistical significance. In (A), *unprocessed PINK1; **processed PINK1.C. TNF-induced PINK1 stabilization does not induce mitophagy. HeLa cells expressing mt-mKeima were treated with TNF (25 ng/ml) for 30 min or 16 h. As a control, mt-mKeima -expressing Hela cells were treated with antimycin A and oligomycin (A/O, 10 µM each) for 1 h. The analysis was done by flow cytometry gating of lysosomal and neutral mt-mKeima.D. Catalytically active HOIP ubiquitinates overexpressed PINK1. HEK293T cells were transfected with the plasmids indicated. After 24 h, the cells were harvested under denaturing conditions and PINK1 was immunoprecipitated via the V5 tag followed by immunoblotting against ubiquitin.E. Recombinant HOIP ubiquitinates PINK1 in vitro. V5-tagged-PINK1 immunoprecipitated from transiently transfected HEK293T cells via the V5 tag was incubated with recombinant mouse Ube1, UBE2L3, C-terminal HOIP and ubiquitin for in vitro ubiquitination. The samples were then analyzed by immunoblotting using V5 antibodies.F. Endogenous PINK1 is modified with M1 ubiquitin chains after TNF treatment. HEK293T cells were treated with TNF (25 ng/ml, 15 min), then endogenous PINK1 was immunoprecipitated from mitochondrial fractions. Cells mildly overexpressing PINK1-V5 were also included (lanes 4, 5, 8), to make sure that the immunoreactive bands seen for endogenous PINK1 indeed correspond to PINK1. As a control for the presence of M1-linked ubiquitin chains, immunoprecipitated PINK1 was treated with recombinant OTULIN. As controls for the specificity of the immunoprecipitation, beads only (lanes 6 - 8) and beads plus IgG (lane 9) were included. The samples were analyzed by immunoblotting using antibodies against PINK1, M1-ubiquitin, and phosphorylated ubiquitin. For immunoprecipitation of overexpressed PINK1, only 50% of cells were used in comparison to the immunoprecipitation of endogenous PINK1.


Figure 3A . PINK1 antagonizes OTULIN activity in cells . HEK293T cells expressing HOIP and HOIL - 1L , were transfected with PINK1 , WT OTULIN or the inactive OTULIN mutant W96A , as indicated . The cells were lysed 24 h later under denaturing conditions , and lysates were subjected to affinity purification using the Strep - tagged UBAN domain of NEMO to enrich proteins modified with M1 - linked ubiquitin . Proteins affinity - purified by Strep - Tactin beads were immunoblotted against ubiquitin . B . Catalytically active PINK1 increases p - S65 - ubiquitin at mitochondria . HEK293T cells were transfected with wildtype PINK1 or kinase - dead ( K / D ) PINK1 . 48 h after transfection , the cells were treated with TNF ( 25 ng / ml , 15 min ) and lysed . Purified mitochondrial fractions were analyzed by immunoblotting using antibodies against p - S65 - ubiquitin , PINK1 , and VDAC ( loading control ) . C . The TNF - induced increase in p - S65 - ubiquitin is abolished in PINK1 - deficient cells . HEK293T cells were transfected with control or PINK1 - specific siRNA and treated with TNF ( 25 ng / ml , 15 min ) or CCCP ( 10 µM , 90 min ) 48 h after transfection . The whole cell lysates were analyzed by immunoblotting using the indicated antibodies . D . TNF - induced M1 ubiquitination is reduced in PINK1 - deficient cells . WT and PINK1 - KO MEFs were treated with TNF ( 25 ng / ml , 15 min ) and then harvested . Purified mitochondrial fractions were subjected to affinity purification using the Strep - tagged UBAN domain , as described in A , followed by immunoblotting using M1 - ubiquitin - specifc antibodies , Strep - Tactin conjugated to horse radish peroxidase ( to control the UBAN pulldown efficiency ) , and TIM23 ( input control ) . Quantification of the M1 - ubiquitin - positive signal intensities is shown in the lower panel . Data represent the mean values with standard deviations of 3 independent experiments ; n . s . not significant , * * * p < 0 . 001 . A two - way ANOVA test was used to analyze statistical significance .

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Figure 3A. PINK1 antagonizes OTULIN activity in cells. HEK293T cells expressing HOIP and HOIL-1L, were transfected with PINK1, WT OTULIN or the inactive OTULIN mutant W96A, as indicated. The cells were lysed 24 h later under denaturing conditions, and lysates were subjected to affinity purification using the Strep-tagged UBAN domain of NEMO to enrich proteins modified with M1-linked ubiquitin. Proteins affinity-purified by Strep-Tactin beads were immunoblotted against ubiquitin.B. Catalytically active PINK1 increases p-S65-ubiquitin at mitochondria. HEK293T cells were transfected with wildtype PINK1 or kinase-dead (K/D) PINK1. 48 h after transfection, the cells were treated with TNF (25 ng/ml, 15 min) and lysed. Purified mitochondrial fractions were analyzed by immunoblotting using antibodies against p-S65-ubiquitin, PINK1, and VDAC (loading control).C. The TNF-induced increase in p-S65-ubiquitin is abolished in PINK1-deficient cells. HEK293T cells were transfected with control or PINK1-specific siRNA and treated with TNF (25 ng/ml, 15 min) or CCCP (10 µM, 90 min) 48 h after transfection. The whole cell lysates were analyzed by immunoblotting using the indicated antibodies.D. TNF-induced M1 ubiquitination is reduced in PINK1-deficient cells. WT and PINK1-KO MEFs were treated with TNF (25 ng/ml, 15 min) and then harvested. Purified mitochondrial fractions were subjected to affinity purification using the Strep-tagged UBAN domain, as described in A, followed by immunoblotting using M1-ubiquitin-specifc antibodies, Strep-Tactin conjugated to horse radish peroxidase (to control the UBAN pulldown efficiency), and TIM23 (input control). Quantification of the M1-ubiquitin-positive signal intensities is shown in the lower panel. Data represent the mean values with standard deviations of 3 independent experiments; n.s. not significant, ***p < 0.001. A two-way ANOVA test was used to analyze statistical significance.


Figure 4A . STS - induced mitochondrial Bax recruitment is reduced by TNF . HeLa cells were treated with STS ( 1 μM , 1 h ) with or without a 15 min pretreatment with TNF ( 25 ng / ml ) and then harvested . Purified mitochondrial fractions were analyzed by immunoblotting using antibodies against Bax and M1 - ubiquitin . The input was immunoblotted for TIM23 ( upper panel ) . Bax - specific signal intensities were quantified and normalized to TIM23 - specific signals ( lower panel ) . B . STS - induced cytochrome C release is decreased by TNF . HeLa cells were treated as described in A . The cytosolic fractions were analyzed by immunoblotting using cytochrome c antibodies . GAPDH was used as a reference . Quantification of 5 biological replicates is shown in the lower panel . Signal intensities were quantified and normalized to that of GAPDH . * p < 0 . 05 . Kolmogorow - Smirnov normality test , paired t - test , two - tailed , n = 5 . C , D . TNF treatment prevents damage of mitochondrial cristae under pro - apoptotic conditions . C . SH - SY5Y cells were treated as described in A , fixed , embedded and the mitochondrial ultrastructure was imaged by electron microscopy . Scale bar : 400 nm . D . Cristae abundance ( average cristae number per mitochondrial area ) and cristae length ( average cristae length per mitochondrium ) were analyzed by Imaris 9 . 8 . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 01 . Cristae abundance : Shapiro - Wilk normality test , One - way ANOVA followed by Tukey ' s multiple comparison test , n = 27 . Cristae length : Kruskal - Wallis test followed by Dunn ' s multiple comparison test , n = 24 - 27 . Bars represent mean ± SEME . The fast anti - apoptotic effect of TNF is not affected by the NF - κΒ inhibitor IκΒα . SH - SY5Y cells were transiently transfected with the NF - κΒ super - repressor IᴋBα - 2S or luciferase as a control . 24 h later , the cells were treated with STS ( 5 µM , 2 h ) with or without a 15 min pretreatment with TNF ( 25 ng / ml ) . Cells were fixed and stained by antibodies against active caspase - 3 . Signal intensities were quantified by immunocytochemistry and fluorescence microscopy . Expression of IᴋBα - 2S was tested by immunoblotting using antibodies against the HA tag . Data represent the standard deviation of three independent experiments ; at least 300 cells were counted per experiment . * p < 0 . 05 , * * p < 0 . 01 . Student ' s t - test , two - tailed , n = 3 . F . The protective effect of TNF is dependent on HOIP expression . HeLa cells were transiently transfected with control or HOIP siRNA . 48 h after transfection , cells were treated with STS ( 1 μM , 1 h ) with or without a 15 min TNF pretreatment ( 25 ng / ml ) and then harvested . The cytosolic fractions were analyzed by immunoblotting using cytochrome c antibodies . HOIP silencing efficiency was analyzed in whole cell lysates using antibodies against HOIP . GAPDH and actin were immunoblotted as input controls . G . The protective effect of TNF is dependent on PINK1 expression . HeLa cells were transiently transfected with control or PINK1 siRNA and treated as described in F . The cytosolic fractions were analyzed by immunoblotting using cytochrome c and PINK1 antibodies . GAPDH was immunoblotted as input 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 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 O O O O O O O O O O O O O O O O O O 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 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

Figure 4A. STS-induced mitochondrial Bax recruitment is reduced by TNF. HeLa cells were treated with STS (1 μM, 1 h) with or without a 15 min pretreatment with TNF (25 ng/ml) and then harvested. Purified mitochondrial fractions were analyzed by immunoblotting using antibodies against Bax and M1-ubiquitin. The input was immunoblotted for TIM23 (upper panel). Bax-specific signal intensities were quantified and normalized to TIM23-specific signals (lower panel).B. STS-induced cytochrome C release is decreased by TNF. HeLa cells were treated as described in A. The cytosolic fractions were analyzed by immunoblotting using cytochrome c antibodies. GAPDH was used as a reference. Quantification of 5 biological replicates is shown in the lower panel. Signal intensities were quantified and normalized to that of GAPDH. *p < 0.05. Kolmogorow-Smirnov normality test, paired t-test, two-tailed, n=5.C, D. TNF treatment prevents damage of mitochondrial cristae under pro-apoptotic conditions. C. SH-SY5Y cells were treated as described in A, fixed, embedded and the mitochondrial ultrastructure was imaged by electron microscopy. Scale bar: 400 nm. D. Cristae abundance (average cristae number per mitochondrial area) and cristae length (average cristae length per mitochondrium) were analyzed by Imaris 9.8. *p < 0.05, **p < 0.01, ***p < 0.01. Cristae abundance: Shapiro-Wilk normality test, One-way ANOVA followed by Tukey's multiple comparison test, n = 27. Cristae length: Kruskal-Wallis test followed by Dunn's multiple comparison test, n=24-27. Bars represent mean ± SEME. The fast anti-apoptotic effect of TNF is not affected by the NF-κΒ inhibitor IκΒα. SH-SY5Y cells were transiently transfected with the NF-κΒ super-repressor IᴋBα-2S or luciferase as a control. 24 h later, the cells were treated with STS (5 µM, 2 h) with or without a 15 min pretreatment with TNF (25 ng/ml). Cells were fixed and stained by antibodies against active caspase-3. Signal intensities were quantified by immunocytochemistry and fluorescence microscopy. Expression of IᴋBα-2S was tested by immunoblotting using antibodies against the HA tag. Data represent the standard deviation of three independent experiments; at least 300 cells were counted per experiment. *p < 0.05, **p < 0.01. Student's t-test, two-tailed, n=3.F. The protective effect of TNF is dependent on HOIP expression. HeLa cells were transiently transfected with control or HOIP siRNA. 48 h after transfection, cells were treated with STS (1 μM, 1 h) with or without a 15 min TNF pretreatment (25 ng/ml) and then harvested. The cytosolic fractions were analyzed by immunoblotting using cytochrome c antibodies. HOIP silencing efficiency was analyzed in whole cell lysates using antibodies against HOIP. GAPDH and actin were immunoblotted as input controls.G. The protective effect of TNF is dependent on PINK1 expression. HeLa cells were transiently transfected with control or PINK1 siRNA and treated as described in F. The cytosolic fractions were analyzed by immunoblotting using cytochrome c and PINK1 antibodies. GAPDH was immunoblotted as input control.


Figure 5A . NF - κB signaling components are recruited to mitochondria upon TNF treatment . A . HEK293T cells were treated with TNF ( 25 ng / ml , 15 min ) , harvested and purified mitochondrial fractions were analyzed by immunoblotting using the antibodies indicated ; p - p65 : phospho - S536 - p65 ; p - TBK1 : phospho - S172 - TBK1 . B . Phosphorylated p65 is in close proximity to TOM20 in response to TNF treatment . Representative immunofluorescence images of SH - SY5Y cells using the proximity ligation assay ( PLA ) between phospho - S536 - p65 ( rabbit ) and TOM20 ( mouse ) couple antibodies . One set of cells were treated with TNF ( 25 ng / ml , 15 min ) before fixation . Nuclei were stained with DAPI , mitochondria were stained with MitoTrackerTM Red CMXRos ( red ) , and the PLA amplification reaction was visualized by green foci . As a positive control , fixed cells were incubated with primary TOM20 ( mouse ) and TOM70 ( rabbit ) antibodies and subjected to the PLA assay . As negative controls , fixed cells were incubated with either TOM20 or p - p65 antibodies prior to the PLA assay . C . Quantification of the phospho - S536 - p65 / TOM20 PLA foci per cells . Data represent the number of PLA foci per cell ( mean ± SD ) ; at least 84 cells in total were analyzed per condition . * * * * p < 0 . 0001 . A two - tailed non - parametric Mann - Whitney test was used to analyze statistical significance .

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 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 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

Figure 5A. NF-κB signaling components are recruited to mitochondria upon TNF treatment. A. HEK293T cells were treated with TNF (25 ng/ml, 15 min), harvested and purified mitochondrial fractions were analyzed by immunoblotting using the antibodies indicated; p-p65: phospho-S536-p65; p-TBK1: phospho-S172-TBK1.B. Phosphorylated p65 is in close proximity to TOM20 in response to TNF treatment. Representative immunofluorescence images of SH-SY5Y cells using the proximity ligation assay (PLA) between phospho-S536-p65 (rabbit) and TOM20 (mouse) couple antibodies. One set of cells were treated with TNF (25 ng/ml, 15 min) before fixation. Nuclei were stained with DAPI, mitochondria were stained with MitoTrackerTM Red CMXRos (red), and the PLA amplification reaction was visualized by green foci. As a positive control, fixed cells were incubated with primary TOM20 (mouse) and TOM70 (rabbit) antibodies and subjected to the PLA assay. As negative controls, fixed cells were incubated with either TOM20 or p-p65 antibodies prior to the PLA assay.C. Quantification of the phospho-S536-p65/TOM20 PLA foci per cells. Data represent the number of PLA foci per cell (mean ±SD); at least 84 cells in total were analyzed per condition. ****p < 0.0001. A two-tailed non-parametric Mann-Whitney test was used to analyze statistical significance.


Figure 6A . Nuclear translocation of p65 is impaired in cells silenced for PINK1 expression . SH - SY5Y cells were transfected with control or PINK1 - specific siRNAs . For rescue experiments , cells were co - transfected with wildtype PINK1 , PINK1Δ77 , or kinase - dead PINK1 - K / D . Two days after transfection the cells were treated with TNF ( 25 ng / ml , 25 min ) , fixed and stained with p65 antibodies . The fraction of cells showing nuclear translocation of p65 was determined for each condition . Data represent the mean ± SEM of 3 independent experiments . The experiment was performed in triplicates and more than 300 cells were quantified per experiment and condition . B . PINK1 phosphorylates ubiquitinated NEMO . HEK293T cells were co - transfected with V5 - tagged wildtype PINK1 or kinase - dead PINK1 - K / D and HA - tagged NEMO . One day after transfection the cells were lysed and subjected to immunoprecipitation using antibodies against HA . Precipitated proteins were then detected by immunoblotting using p - S65 - ubiquitin antibodies . The input was immunoblotted for NEMO , PINK1 , p - S65 - ubiquitin , and GAPDH . C . Linear ubiquitination of NEMO is reduced in PINK1 - deficient cells . HEK293T cells were co - transfected with HA - NEMO and control or PINK1 - specific siRNAs . 48 h after transfection the cells were lysed and subjected to immunoprecipitation using antibodies against HA . Precipitated proteins were then detected by immunoblotting using M1 - ubiquitin antibodies . The input was immunoblotted for NEMO and actin . PINK1 silencing efficiency was determined by real - time RT - PCR . Bars represent mean ± SD with three technical replicates .

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Figure 6A. Nuclear translocation of p65 is impaired in cells silenced for PINK1 expression. SH-SY5Y cells were transfected with control or PINK1-specific siRNAs. For rescue experiments, cells were co-transfected with wildtype PINK1, PINK1Δ77, or kinase-dead PINK1-K/D. Two days after transfection the cells were treated with TNF (25 ng/ml, 25 min), fixed and stained with p65 antibodies. The fraction of cells showing nuclear translocation of p65 was determined for each condition. Data represent the mean ± SEM of 3 independent experiments. The experiment was performed in triplicates and more than 300 cells were quantified per experiment and condition.B. PINK1 phosphorylates ubiquitinated NEMO. HEK293T cells were co-transfected with V5-tagged wildtype PINK1 or kinase-dead PINK1-K/D and HA-tagged NEMO. One day after transfection the cells were lysed and subjected to immunoprecipitation using antibodies against HA. Precipitated proteins were then detected by immunoblotting using p-S65-ubiquitin antibodies. The input was immunoblotted for NEMO, PINK1, p-S65-ubiquitin, and GAPDH.C. Linear ubiquitination of NEMO is reduced in PINK1-deficient cells. HEK293T cells were co-transfected with HA-NEMO and control or PINK1-specific siRNAs. 48 h after transfection the cells were lysed and subjected to immunoprecipitation using antibodies against HA. Precipitated proteins were then detected by immunoblotting using M1-ubiquitin antibodies. The input was immunoblotted for NEMO and actin. PINK1 silencing efficiency was determined by real-time RT-PCR. Bars represent mean± SD with three technical replicates.


Figure 7A . TNF induces the movement of peripheral mitochondria towards the nucleus . Primary macrophages were stained by MitotrackerTM Green to visualize mitochondria and by Hoechst 33342 to visualize the nucleus and monitored every 30 s for 15 min after treatment with TNF ( 25 ng / ml ) . The motility of mitochondria was analyzed using Imaris 9 . 8 spot function . The graphical representation of the top 25 % peripheral mitochondrial indicates a decrease of the average mitochondrial distance to the nucleus after TNF treatment . Exemplified dataset shows the median distance with the upper and lower quartile ( shaded area ) of n = 105 - 187 mitochondrial spots per time point . B . Nuclear p65 translocation upon TNF treatment is reduced cells silenced for Miro1 / 2 expression . HeLa cells were transfected with control or Miro1 and Miro2 siRNAs . 48 h later , cells were treated with TNF ( 25 ng / ml , 15 min ) , fixed and stained using antibodies against p65 and tubulin and DAPI . Images were segmented in a cytoplasmic and nuclear compartment and the fluorescence signal intensity ratio of p65 was measured using CellProfiler 4 . 2 . 1 . Quantification is based on 3 biological replicates . Kruskal - Wallis test followed by Dunn ' s multiple comparison test , n = 79 - 157 , * p < 0 . 05 . E , F , G . TNF increases mitochondria - nucleus contact sites . Representative immunofluorescence images ( E , F ) and quantification ( G ) of mitochondria - nucleus contact sites in HeLa cells transfected with the SPLICSNU - MT and treated with 25 ng / ml TNF or PBS for 15 min . TOM20 antibodies were used to stain mitochondria and p65 antibodies to assess p65 nuclear translocation . Nuclei were stained with Hoechst33342 ( ThermoFisher , 1 μg / ml ) . Unpaired two - tailed t - test , n = 43 ( control ) or 51 ( TNF ) , H , I , J . TNF increases mitochondria - nucleus contact sites in a HOIP - dependent manner . Representative immunofluorescence images ( H , I ) and quantification ( J ) of mitochondria - nucleus contact sites in control or HOIP - silenced ( HOIP siRNA ) HeLa cells transfected with the SPLICSNU - MT probe treated with 25 ng / ml TNF or PBS for 15 minutes . Mitochondria and nuclei were stained as described in E and F .

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Figure 7A. TNF induces the movement of peripheral mitochondria towards the nucleus. Primary macrophages were stained by MitotrackerTM Green to visualize mitochondria and by Hoechst 33342 to visualize the nucleus and monitored every 30 s for 15 min after treatment with TNF (25 ng/ml). The motility of mitochondria was analyzed using Imaris 9.8 spot function. The graphical representation of the top 25% peripheral mitochondrial indicates a decrease of the average mitochondrial distance to the nucleus after TNF treatment. Exemplified dataset shows the median distance with the upper and lower quartile (shaded area) of n=105-187 mitochondrial spots per time point.B. Nuclear p65 translocation upon TNF treatment is reduced cells silenced for Miro1/2 expression. HeLa cells were transfected with control or Miro1 and Miro2 siRNAs. 48 h later, cells were treated with TNF (25 ng/ml, 15 min), fixed and stained using antibodies against p65 and tubulin and DAPI. Images were segmented in a cytoplasmic and nuclear compartment and the fluorescence signal intensity ratio of p65 was measured using CellProfiler 4.2.1. Quantification is based on 3 biological replicates. Kruskal-Wallis test followed by Dunn's multiple comparison test, n=79-157, *p < 0.05.E, F, G. TNF increases mitochondria-nucleus contact sites. Representative immunofluorescence images (E, F) and quantification (G) of mitochondria-nucleus contact sites in HeLa cells transfected with the SPLICSNU-MT and treated with 25 ng/ml TNF or PBS for 15 min. TOM20 antibodies were used to stain mitochondria and p65 antibodies to assess p65 nuclear translocation. Nuclei were stained with Hoechst33342 (ThermoFisher, 1 μg/ml). Unpaired two-tailed t-test, n=43 (control) or 51 (TNF),H, I, J. TNF increases mitochondria-nucleus contact sites in a HOIP-dependent manner. Representative immunofluorescence images (H, I) and quantification (J) of mitochondria-nucleus contact sites in control or HOIP-silenced (HOIP siRNA) HeLa cells transfected with the SPLICSNU-MT probe treated with 25 ng/ml TNF or PBS for 15 minutes. Mitochondria and nuclei were stained as described in E and F.


Figure 1The heatmap shows the dynamic expression of genes in the SF3A / 3B complex ( E ) and SR family ( F ) at each indicated differentiation stage . Both heatmaps were row normalized . The heatmaps were scaled with Z - Score using the log2 ( FPKM ) expression of indicated components . n = 3 technical replicates . The mRNA expression of splicing regulator SRSF2 and splicing factor PCBP2 during hematopoietic differentiation were measured by RT - qPCR . The ACTB gene was used as a control . Results given are mean ± standard deviation ( SD ) . P - values were determined by an unpaired two - tailed Student ' s t - test . n ≥ 3 biological replicates .

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Figure 1The heatmap shows the dynamic expression of genes in the SF3A/3B complex (E) and SR family (F) at each indicated differentiation stage. Both heatmaps were row normalized. The heatmaps were scaled with Z-Score using the log2(FPKM) expression of indicated components. n=3 technical replicates.The mRNA expression of splicing regulator SRSF2 and splicing factor PCBP2 during hematopoietic differentiation were measured by RT-qPCR. The ACTB gene was used as a control. Results given are mean ± standard deviation (SD). P-values were determined by an unpaired two-tailed Student's t-test. n ≥ 3 biological replicates.


Figure 2The experimental schematic ( upper panel ) . APLNR + cells on day 2 were FACS - purified and treated with various dosages of PLB from day 2 . 5 to 5 during inducing hematopoietic differentiation . On day 5 , the generation of CD31 + CD34 + EPCs and CD31 + CD34 + CD73 - HEPs were assessed . The representative FACS plots showed the frequency of APLNR + cells , CD31 + CD34 + EPCs and CD31 + CD34 + CD73 - HEPs without or with PLB treatment . The bar graphs show the percentage of CD31 + CD34 + EPCs and CD31 + CD34 + CD73 - HEPs without or with PLB treatment .

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Figure 2The experimental schematic (upper panel). APLNR+ cells on day 2 were FACS-purified and treated with various dosages of PLB from day 2.5 to 5 during inducing hematopoietic differentiation. On day 5, the generation of CD31+CD34+ EPCs and CD31+CD34+CD73- HEPs were assessed. The representative FACS plots showed the frequency of APLNR+ cells, CD31+CD34+ EPCs and CD31+CD34+CD73- HEPs without or with PLB treatment. The bar graphs show the percentage of CD31+CD34+ EPCs and CD31+CD34+CD73- HEPs without or with PLB treatment.


Figure 3The bar graph represents the percentage of CD31 + CD34 + cells from ( H ) . P - values were calculated by one - way ANOVA followed by Tukey ' s test . The bar graph representing the percentage of CD31 + CD34 + cells from ( K ) . P - values were determined by an unpaired two - tailed Student ' s t - test .

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Figure 3The bar graph represents the percentage of CD31+CD34+ cells from (H). P-values were calculated by one-way ANOVA followed by Tukey's test.The bar graph representing the percentage of CD31+CD34+ cells from (K). P-values were determined by an unpaired two-tailed Student's t-test.


Figure 4The representative FACS plots show the frequency of CD31 + CD34 + EPCs in the GFP + population at day 5 with 1 . 25 nM PLB supplementation alone or in combination with NUMB _ S ectopic expression from day 2 . 5 . The bar graph denotes the percentage of CD31 + CD34 + in GFP + cells as described in ( F ) . P - values were determined by an unpaired two - tailed Student ' s t - test .

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Figure 4The representative FACS plots show the frequency of CD31+CD34+ EPCs in the GFP+ population at day 5 with 1.25 nM PLB supplementation alone or in combination with NUMB_S ectopic expression from day 2.5. The bar graph denotes the percentage of CD31+CD34+ in GFP+ cells as described in (F). P-values were determined by an unpaired two-tailed Student's t-test.


Figure 5On day 5 of hematopoietic differentiation , the inclusion or exclusion of NUMB exon 9 were detected by RT - PCR with 1 . 25 nM PLB supplementation or SRSF2 exogenous expression alone or their combinational treatment from day 2 . 5 . The bar graph showing the signaling intensity of each specific band from the RT - PCR electropherogram . The top panel depicts the location of specific primers targeting NUMB _ S and NUMB _ L isoform . The bottom bar graph showing the expression of NUMB _ S and NUMB _ L in cells on day 5 of differentiation with 1 . 25 nM PLB supplementation or SRSF2 exogenous expression alone or their combinational treatment from day 2 . 5 with RT - qPCR .

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Figure 5On day 5 of hematopoietic differentiation, the inclusion or exclusion of NUMB exon 9 were detected by RT-PCR with 1.25 nM PLB supplementation or SRSF2 exogenous expression alone or their combinational treatment from day 2.5. The bar graph showing the signaling intensity of each specific band from the RT-PCR electropherogram.The top panel depicts the location of specific primers targeting NUMB_S and NUMB_L isoform. The bottom bar graph showing the expression of NUMB_S and NUMB_L in cells on day 5 of differentiation with 1.25 nM PLB supplementation or SRSF2 exogenous expression alone or their combinational treatment from day 2.5 with RT-qPCR.


Figure 6The heatmap plotting the expression of NUMB _ S and the key components in NOTCH signaling in day 2 - APLNR + cells , day 5 - DMSO treated and day 5 - PLB treated cells ( 2 . 5 nM ) , respectively . The NOTCH components were divided into groups of upregulated , downregulated , and unchanged by comparison between DMSO and PLB treated cells . The heatmap was scaled with Z - Score using the log2 ( FPKM ) expression of indicated genes . n = 2 technical replicatesThe representative FACS plots indicating the generation of CD31 + CD34 + EPCs on day 5 of differentiation with or without HES1 overexpression . DOX was treated from day 2 . 5 to 5 to induce HES1 overexpression . Statistical analysis of the percentage of CD31 + CD34 + EPCs of ( H ) .

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Figure 6The heatmap plotting the expression of NUMB_S and the key components in NOTCH signaling in day 2-APLNR+ cells, day 5-DMSO treated and day 5-PLB treated cells (2.5 nM), respectively. The NOTCH components were divided into groups of upregulated, downregulated, and unchanged by comparison between DMSO and PLB treated cells. The heatmap was scaled with Z-Score using the log2(FPKM) expression of indicated genes. n=2 technical replicatesThe representative FACS plots indicating the generation of CD31+CD34+ EPCs on day 5 of differentiation with or without HES1 overexpression. DOX was treated from day 2.5 to 5 to induce HES1 overexpression. Statistical analysis of the percentage of CD31+CD34+ EPCs of (H).


f1 ( b ) AlPcS2a can be specifically targeted onto lysosomes . Using our staining procedure , AlPcS2a signals colocalized with Lysotracker Red ( top panels ) and lamp1 - RFP ( middle panels ) , but not with other cellular organelle markers such as EGFP - LC3B ( bottom panels , marker for autophagic structures ) , in HeLa cells . Scale bars , 10 μm .

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f1(b) AlPcS2a can be specifically targeted onto lysosomes. Using our staining procedure, AlPcS2a signals colocalized with Lysotracker Red (top panels) and lamp1-RFP (middle panels), but not with other cellular organelle markers such as EGFP-LC3B (bottom panels, marker for autophagic structures), in HeLa cells. Scale bars, 10 μm.


f2 ( a ) Simultaneous loss of Lysotracker and AlPcS2a fluorescence immediately following light activation . A HeLa cell labelled with Lysotracker Red and AlPcS2a was 635 - nm illuminated within the white square region ( left panels ) . Lysotracker fluorescence within the illuminated region was immediately lost after light illumination ( middle panels ) , indicating the successful triggering of LMP . Right panels : magnified view of Lysotracker fluorescence within the 635 - nm illuminated region pre - and post light activation . ( b ) 635 nm illumination did not lead to complete lysosome rupture . A HeLa cell labelled with FITC - dextran ( molecular weight = 10 , 000 ) and AlPcS2a was 635 - nm illuminated within the white square region ( left panels ) . FITC - dextran remained within illuminated lysosomes ( an increase in FITC fluorescence was observed , in accordance with an increase in pH in damaged lysosomes ; middle panels ) . Right panels : magnified view of FITC - dextran fluorescence within the 635 - nm illuminated region pre - and post light activation . ( c ) Light activation did not affect other cellular structures , such as mitochondria . A HeLa cell labelled with TMRE and AlPcS2a was 635 - nm illuminated within the white square region ( left panels ) . Illumination did not affect TMRE fluorescence , indicating that mitochondria within the same region were not affected / impaired . Right panels : magnified view of TMRE fluorescence within the 635 - nm illuminated region pre - and post light activation . Scale bars , 10 μm .

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f2(a) Simultaneous loss of Lysotracker and AlPcS2a fluorescence immediately following light activation. A HeLa cell labelled with Lysotracker Red and AlPcS2a was 635-nm illuminated within the white square region (left panels). Lysotracker fluorescence within the illuminated region was immediately lost after light illumination (middle panels), indicating the successful triggering of LMP. Right panels: magnified view of Lysotracker fluorescence within the 635-nm illuminated region pre- and post light activation. (b) 635 nm illumination did not lead to complete lysosome rupture. A HeLa cell labelled with FITC-dextran (molecular weight=10,000) and AlPcS2a was 635-nm illuminated within the white square region (left panels). FITC-dextran remained within illuminated lysosomes (an increase in FITC fluorescence was observed, in accordance with an increase in pH in damaged lysosomes; middle panels). Right panels: magnified view of FITC-dextran fluorescence within the 635-nm illuminated region pre- and post light activation. (c) Light activation did not affect other cellular structures, such as mitochondria. A HeLa cell labelled with TMRE and AlPcS2a was 635-nm illuminated within the white square region (left panels). Illumination did not affect TMRE fluorescence, indicating that mitochondria within the same region were not affected/impaired. Right panels: magnified view of TMRE fluorescence within the 635-nm illuminated region pre- and post light activation. Scale bars, 10 μm.


f3 ( a ) A HeLa cell expressing EGFP - Ub and lamp1 - RFP , and labelled with AlPcS2a , was 635 - nm illuminated within the white square regionb ) Punctate structures containing ubiquitin appeared within the 635 - nm illuminated region ( white arrow ) 30 - 50 min after light activation . ( c ) Ubiquitin puncta colocalized with damaged lysosomes . Magnified view of the 635 - nm illuminated cell region showed that ubiquitin signals localized on lysosomes ( lamp1 - labelled ) lacking AlPcS2a fluorescence ( indicating that it had been 635 - nm illuminated ) . White arrows indicate examples of Ub on damaged lysosomes . ( d ) Quantification of ubiquitin signals within three independent cellular regions ( A1 , A2 and A3 ) revealed a specific recruitment of ubiquitin to the site of light activation ( inset , right , A1 ) . Scale bars , 10 μm ( a , b , d ) ; 3 μm ( c ) .

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f3(a) A HeLa cell expressing EGFP-Ub and lamp1-RFP, and labelled with AlPcS2a, was 635-nm illuminated within the white square regionb) Punctate structures containing ubiquitin appeared within the 635-nm illuminated region (white arrow) 30-50 min after light activation.(c) Ubiquitin puncta colocalized with damaged lysosomes. Magnified view of the 635-nm illuminated cell region showed that ubiquitin signals localized on lysosomes (lamp1-labelled) lacking AlPcS2a fluorescence (indicating that it had been 635-nm illuminated). White arrows indicate examples of Ub on damaged lysosomes.(d) Quantification of ubiquitin signals within three independent cellular regions (A1, A2 and A3) revealed a specific recruitment of ubiquitin to the site of light activation (inset, right, A1). Scale bars, 10 μm (a,b,d); 3 μm (c).


f4 ( a ) A HeLa cell expressing EGFP - Ub and TagRFP - p62 , and labelled with AlPcS2a , was 635 - nm illuminated within the white square region , resulting in ubiquitination of and p62 recruitment to the photodamaged lysosomes , as shown in b . ( b ) Magnified view of the 635 - nm illuminated cell region in a . Ubiquitin and p62 initially distributed uniformly within the region ( t = 0 min , top panels ) , but formed distinct foci after light activation ( bottom panels ) . ( c ) A HeLa cell expressing EGFP - LC3B and lamp1 - RFP , and labelled with AlPcS2a , was 635 - nm illuminated within the white square region , resulting in LC3 recruitment onto photodamaged lysosomes , as shown in d . ( d ) Magnified view of the 635 - nm illuminated cell region in c . LC3 is initially distributed away from lysosomes ( lamp1 , t = 0 min , top panels ) , but appears on lamp1 - positive structures 40 min after light activation ( bottom panels ) . Scale bars , 10 μm ( a , c ) ; 3 μm ( b , d ) .

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f4(a) A HeLa cell expressing EGFP-Ub and TagRFP-p62, and labelled with AlPcS2a, was 635-nm illuminated within the white square region, resulting in ubiquitination of and p62 recruitment to the photodamaged lysosomes, as shown in b. (b) Magnified view of the 635-nm illuminated cell region in a. Ubiquitin and p62 initially distributed uniformly within the region (t=0 min, top panels), but formed distinct foci after light activation (bottom panels).(c) A HeLa cell expressing EGFP-LC3B and lamp1-RFP, and labelled with AlPcS2a, was 635-nm illuminated within the white square region, resulting in LC3 recruitment onto photodamaged lysosomes, as shown in d. (d) Magnified view of the 635-nm illuminated cell region in c. LC3 is initially distributed away from lysosomes (lamp1, t=0 min, top panels), but appears on lamp1-positive structures 40 min after light activation (bottom panels). Scale bars, 10 μm (a,c); 3 μm (b,d).


f5 ( a ) Two HeLa cells expressing RFP - EGFP - LC3 and labelled with AlPcS2a was 635 - nm illuminated within the white rectangular region , resulting in local LC3 puncta formation , as shown in b . ( b ) LC3 puncta were specifically induced within the 635 - nm illuminated cell regions in a ( LC3 initially distributed uniformly at t = 0 ) . ( c ) The loss of EGFP fluorescence from light - induced LC3 puncta preceded the disappearance of RFP fluorescence ( white arrows indicate examples of LC3 puncta that exhibited diminished EGFP fluorescence ) , indicating their successful transition from autophagosomes to autolysosomes . Scale bars , 10 μm .

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f5(a) Two HeLa cells expressing RFP-EGFP-LC3 and labelled with AlPcS2a was 635-nm illuminated within the white rectangular region, resulting in local LC3 puncta formation, as shown in b. (b) LC3 puncta were specifically induced within the 635-nm illuminated cell regions in a (LC3 initially distributed uniformly at t=0). (c) The loss of EGFP fluorescence from light-induced LC3 puncta preceded the disappearance of RFP fluorescence (white arrows indicate examples of LC3 puncta that exhibited diminished EGFP fluorescence), indicating their successful transition from autophagosomes to autolysosomes. Scale bars, 10 μm.


Figure 1 ( A ) β - Catenin is hyperphosphorylated during the mitotic phase . HeLa CCL2 cells were synchronized at the G1 / S phase using a double thymidine block and released using medium containing 200 ng / mL nocodazole ( Noc . ) . Cells were harvested at the indicated time points and treated with ( + ) or without ( - ) λ - phosphatase ( λ - PPase ) . The resulting cell lysates were used for immunoblot analysis . Hyperphosphorylated β - catenin ( red arrowhead ) was distinguished from non - / less - phosphorylated β - catenin ( blue arrowhead ) by slower migration during SDS - PAGE . Asterisk indicates the phosphorylated form of Cdc25C . ( F ) Total cell lysates from nocodazole ( Noc . ) - treated HeLa CCL2 cells were immunoprecipitated with two different Plk1 antibodies . Each antibody ( gifted from Kyung S . Lee , NIH / NCI ) recognized either Plk1 N - terminus ( N - term . Ab . ) or C - terminus ( C - term . Ab . ) . The resulting precipitates were subjected to immunoblot analysis . Hyperphosphorylated β - catenin ( red arrowhead ) was distinguished from non - / less - phosphorylated β - catenin ( blue arrowhead ) by slower migration during SDS - PAGE . ( G ) Lysates from nocodazole ( Noc . ) - treated HeLa CCL2 cells were mixed with either control ( phosphate - buffered saline only , + PBS ) , 10 μg / ml of high - affinity Plk1 PBD binding phospho - peptide ( PLHSpT , + P ) , or 10 μg / ml of non - phospho - peptide ( PLHST , + NP ) . The resulting cell lysates were immunoprecipitated with anti - Plk1 antibody . Precipitates were subjected to immunoblot analysis . Immunoblotting with anti - Dvl2 antibody represents a positive control of phospho - peptide ( PLHSpT , + P ) competition for Plk1 binding . Hyperphosphorylated β - catenin ( red arrowhead ) was distinguished from non - / less - phosphorylated β - catenin ( blue arrowhead ) by slower migration during SDS - PAGE .

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Figure 1(A) β-Catenin is hyperphosphorylated during the mitotic phase. HeLa CCL2 cells were synchronized at the G1/S phase using a double thymidine block and released using medium containing 200 ng/mL nocodazole (Noc.). Cells were harvested at the indicated time points and treated with (+) or without (-) λ-phosphatase (λ-PPase). The resulting cell lysates were used for immunoblot analysis. Hyperphosphorylated β-catenin (red arrowhead) was distinguished from non-/less-phosphorylated β-catenin (blue arrowhead) by slower migration during SDS-PAGE. Asterisk indicates the phosphorylated form of Cdc25C.(F) Total cell lysates from nocodazole (Noc.)-treated HeLa CCL2 cells were immunoprecipitated with two different Plk1 antibodies. Each antibody (gifted from Kyung S. Lee, NIH/NCI) recognized either Plk1 N-terminus (N-term. Ab.) or C-terminus (C-term. Ab.). The resulting precipitates were subjected to immunoblot analysis. Hyperphosphorylated β-catenin (red arrowhead) was distinguished from non-/less-phosphorylated β-catenin (blue arrowhead) by slower migration during SDS-PAGE.(G) Lysates from nocodazole (Noc.)-treated HeLa CCL2 cells were mixed with either control (phosphate-buffered saline only, +PBS), 10 μg/ml of high-affinity Plk1 PBD binding phospho-peptide (PLHSpT, +P), or 10 μg/ml of non-phospho-peptide (PLHST, +NP). The resulting cell lysates were immunoprecipitated with anti-Plk1 antibody. Precipitates were subjected to immunoblot analysis. Immunoblotting with anti-Dvl2 antibody represents a positive control of phospho-peptide (PLHSpT, +P) competition for Plk1 binding. Hyperphosphorylated β-catenin (red arrowhead) was distinguished from non-/less-phosphorylated β-catenin (blue arrowhead) by slower migration during SDS-PAGE.


Figure 2 ( C ) Bacterially produced β - catenin T1 WT , T3 WT , and corresponding alanine ( " A " ) mutants were purified and reacted with Flag - Plk1 WT in the presence of [ γ - 32P ] ATP . Incorporation of 32P onto each " A " mutant of β - catenin was confirmed by SDS - PAGE followed by autoradiography analysis . ( H ) HeLa CCL2 cells were treated for 18 h with 200 ng / mL nocodazole followed by treatment with either DMSO ( control ) or BI 2536 for an additional 1 h . The resulting cells were lysed with 2 × Laemmli SDS sample buffer and used for immunoblot analysis with the indicated antibodies .

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Figure 2(C) Bacterially produced β-catenin T1 WT, T3 WT, and corresponding alanine ("A") mutants were purified and reacted with Flag-Plk1 WT in the presence of [γ-32P]ATP. Incorporation of 32P onto each "A" mutant of β-catenin was confirmed by SDS-PAGE followed by autoradiography analysis.(H) HeLa CCL2 cells were treated for 18 h with 200 ng/mL nocodazole followed by treatment with either DMSO (control) or BI 2536 for an additional 1 h. The resulting cells were lysed with 2× Laemmli SDS sample buffer and used for immunoblot analysis with the indicated antibodies.


Figure 3 ( C ) Asynchronously growing HeLa CCL2 cells were immunostained with the indicated antibodies as described in ( A ) , and the midbody localization of β - catenin / β - catenin p - S60 in late telophase cells was observed using a confocal laser microscope ( Zeiss LSM 700 ) . Scale bars , 10 μm .

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Figure 3(C) Asynchronously growing HeLa CCL2 cells were immunostained with the indicated antibodies as described in (A), and the midbody localization of β-catenin/β-catenin p-S60 in late telophase cells was observed using a confocal laser microscope (Zeiss LSM 700). Scale bars, 10 μm.


Figure 4 ( A ) Asynchronously growing HeLa CCL2 cells were immunostained with the indicated antibodies , and early telophase cells were observed using a super - resolution confocal laser scanning microscope ( LSM - 880 with Airyscan ) ( left panel ) . Scale bars , 10 μm . Linear signal profiles of β - catenin / Plk1 / β - catenin p - S60 / F - actin in the contractile ring region from the XY plane of immunostained early telophase cells ( right panel ) . All immunofluorescence images and linear signal profiles show representative data of more than 50 telophase cells obtained from three independent experiments . The value of the relative neck thickness is shown in the lower left corner of the merged images ( A ) .

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Figure 4(A) Asynchronously growing HeLa CCL2 cells were immunostained with the indicated antibodies, and early telophase cells were observed using a super-resolution confocal laser scanning microscope (LSM-880 with Airyscan) (left panel). Scale bars, 10 μm. Linear signal profiles of β-catenin/Plk1/β-catenin p-S60/F-actin in the contractile ring region from the XY plane of immunostained early telophase cells (right panel). All immunofluorescence images and linear signal profiles show representative data of more than 50 telophase cells obtained from three independent experiments. The value of the relative neck thickness is shown in the lower left corner of the merged images (A).


Figure 5HeLa CCL2 cells stably expressing control vector only ( + vector ) , β - catenin WT ( + WTR ) , S60A mutant ( + S60AR ) , or S60D mutant ( + S60DR ) were generated using lentiviral infection and then infected with β - catenin knockdown lentivirus ( shβ - catenin ) . A dot density graph with a box plot shows the percentage ( % ) of cells with cytokinesis - defective phenotypes ( C ) . The box plot represents : central blue band , Median ; box , Interquartile range ; upper whisker , Maximum observation below upper fence ; lower whisker , Minimum observation above lower fence ; dot , Outlier . Each symbol in the graph represents a percentage of more than 30 cells from three independent experiments . * * * , p < 0 . 001 ( one - way ANOVA ) ; Scale bars , 10 μm . HeLa CCL2 cells transiently transfected with either GFP - β - catenin WT or GFP - β - catenin S60A were monitored during cell cycle progression by live cell imaging ( E ) . The subcellular localizations of GFP - tagged β - catenin WT or S60A mutant construct were confirmed by immunofluorescence with anti - GFP antibody . The cells were additionally stained for Plk1 ( see Appendix Figure S5A ) . The specific localization of GFP - β - catenin WT at the midbody was observed under a confocal laser microscope with high magnification ( E , left panel ) . Cytokinesis - defective cellular phenotypes in each transfected HeLa cell were monitored ( E , right panel )

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Figure 5HeLa CCL2 cells stably expressing control vector only (+vector), β-catenin WT (+WTR), S60A mutant (+S60AR), or S60D mutant (+S60DR) were generated using lentiviral infection and then infected with β-catenin knockdown lentivirus (shβ-catenin). A dot density graph with a box plot shows the percentage (%) of cells with cytokinesis-defective phenotypes (C). The box plot represents: central blue band, Median; box, Interquartile range; upper whisker, Maximum observation below upper fence; lower whisker, Minimum observation above lower fence; dot, Outlier. Each symbol in the graph represents a percentage of more than 30 cells from three independent experiments. ***, p<0.001 (one-way ANOVA); Scale bars, 10 μm.HeLa CCL2 cells transiently transfected with either GFP-β-catenin WT or GFP-β-catenin S60A were monitored during cell cycle progression by live cell imaging (E). The subcellular localizations of GFP-tagged β-catenin WT or S60A mutant construct were confirmed by immunofluorescence with anti-GFP antibody. The cells were additionally stained for Plk1 (see Appendix Figure S5A). The specific localization of GFP-β-catenin WT at the midbody was observed under a confocal laser microscope with high magnification (E, left panel). Cytokinesis-defective cellular phenotypes in each transfected HeLa cell were monitored (E, right panel)


Figure 6 ( E , F ) HeLa CCL2 cells infected with the indicated lentiviruses were co - transfected with HA - Ect2 and Flag - Plk1 . Cells were then treated with nocodazole ( 200 ng / mL ) for 18 h and released from cell cycle arrest by incubation in fresh medium for 6 h . The resulting cells were used for immunoprecipitation analysis using anti - Flag ( E ) or anti - HA ( F ) antibodies . Precipitated beads were used for immunoblot analysis using the indicated antibodies . ( G , H ) HeLa CCl2 cells infected with either sh - control luciferase ( shGL ) or shβ - catenin lentiviruses were co - immunostained with anti - β - catenin and anti - Ect2 antibodies . Localization of β - catenin and Ect2 in the midbody of telophase cells was observed using a confocal microscope ( G ) and the signal intensity of Ect2 was monitored in the midbody of each knockdown telophase cell ( H ) . Arrows indicate total β - catenin and Ect2 located at the midbody in telophase HeLa CCL2 cells ( G ) . A dot density graph with a box plot shows the signal intensity of midbody - localized Ect2 in telophase cells . The box plot represents : central blue band , Median ; box , Interquartile range ; upper whisker , Maximum observation below upper fence ; lower whisker , Minimum observation above lower fence ; dot , Outlier . Approximately 70 cells from three independent experiments were measured . * * * , p < 0 . 001 ( unpaired two - tailed t test ) ; Scale bars , 10 μm . The value of the relative neck thickness is shown in the lower left corner of the merged images ( G ) .

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Figure 6(E, F) HeLa CCL2 cells infected with the indicated lentiviruses were co-transfected with HA-Ect2 and Flag-Plk1. Cells were then treated with nocodazole (200 ng/mL) for 18 h and released from cell cycle arrest by incubation in fresh medium for 6 h. The resulting cells were used for immunoprecipitation analysis using anti-Flag (E) or anti-HA (F) antibodies. Precipitated beads were used for immunoblot analysis using the indicated antibodies.(G, H) HeLa CCl2 cells infected with either sh-control luciferase (shGL) or shβ-catenin lentiviruses were co-immunostained with anti-β-catenin and anti-Ect2 antibodies. Localization of β-catenin and Ect2 in the midbody of telophase cells was observed using a confocal microscope (G) and the signal intensity of Ect2 was monitored in the midbody of each knockdown telophase cell (H). Arrows indicate total β-catenin and Ect2 located at the midbody in telophase HeLa CCL2 cells (G). A dot density graph with a box plot shows the signal intensity of midbody-localized Ect2 in telophase cells. The box plot represents: central blue band, Median; box, Interquartile range; upper whisker, Maximum observation below upper fence; lower whisker, Minimum observation above lower fence; dot, Outlier. Approximately 70 cells from three independent experiments were measured. ***, p<0.001 (unpaired two-tailed t test); Scale bars, 10 μm. The value of the relative neck thickness is shown in the lower left corner of the merged images (G).


Figure 7HeLa CCL2 cells treated with DMSO ( control ) or the Plk1 inhibitor , BI 2536 ( 1 μM ) for 20 min were immunostained with a combination of either anti - total β - catenin and anti - Ect2 antibodies or anti - β - catenin p - S60 and anti - Ect2 antibodies ( A ) . Scale bars , 10 μm . ( C ) After 18 h of nocodazole ( 200 ng / mL ) - induced cell cycle arrest , HeLa CCL2 cells transfected with HA - Ect2 were released by incubation in fresh medium for 2 h and treated with either DMSO ( control ) or Plk1 inhibitor ( BI 2536 ) for the indicated times . The resulting cells were used for immunoprecipitation analysis using anti - HA antibody , and immunoprecipitates were separated by SDS - PAGE followed by immunoblot analysis with the indicated antibodies . The band intensity of β - catenin bound to HA - Ect2 was quantified with ImageJ and normalized to the signal intensity of immunoprecipitated HA - Ect2 , and the relative values are shown below the bands . ( G ) HeLa CCL2 cells were transfected with either wild type Flag − β - catenin ( + WT ) , Flag - β - catenin S60A mutant ( + S60A ) , or Flag - β - catenin S60D mutant ( + S60D ) , then treated with 200 ng / mL nocodazole for 18 h ( metaphase ) and released by incubation in fresh medium for 2 h ( telophase ) . Cell lysates were used for pull - down analysis with rhotekin - RBD beads , and the pulled down beads were separated by 12 % SDS - PAGE followed by immunoblot analysis with the indicated antibodies . The membrane was then stained with Coomassie ( CBB ) to determine the amount of RBD precipitated .

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Figure 7HeLa CCL2 cells treated with DMSO (control) or the Plk1 inhibitor, BI 2536 (1 μM) for 20 min were immunostained with a combination of either anti-total β-catenin and anti-Ect2 antibodies or anti-β-catenin p-S60 and anti-Ect2 antibodies (A). Scale bars, 10 μm.(C) After 18 h of nocodazole (200 ng/mL)-induced cell cycle arrest, HeLa CCL2 cells transfected with HA-Ect2 were released by incubation in fresh medium for 2 h and treated with either DMSO (control) or Plk1 inhibitor (BI 2536) for the indicated times. The resulting cells were used for immunoprecipitation analysis using anti-HA antibody, and immunoprecipitates were separated by SDS-PAGE followed by immunoblot analysis with the indicated antibodies. The band intensity of β-catenin bound to HA-Ect2 was quantified with ImageJ and normalized to the signal intensity of immunoprecipitated HA-Ect2, and the relative values are shown below the bands.(G) HeLa CCL2 cells were transfected with either wild type Flag−β-catenin (+WT), Flag-β-catenin S60A mutant (+S60A), or Flag-β-catenin S60D mutant (+S60D), then treated with 200 ng/mL nocodazole for 18 h (metaphase) and released by incubation in fresh medium for 2 h (telophase). Cell lysates were used for pull-down analysis with rhotekin-RBD beads, and the pulled down beads were separated by 12 % SDS-PAGE followed by immunoblot analysis with the indicated antibodies. The membrane was then stained with Coomassie (CBB) to determine the amount of RBD precipitated.


figf1 ( a ) Representative differential interference contrast microscopy images of midgut cells from wild - type animals at the early third instar larval ( Early 3rd ) , late third instar larval ( Late 3rd ) and at puparium formation ( white prepupal , WPP ) stages . ( b ) Autophagy detected by formation of mCherry - Atg8a punctate spots in midgut cells from wild - type animals at indicated stages . Representative images are shown . ( c ) Midguts from control Atg18KG03090 / wild - type ( Atg18 / + ) , n = 14 , and Atg18KG03090 / Df ( 3L ) 6112 mutant ( Atg18 / Df ) , n = 11 , animals at puparium formation analysed by differential interference contrast microscopy . Representative images are shown . ( d ) Wild - type , control ( Atg18KG03090 / + ) and Atg18 mutant ( Atg18 / Df ) midgut cell size quantification ( μm2 ) at indicated stages ; n = 10 animal intestines per genotype with 5 cells measured per intestine per stage . ( e ) Differential interference contrast images of midgut cells from Atg2EP3697 / wild - type control ( Atg2 / + ) and Atg2EP3697 / Df ( 3L ) 6091 mutant ( Atg2 / Df ) animals at puparium formation . Representative images are shown . ( f ) Cell size quantification ( μm2 ) from e , n = 12 ( Atg2 / + ) and n = 7 ( Atg2 / Df ) animal intestines per genotype with 5 cells measured per intestine . ( g - j ) Representative TEM images of intestine cells 2 h after puparium formation . ( g , i ) Control Atg18KG03090 / wild - type ( Atg18 / + , g ) and control Atg2EP3697 / wild - type ( Atg2 / + , i ) cells with an enlarged image showing a double - membrane structure ( arrowhead ) that surrounds a mitochondrion and endoplasmic reticulum . Arrows indicate autolysosomes . ( h , j ) Atg18KG03090 / Df ( 3L ) 6112mutant ( Atg18 / Df , h ) and Atg2EP3697 / Df ( Atg2 / Df , j ) mutant cells lacking autophagic structures . Quantification is shown as mean ± s . d . Scale bars , 20 μm ( a - c and e ) and 1 μm ( g - j ) .

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figf1(a) Representative differential interference contrast microscopy images of midgut cells from wild-type animals at the early third instar larval (Early 3rd), late third instar larval (Late 3rd) and at puparium formation (white prepupal, WPP) stages.(b) Autophagy detected by formation of mCherry-Atg8a punctate spots in midgut cells from wild-type animals at indicated stages. Representative images are shown.(c) Midguts from control Atg18KG03090/wild-type (Atg18/+), n = 14, and Atg18KG03090/Df(3L)6112 mutant (Atg18/Df), n = 11, animals at puparium formation analysed by differential interference contrast microscopy. Representative images are shown. (d) Wild-type, control (Atg18KG03090/+) and Atg18 mutant (Atg18/Df) midgut cell size quantification (μm2) at indicated stages; n = 10 animal intestines per genotype with 5 cells measured per intestine per stage.(e) Differential interference contrast images of midgut cells from Atg2EP3697/wild-type control (Atg2/+) and Atg2EP3697/Df(3L)6091 mutant (Atg2/Df) animals at puparium formation. Representative images are shown. (f) Cell size quantification (μm2) from e, n = 12 (Atg2/+) and n = 7 (Atg2/Df) animal intestines per genotype with 5 cells measured per intestine.(g-j) Representative TEM images of intestine cells 2 h after puparium formation. (g,i) Control Atg18KG03090/wild-type (Atg18/+, g) and control Atg2EP3697/wild-type (Atg2/+, i) cells with an enlarged image showing a double-membrane structure (arrowhead) that surrounds a mitochondrion and endoplasmic reticulum. Arrows indicate autolysosomes. (h,j) Atg18KG03090/Df(3L)6112mutant (Atg18/Df, h) and Atg2EP3697/Df (Atg2/Df, j) mutant cells lacking autophagic structures. Quantification is shown as mean ± s.d. Scale bars, 20 μm (a-c and e) and 1 μm (g-j).


figf2 ( a ) Midguts dissected from animals expressing Atg18IR specifically in DsRed - marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast ( DIC ) microscopy . Representative images are shown . ( b ) Quantification ( μm2 ) from a , n = 12 animal intestines per genotype with 1 - 5 cells measured per intestine . ( c ) Midguts expressing mCherry - Atg8a in all cells , and expressing Atg18IR specifically in GFP - marked cell clones dissected from animals at 2 h after puparium formation . Representative images are shown . ( d ) Midguts dissected from animals at puparium formation that contain an Atg1Δ3D loss - of - function mutant cell clone ( lacking GFP ) and analysed by fluorescence and differential interference contrast microscopy . Wild - type ( + / + ) control cell possesses stronger GFP and heterozygous Atg1Δ3D / wild - type ( Δ3D / + ) cells have weaker GFP . Representative images are shown . ( e ) Quantification ( μm2 ) from d , n = 7 ( + / + ) , n = 8 ( Δ3D / + ) and n = 8 ( Δ3D / Δ3D ) animal intestines per genotype with 1 - 5 cells measured per intestine . ( f ) Midguts dissected from animals expressing mCherry - Atg8a in all cells , and expressing Atg1IR specifically in GFP - marked clones of cells at 2 h after puparium formation . Representative images are shown . ( g - j ) Representative cut views from three - dimensional reconstitution images from wild - type ( g , h ) and Atg1 knockdown ( i , j ) cells at early third instar larval stage ( g , i ) and at puparium formation ( h , j ) . ( k ) Quantification of cell volume ( μm3 ) from g - j , n = 5 animal intestines per genotype with 1 - 5 cells measured per intestine per stage . Black columns : early third instar larval stage . White columns : puparium formation stage . Quantification is shown as mean ± s . d . NS , not significant . Scale bars , 20 μm .

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figf2(a) Midguts dissected from animals expressing Atg18IR specifically in DsRed-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast (DIC) microscopy. Representative images are shown. (b) Quantification (μm2) from a, n = 12 animal intestines per genotype with 1-5 cells measured per intestine.(c) Midguts expressing mCherry-Atg8a in all cells, and expressing Atg18IR specifically in GFP-marked cell clones dissected from animals at 2 h after puparium formation. Representative images are shown.(d) Midguts dissected from animals at puparium formation that contain an Atg1Δ3D loss-of-function mutant cell clone (lacking GFP) and analysed by fluorescence and differential interference contrast microscopy. Wild-type (+/+) control cell possesses stronger GFP and heterozygous Atg1Δ3D/wild-type (Δ3D/+) cells have weaker GFP. Representative images are shown. (e) Quantification (μm2) from d, n = 7 (+/+), n = 8 (Δ3D/+) and n = 8 (Δ3D/Δ3D) animal intestines per genotype with 1-5 cells measured per intestine.(f) Midguts dissected from animals expressing mCherry-Atg8a in all cells, and expressing Atg1IR specifically in GFP-marked clones of cells at 2 h after puparium formation. Representative images are shown.(g-j) Representative cut views from three-dimensional reconstitution images from wild-type (g,h) and Atg1 knockdown (i,j) cells at early third instar larval stage (g,i) and at puparium formation (h,j). (k) Quantification of cell volume (μm3) from g-j, n = 5 animal intestines per genotype with 1-5 cells measured per intestine per stage. Black columns: early third instar larval stage. White columns: puparium formation stage. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 20 μm.


figf3 ( a ) Midguts dissected from animals at puparium formation that contain Vps34Δm22 loss - of - function mutant cell clones ( lacking GFP ) and analysed by fluorescence and differential interference contrast ( DIC ) microscopy . Wild - type ( + / + ) control cells possess stronger GFP and heterozygous Vps34Δm22 / wild - type ( Δm22 / + ) cells have weaker GFP . Representative images are shown . ( b ) Quantification ( μm2 ) from a , n = 14 animal intestines per genotype with 1 - 5 cells measured per intestine . ( c ) Midguts dissected from animals expressing Atg8aIR specifically in GFP - marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy . Representative images are shown . ( d ) Cell size quantification ( μm2 ) from c , n = 10 animal intestines per genotype with 1 - 5 cells measured per intestine . ( e ) Midguts dissected from animals expressing Atg12IR specifically in DsRed - marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy . Representative images are shown . ( f ) Cell size quantification ( μm2 ) from e , n = 14 animal intestines per genotype with 1 - 5 cells measured per intestine . ( g - i ) Midguts dissected from early third instar larvae that mis - express Atg1GS10797 ( Atg1 ; g , GFP in nucleus and cytoplasm ) , both Atg1 + Atg8aIR ( h ) , or both Atg1 + Atg7IR ( i ) and stained to detect Discs large ( green ) on the cortex of all cells . Representative images are shown . ( g ' and i ' ) DIC images of midguts expressing mCherry - Atg8a in all cells , Atg1 ( g ' ) , and Atg1 + Atg7IR ( i ' ) in GFP - marked clones of cells . mCherry - Atg8a puncta are shown in insets . Representative images are shown . Quantification is shown as mean ± s . d . Scale bars , 20 μm .

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 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 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 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

figf3(a) Midguts dissected from animals at puparium formation that contain Vps34Δm22 loss-of-function mutant cell clones (lacking GFP) and analysed by fluorescence and differential interference contrast (DIC) microscopy. Wild-type (+/+) control cells possess stronger GFP and heterozygous Vps34Δm22/wild-type (Δm22/+) cells have weaker GFP. Representative images are shown. (b) Quantification (μm2) from a, n = 14 animal intestines per genotype with 1-5 cells measured per intestine.(c) Midguts dissected from animals expressing Atg8aIR specifically in GFP-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (d) Cell size quantification (μm2) from c, n = 10 animal intestines per genotype with 1-5 cells measured per intestine.(e) Midguts dissected from animals expressing Atg12IR specifically in DsRed-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (f) Cell size quantification (μm2) from e, n = 14 animal intestines per genotype with 1-5 cells measured per intestine.(g-i) Midguts dissected from early third instar larvae that mis-express Atg1GS10797 (Atg1; g, GFP in nucleus and cytoplasm), both Atg1 +Atg8aIR (h), or both Atg1 +Atg7IR (i) and stained to detect Discs large (green) on the cortex of all cells. Representative images are shown. (g' and i') DIC images of midguts expressing mCherry-Atg8a in all cells, Atg1 (g'), and Atg1+Atg7IR (i') in GFP-marked clones of cells. mCherry-Atg8a puncta are shown in insets. Representative images are shown. Quantification is shown as mean ± s.d. Scale bars, 20 μm.


figf4 ( a ) Midguts dissected from animals at puparium formation that contain Atg7d4 loss - of - function mutant cell clones ( lacking GFP ) and analysed by fluorescence and differential interference contrast ( DIC ) microscopy . Wild - type ( + / + ) control cells possess stronger GFP and heterozygous Atg7d4 / wild - type ( d4 / + ) cells have weaker GFP . Representative images are shown . ( b ) Cell size quantification ( μm2 ) from a , n = 8 animal intestines per genotype with 1 - 5 cells measured per intestine . ( c ) Midguts expressing mCherry - Atg8a in all cells , and with Atg7d4 loss - of - function clone cells ( lacking GFP ) at 2 h after puparium formation . Representative images are shown . ( e ) Midguts dissected from animals expressing Atg12IR specifically in DsRed - marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy . Representative images are shown . ( f ) Cell size quantification ( μm2 ) from e , n = 14 animal intestines per genotype with 1 - 5 cells measured per intestine . ( f ) Midguts expressing GFP - Atg5 in enterocytes ( larger nuclei ) with an Atg7d4 loss - of - function clone cell ( lacking RFP ) at puparium formation . Representative images are shown . ( g , h ) Representative TEM images of intestine cells 2 h after puparium formation . Arrows indicate autolysosomes . ( g , h ) Control ( Atg7d30 / d14 ; g ) and Atg7 mutant ( Atg7d77 / d14 ; h ) cells both contain autophagic structures . ( h , right ) Enlarged Atg7 mutant cell image of a double - membrane autophagosome ( arrowhead ) surrounding a mitochondrion . Quantification is shown as mean ± s . d . NS , not significant . Scale bars , 20 μm ( a , c , d and f ) and 1 μm ( g , h ) .

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 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 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 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

figf4(a) Midguts dissected from animals at puparium formation that contain Atg7d4 loss-of-function mutant cell clones (lacking GFP) and analysed by fluorescence and differential interference contrast (DIC) microscopy. Wild-type (+/+) control cells possess stronger GFP and heterozygous Atg7d4/wild-type (d4/+) cells have weaker GFP. Representative images are shown. (b) Cell size quantification (μm2) from a, n = 8 animal intestines per genotype with 1-5 cells measured per intestine.(c) Midguts expressing mCherry-Atg8a in all cells, and with Atg7d4 loss-of-function clone cells (lacking GFP) at 2 h after puparium formation. Representative images are shown.(e) Midguts dissected from animals expressing Atg12IR specifically in DsRed-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (f) Cell size quantification (μm2) from e, n = 14 animal intestines per genotype with 1-5 cells measured per intestine.(f) Midguts expressing GFP-Atg5 in enterocytes (larger nuclei) with an Atg7d4 loss-of-function clone cell (lacking RFP) at puparium formation. Representative images are shown.(g,h) Representative TEM images of intestine cells 2 h after puparium formation. Arrows indicate autolysosomes. (g,h) Control (Atg7d30/d14; g) and Atg7 mutant (Atg7d77/d14; h) cells both contain autophagic structures. (h, right) Enlarged Atg7 mutant cell image of a double-membrane autophagosome (arrowhead) surrounding a mitochondrion. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 20 μm (a,c,d and f) and 1 μm (g,h).


figf5 ( a ) Quantification of mitochondria numbers from Atg18 control ( Atg18KG03090 / wild type ) and mutant ( Atg18KG03090 / Df ( 3L ) 6112 ) or Atg7 control ( Atg7d30 / d14 ) and mutant ( Atg7d77 / d14 ) TEM images . Mitochondria were quantified from 2 distinct 25 μm2 regions per cell from 2 cells per animal from at least 3 different animals per genotype . ( b , c ) Midguts dissected from either early third instar larvae ( b ) or at puparium formation ( c ) that express GFP - labelled mitochondria in all cells and contain an Atg1Δ3D loss - of - function mutant cell clone ( lacking RFP ) . Wild - type control cells possess stronger RFP and heterozygous cells have weaker RFP . Representative images are shown . Quantification is shown as mean ± s . d . NS , not significant . Scale bars , 0 μm .

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

figf5(a) Quantification of mitochondria numbers from Atg18 control (Atg18KG03090/wild type) and mutant (Atg18KG03090/Df(3L)6112) or Atg7 control (Atg7d30/d14) and mutant (Atg7d77/d14) TEM images. Mitochondria were quantified from 2 distinct 25 μm2 regions per cell from 2 cells per animal from at least 3 different animals per genotype.(b,c) Midguts dissected from either early third instar larvae (b) or at puparium formation (c) that express GFP-labelled mitochondria in all cells and contain an Atg1Δ3D loss-of-function mutant cell clone (lacking RFP). Wild-type control cells possess stronger RFP and heterozygous cells have weaker RFP. Representative images are shown. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 0 μm.


figf6 ( a ) Midguts dissected from animals expressing Uba1IR specifically in GFP - marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast ( DIC ) microscopy . Representative images are shown . ( b ) Cell size quantification ( μm2 ) from a ; n = 16 animal intestines per genotype with 1 - 5 cells measured per intestine . ( c ) Midguts dissected from animals at puparium formation that contain Uba1H33 loss - of - function mutant cell clones ( lacking RFP ) and analysed by fluorescence and differential interference contrast microscopy . Wild - type ( + / + ) control cells possess stronger RFP and heterozygous Uba1H33 / wild - type ( H33 / + ) cells have weaker RFP . Representative images are shown . ( d ) Quantification ( μm2 ) from c , n = 8 ( + / + ) , n = 11 ( H33 / + ) and n = 11 ( H33 / H33 ) animal intestines per genotype with 1 - 5 cells measured per intestine . ( e ) Midguts dissected from animals at puparium formation that contain Uba1H33 loss - of - function MARCM mutant cell clones ( GFP - positive ) that also have mCherry - Atg8a expressed in all cells and analysed by fluorescence microscopy . Control wild - type and heterozygous cells have no GFP . Representative images are shown . ( f ) Midguts expressing GFP - Atg5 in enterocytes ( larger nuclei ) , and with an Uba1H33 loss - of - function clone cell ( lacking RFP ) at puparium formation analysed by fluorescence microscopy . Representative images are shown . ( g ) Midguts dissected from animals at puparium formation that contain Uba1H33 loss - of - function MARCM mutant cell clones ( GFP - positive ) that are stained with p62 antibody and analysed by fluorescence microscopy . Control wild - type and heterozygous cells have no GFP . Representative images are shown . ( h ) Midguts dissected from animals expressing Dts7 , a dominant temperature - sensitive mutant of the β2 subunit of the proteasome , specifically in GFP - marked cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy . Representative images are shown . ( i ) Cell size quantification ( μm2 ) from h , n = 6 animal intestines per genotype with 1 - 5 cells measured per intestine . Quantification is shown as mean ± s . d . NS , not significant . Scale bars , 20 μm .

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 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 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 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 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

figf6(a) Midguts dissected from animals expressing Uba1IR specifically in GFP-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast (DIC) microscopy. Representative images are shown. (b) Cell size quantification (μm2) from a; n = 16 animal intestines per genotype with 1-5 cells measured per intestine.(c) Midguts dissected from animals at puparium formation that contain Uba1H33 loss-of-function mutant cell clones (lacking RFP) and analysed by fluorescence and differential interference contrast microscopy. Wild-type (+/+) control cells possess stronger RFP and heterozygous Uba1H33/wild-type (H33/+) cells have weaker RFP. Representative images are shown. (d) Quantification (μm2) from c, n = 8 (+/+), n = 11 (H33/+) and n = 11 (H33/H33) animal intestines per genotype with 1-5 cells measured per intestine.(e) Midguts dissected from animals at puparium formation that contain Uba1H33 loss-of-function MARCM mutant cell clones (GFP-positive) that also have mCherry-Atg8a expressed in all cells and analysed by fluorescence microscopy. Control wild-type and heterozygous cells have no GFP. Representative images are shown.(f) Midguts expressing GFP-Atg5 in enterocytes (larger nuclei), and with an Uba1H33 loss-of-function clone cell (lacking RFP) at puparium formation analysed by fluorescence microscopy. Representative images are shown.(g) Midguts dissected from animals at puparium formation that contain Uba1H33 loss-of-function MARCM mutant cell clones (GFP-positive) that are stained with p62 antibody and analysed by fluorescence microscopy. Control wild-type and heterozygous cells have no GFP. Representative images are shown.(h) Midguts dissected from animals expressing Dts7, a dominant temperature-sensitive mutant of the β2 subunit of the proteasome, specifically in GFP-marked cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (i) Cell size quantification (μm2) from h, n = 6 animal intestines per genotype with 1-5 cells measured per intestine. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 20 μm.