Figure 4 ( a ) Effect of compound WX - 340 on binding of recombinant murine uPA to PAI - 1 protein as assessed by ELISA , quantitative data are shown ( mean ± SEM for n = 3 experiments per group ; # p < 0 . 05 vs . drug vehicle ; One - way ANOVA ) . ( d ) Relative development rates and tumor weight in animals treated with WX - 340 a priori or therapeutically after 1 week after tumor cell injection on a daily basis as assessed in an orthotopic model of 4T1 breast cancer in WT mice ( mean ± SEM for n = 4 - 6 mice per group ; * p < 0 . 05 vs . drug vehicle ; One - way ANOVA ) . ( f ) Numbers of neutrophils and 4T1 tumor cells in lungs and brain of WT mice receiving WX - 340 or vehicle therapeutically after 1 week after tumor cell injection on a daily basis as assessed 14 days after intravenous injection of 4T1 tumor cells by multi - channel flow cytometry , quantitative data are shown ( mean ± SEM for n = 4 - 6 mice per group ; * p < 0 . 05 vs . drug vehicle ; t test ) .

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 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 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

Figure 4(a) Effect of compound WX-340 on binding of recombinant murine uPA to PAI-1 protein as assessed by ELISA, quantitative data are shown (mean±SEM for n=3 experiments per group; #p<0.05 vs. drug vehicle; One-way ANOVA).(d) Relative development rates and tumor weight in animals treated with WX-340 a priori or therapeutically after 1 week after tumor cell injection on a daily basis as assessed in an orthotopic model of 4T1 breast cancer in WT mice (mean±SEM for n=4-6 mice per group; *p<0.05 vs. drug vehicle; One-way ANOVA).(f) Numbers of neutrophils and 4T1 tumor cells in lungs and brain of WT mice receiving WX-340 or vehicle therapeutically after 1 week after tumor cell injection on a daily basis as assessed 14 days after intravenous injection of 4T1 tumor cells by multi-channel flow cytometry, quantitative data are shown (mean±SEM for n=4-6 mice per group; *p<0.05 vs. drug vehicle; t test).


Figure 1C Immunofluorescent analyses of USP33 ( red ) , the hypoxia marker CA9 ( green ) , and the stem cell marker SOX2 ( gray ) in human primary GBMs . Arrows indicate the USP33 + / CA9 + / SOX2 + cells . Scale bar , 80 µm . E Immunofluorescent analyses of USP33 ( red ) and the hypoxic cell population labeled by pimonidazole ( green ) in intracranial xenografts derived from T3359 GSCs . Mice bearing intracranial xenografts were injected intravenously ( tail vein ) with 60 mg / kg of pimonidazole and were sacrificed 2 hours post injection . Hypoxic cells were labeled with the FITC - conjugated mouse monoclonal anti - pimonidazole antibody . Scale bar , 80 µm . G Immunoblot analysis of USP33 , HIF2α and HIF1α protein levels in GSCs , NSTCs and NPCs in normoxic and hypoxic conditions . Hypoxia ( 5 % O2 ) relative to normoxia ( 20 % O2 ) clearly induced USP33 and HIF2α expression in GSCs but not NSTCs or NPCs . The mild hypoxia of 5 % O2 showed negligible induction of HIF1α in all cells .

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Figure 1C Immunofluorescent analyses of USP33 (red), the hypoxia marker CA9 (green), and the stem cell marker SOX2 (gray) in human primary GBMs. Arrows indicate the USP33+/CA9+/SOX2+ cells. Scale bar, 80 µm.E Immunofluorescent analyses of USP33 (red) and the hypoxic cell population labeled by pimonidazole (green) in intracranial xenografts derived from T3359 GSCs. Mice bearing intracranial xenografts were injected intravenously (tail vein) with 60 mg/kg of pimonidazole and were sacrificed 2 hours post injection. Hypoxic cells were labeled with the FITC-conjugated mouse monoclonal anti-pimonidazole antibody. Scale bar, 80 µm.G Immunoblot analysis of USP33, HIF2α and HIF1α protein levels in GSCs, NSTCs and NPCs in normoxic and hypoxic conditions. Hypoxia (5% O2) relative to normoxia (20% O2) clearly induced USP33 and HIF2α expression in GSCs but not NSTCs or NPCs. The mild hypoxia of 5% O2 showed negligible induction of HIF1α in all cells.


Figure 2A , Representative images of immunofluorescent analyses of USP33 ( red ) and HIF2α ( green ) in human primary GBMs . Frozen sections of human GBMs were immunostained with antibodies against USP33 and HIF2α , and counterstained with DAPI to show nuclei ( blue ) . Scale bar , 40 μm . ( n = 5 sections ; mean ± s . e . m . ) . F Immunoblot analysis of ectopic HIF2α protein levels in 293T cells with gradually increased amount of ectopic USP33 . 0 . 5 µg of HA - tagged HIF2α plasmids together with the increased amounts of GFP - tagged USP33 plasmids ( 0 , 0 . 1 , 0 . 2 or 0 . 3 μg ) were introduced into 293T cells . Forty - eight hours post transfection , levels of ectopic HIF2α - HA and GFP - USP33 were analyzed by immunoblot analysis . Concomitantly increased levels of ectopic HIF2α and USP33 expression indicated the positive regulation of HIF2α protein by USP33 . G Immunoblot analysis of ectopic HIF2α protein levels in 293T cells expressing ectopic wild type ( WT ) USP33 or the catalytically dead ( CD ) USP33 . HA - tagged HIF2α plasmids ( 0 . 5 µg ) were introduced into 293T cells alone or together with GFP - tagged WT or CD USP33 plasmids ( 0 . 2 µg ) . Levels of ectopic HIF2α - HA and GFP - USP33 were analyzed 48 hours post transfection . Expression of the wild type USP33 but not the catalytically dead USP33 increased the ectopic HIF2α protein levels .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Representative images of immunofluorescent analyses of USP33 (red) and HIF2α (green) in human primary GBMs. Frozen sections of human GBMs were immunostained with antibodies against USP33 and HIF2α, and counterstained with DAPI to show nuclei (blue). Scale bar, 40 μm. (n = 5 sections; mean ± s.e.m.).F Immunoblot analysis of ectopic HIF2α protein levels in 293T cells with gradually increased amount of ectopic USP33. 0.5 µg of HA-tagged HIF2α plasmids together with the increased amounts of GFP-tagged USP33 plasmids (0, 0.1, 0.2 or 0.3 μg) were introduced into 293T cells. Forty-eight hours post transfection, levels of ectopic HIF2α-HA and GFP-USP33 were analyzed by immunoblot analysis. Concomitantly increased levels of ectopic HIF2α and USP33 expression indicated the positive regulation of HIF2α protein by USP33. G Immunoblot analysis of ectopic HIF2α protein levels in 293T cells expressing ectopic wild type (WT) USP33 or the catalytically dead (CD) USP33. HA-tagged HIF2α plasmids (0.5 µg) were introduced into 293T cells alone or together with GFP-tagged WT or CD USP33 plasmids (0.2 µg). Levels of ectopic HIF2α-HA and GFP-USP33 were analyzed 48 hours post transfection. Expression of the wild type USP33 but not the catalytically dead USP33 increased the ectopic HIF2α protein levels.


Figure 3A , Co - immunoprecipitation ( Co - IP ) to detect the interaction between endogenous HIF2α and USP33 in GSCs in response to low oxygen . GSCs were cultured under 20 % or 5 % oxygen for 24 hours before harvest for the Co - IP . Cell lysate was subjected to immunoprecipitation with anti - HIF2α antibodies . Co - immunoprecipitated products were immunoblotted with the indicated antibodies . An increased interaction between HIF2α and USP33 was detected in GSCs cultured under 5 % oxygen relative to 20 % oxygen . D Co - immunoprecipitation ( Co - IP ) to detect the interaction between endogenous HIF2α and USP33 in GSCs in response to CoCl2 treatment . GSCs were treated with CoCl2 ( 300 µM ) for 12 hours before harvest for the Co - IP . Cell lysate was subjected to immunoprecipitation with anti - USP33 antibodies . Co - immunoprecipitated products were immunoblotted with the indicated antibodies . An increased interaction between HIF2α and USP33 was detected in GSCs treated with CoCl2 mimicking hypoxia . G Co - immunoprecipitation to detect the interaction between HIF2α and the wild - type ( WT ) or the catalytically dead ( CD ) USP33 . Ectopic HA - tagged HIF2α were expressed in 293T cells with GFP - tagged wild - type or catalytically dead USP33 . Cell lysates were subjected to immunoprecipitation with anti - HA antibodies . Co - immunoprecipitated products immunoblotted with the indicated antibodies . The catalytically dead USP33 relative to the wild type USP33 showed a stronger affinity to HIF2α .

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 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 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

Figure 3A, Co-immunoprecipitation (Co-IP) to detect the interaction between endogenous HIF2α and USP33 in GSCs in response to low oxygen. GSCs were cultured under 20% or 5% oxygen for 24 hours before harvest for the Co-IP. Cell lysate was subjected to immunoprecipitation with anti-HIF2α antibodies. Co-immunoprecipitated products were immunoblotted with the indicated antibodies. An increased interaction between HIF2α and USP33 was detected in GSCs cultured under 5% oxygen relative to 20% oxygen.D Co-immunoprecipitation (Co-IP) to detect the interaction between endogenous HIF2α and USP33 in GSCs in response to CoCl2 treatment. GSCs were treated with CoCl2 (300 µM) for 12 hours before harvest for the Co-IP. Cell lysate was subjected to immunoprecipitation with anti-USP33 antibodies. Co-immunoprecipitated products were immunoblotted with the indicated antibodies. An increased interaction between HIF2α and USP33 was detected in GSCs treated with CoCl2 mimicking hypoxia.G Co-immunoprecipitation to detect the interaction between HIF2α and the wild-type (WT) or the catalytically dead (CD) USP33. Ectopic HA-tagged HIF2α were expressed in 293T cells with GFP-tagged wild-type or catalytically dead USP33. Cell lysates were subjected to immunoprecipitation with anti-HA antibodies. Co-immunoprecipitated products immunoblotted with the indicated antibodies. The catalytically dead USP33 relative to the wild type USP33 showed a stronger affinity to HIF2α.


Figure 4A Immunoblot analysis of the serine / threonine phosphorylation ( pS / T ) status of HIF2α in GSCs in response to 5 % oxygen . GSCs were cultured under 20 % or 5 % oxygen for 24 hours , and then harvested for immunoprecipitation with anti - HIF2α antibodies or IgG control . Immunoprecipitated endogenous HIF2α were immunoblotted with anti - pan - pS / T antibodies . An increased pS / T of HIF2α was detected in GSCs cultured under 5 % oxygen relative to 20 % oxygen . D Co - immunoprecipitation to detect the interaction between endogenous HIF2α and USP33 in GSCs after inhibition of ERK1 / 2 activity . GSCs were cultured under 20 % or 5 % oxygen for 24 hours , then treated with the MEK inhibitor U0126 ( 10µM ) for 30 minutes to inhibit ERK1 / 2 activity , and harvested for immunoprecipitation with anti - USP33 antibodies . Co - immunoprecipitated products were immunoblotted with the indicated antibodies . Relative to 20 % oxygen treatment , 5 % oxygen treatment promoted the interaction between HIF2α and USP33 in GSCs . However , inhibition of ERK1 / 2 activity by U0126 treatment suppressed the interaction between HIF2α and USP33 . G Co - immunoprecipitation to determine the serine phosphorylation sites in human HIF2α responsible for its binding to USP33 . Ectopic GFP - tagged USP33 along with HA - tagged wild type HIF2α or HIF2α mutants with indicated serine to alanine point mutations were expressed in 293T cells . Cell lysates were subjected to immunoprecipitation with anti - HA agaroses Co - immunoprecipitated products were immunoblotted with the indicated antibodies . The S484A mutation largely attenuated the interaction of HIF2α with USP33 , indicating that the serine 484 site in HIF2α was the most important phosphorylation site for the interaction between HIF2α and USP33 .

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 O O O O 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

Figure 4A Immunoblot analysis of the serine/threonine phosphorylation (pS/T) status of HIF2α in GSCs in response to 5% oxygen. GSCs were cultured under 20% or 5% oxygen for 24 hours, and then harvested for immunoprecipitation with anti-HIF2α antibodies or IgG control. Immunoprecipitated endogenous HIF2α were immunoblotted with anti-pan-pS/T antibodies. An increased pS/T of HIF2α was detected in GSCs cultured under 5% oxygen relative to 20% oxygen.D Co-immunoprecipitation to detect the interaction between endogenous HIF2α and USP33 in GSCs after inhibition of ERK1/2 activity. GSCs were cultured under 20% or 5% oxygen for 24 hours, then treated with the MEK inhibitor U0126 (10µM) for 30 minutes to inhibit ERK1/2 activity, and harvested for immunoprecipitation with anti-USP33 antibodies. Co-immunoprecipitated products were immunoblotted with the indicated antibodies. Relative to 20% oxygen treatment, 5% oxygen treatment promoted the interaction between HIF2α and USP33 in GSCs. However, inhibition of ERK1/2 activity by U0126 treatment suppressed the interaction between HIF2α and USP33.G Co-immunoprecipitation to determine the serine phosphorylation sites in human HIF2α responsible for its binding to USP33. Ectopic GFP-tagged USP33 along with HA-tagged wild type HIF2α or HIF2α mutants with indicated serine to alanine point mutations were expressed in 293T cells. Cell lysates were subjected to immunoprecipitation with anti-HA agaroses Co-immunoprecipitated products were immunoblotted with the indicated antibodies. The S484A mutation largely attenuated the interaction of HIF2α with USP33, indicating that the serine 484 site in HIF2α was the most important phosphorylation site for the interaction between HIF2α and USP33.


Figure 5B Representative images of tumorsphere formation of T387 GSCs expressing USP33 - targeting shRNA ( shUSP33 ) or non - targeting shRNA ( shNT ) . Twenty - four hours after lentiviral infection , T387 GSCs expressing shUSP33 or shNT were planted in 96 well plates at the density of 2 , 000 cells per well and cultured for 48 hours under normoxia ( 20 % O2 ) or hypoxia ( 5 % O2 ) . Disruption of USP33 inhibited GSC tumorsphere formation in normoxia and dramatically suppressed GSC tumorsphere formation in hypoxia . Scale bar , 200 μm . E Cell titer assays showing cell growth of T387 GSCs expressing shUSP33 or shNT in normoxia or hypoxia . GSCs were transduced with shNT or shUSP33 through lentiviral infection for 24 hours , and then split into 96 well plates at the concentration of 2 , 000 cells per well . Cell titer was determined by the Glo luminescent cell viability assay kit ( Promega ) at the indicated time points . Disruption of USP33 inhibited GSC growth in normoxia and hypoxia . The experiment has been repeated three times . The presented curves were based on three technical replicates . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ( mean ± s . e . m . ; two tailed unpaired t - test ) . F Representative immunofluorescent staining of the apoptotic marker cleaved caspase 3 in T387 GSCs expressing shUSP33 or shNT in normoxia or hypoxia . GSCs were attached to hESC - Qualified Matrix ( Corning ) in 6 - well plates at the concentration of 50 , 000 cells per well . Forty - eight hours post infection with shUSP33 or shNT lentiviruses , cells were stained for cleaved caspase 3 ( green ) and nuclei ( blue ) . Scale bar , 80 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 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 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

Figure 5B Representative images of tumorsphere formation of T387 GSCs expressing USP33-targeting shRNA (shUSP33) or non-targeting shRNA (shNT). Twenty-four hours after lentiviral infection, T387 GSCs expressing shUSP33 or shNT were planted in 96 well plates at the density of 2,000 cells per well and cultured for 48 hours under normoxia (20% O2) or hypoxia (5% O2). Disruption of USP33 inhibited GSC tumorsphere formation in normoxia and dramatically suppressed GSC tumorsphere formation in hypoxia. Scale bar, 200 μm.E Cell titer assays showing cell growth of T387 GSCs expressing shUSP33 or shNT in normoxia or hypoxia. GSCs were transduced with shNT or shUSP33 through lentiviral infection for 24 hours, and then split into 96 well plates at the concentration of 2,000 cells per well. Cell titer was determined by the Glo luminescent cell viability assay kit (Promega) at the indicated time points. Disruption of USP33 inhibited GSC growth in normoxia and hypoxia. The experiment has been repeated three times. The presented curves were based on three technical replicates. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (mean ± s.e.m.; two tailed unpaired t-test).F Representative immunofluorescent staining of the apoptotic marker cleaved caspase 3 in T387 GSCs expressing shUSP33 or shNT in normoxia or hypoxia. GSCs were attached to hESC-Qualified Matrix (Corning) in 6-well plates at the concentration of 50,000 cells per well. Forty-eight hours post infection with shUSP33 or shNT lentiviruses, cells were stained for cleaved caspase 3 (green) and nuclei (blue). Scale bar, 80 μm.


Figure 6C , Representative images of autonomous fluorescent signals of 4HRE - d2GFP in xenografts derived from or shUSP33 - or shNT - expressing T387 GSCs transduced with 4HRE - d2GFP . Scale bar , 200 μm . E , Representative images of autonomous fluorescent signals of 4HRE - d2GFP ( green ) and immunofluorescent staining of the stem cell marker SOX2 ( red ) in xenografts derived from T387 GSCs transduced with 4HRE - d2GFP . More GFP signals were detected in the SOX2 + cells compared with the SOX2 - cells , suggesting a stronger hypoxia response in GSCs relative to NSTCs . Scale bar , 200 μm . G , Representative images of immunofluorescent staining of the apoptotic marker cleaved caspase 3 ( red ) and autonomous fluorescent signals of 4HRE - d2GFP ( green ) in xenografts derived from T387 GSCs transduced with 4HRE - d2GFP . The GFP + population had much less apoptosis relative to the GFP - population , suggesting that hypoxia response inhibited cell apoptosis . Scale bar , 200 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 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 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

Figure 6C, Representative images of autonomous fluorescent signals of 4HRE-d2GFP in xenografts derived from or shUSP33- or shNT- expressing T387 GSCs transduced with 4HRE-d2GFP. Scale bar, 200 μm.E, Representative images of autonomous fluorescent signals of 4HRE-d2GFP (green) and immunofluorescent staining of the stem cell marker SOX2 (red) in xenografts derived from T387 GSCs transduced with 4HRE-d2GFP. More GFP signals were detected in the SOX2+ cells compared with the SOX2- cells, suggesting a stronger hypoxia response in GSCs relative to NSTCs. Scale bar, 200 μm.G, Representative images of immunofluorescent staining of the apoptotic marker cleaved caspase 3 (red) and autonomous fluorescent signals of 4HRE-d2GFP (green) in xenografts derived from T387 GSCs transduced with 4HRE-d2GFP. The GFP+ population had much less apoptosis relative to the GFP- population, suggesting that hypoxia response inhibited cell apoptosis. Scale bar, 200 μm.


Figure 7B Kaplan - Meier survival curves of mice bearing GBMs derived from T387 GSCs expressing shUSP33 or shNT from ( A ) . Disruption of USP33 extended the survival of mice bearing GSC - derived GBM tumors . ( n = 5 mice for each group ; shUSP33 # 42 vs shNT , p = 0 . 0185 ; shUSP33 # 45 vs shNT , p = 0 . 0027 ; two - tailed log - rank test ) . E , F Immunofluorescent analysis ( E ) and statistical quantifications ( F ) of the cell proliferation marker Ki67 in xenografts derived from T387 GSCs expressing shUSP33 or shNT . Disruption of USP33 in GSCs reduced cell proliferation in GBM xenografts . Scale bar , 80 μm . ( n = 5 tumors for each group ; * * * , p < 0 . 001 ; mean ± s . e . m . ; two tailed unpaired t - 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 O O O O O O O O O O O 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

Figure 7B Kaplan-Meier survival curves of mice bearing GBMs derived from T387 GSCs expressing shUSP33 or shNT from (A). Disruption of USP33 extended the survival of mice bearing GSC-derived GBM tumors. (n= 5 mice for each group; shUSP33#42 vs shNT, p = 0.0185; shUSP33#45 vs shNT, p = 0.0027; two-tailed log-rank test).E, F Immunofluorescent analysis (E) and statistical quantifications (F) of the cell proliferation marker Ki67 in xenografts derived from T387 GSCs expressing shUSP33 or shNT. Disruption of USP33 in GSCs reduced cell proliferation in GBM xenografts. Scale bar, 80 μm. (n = 5 tumors for each group; ***, p < 0.001; mean ± s.e.m.; two tailed unpaired t-test).


Figure 1 A . HeLa cells released from double thymidine synchronization in early S phase were treated with 5 - azadC for 30 min , washed and collected at the indicated times . Soluble and chromatin - enriched fractions were immunoblotted with indicated antibodies . E . HeLa / GFP - DNMT1 cells transfected with indicated siRNAs were processed as in ( D ) . H . Representative images of U2OS cells transfected with indicated siRNAs followed by mCherry - RNF4 expression plasmid . Cells were treated with 5 - azadC in the presence or absence of SUMOi , fixed 2 h later , pre - extracted and co - immunostained with DNMT1 and SUMO2 / 3 antibodies . Scale bar , 5 μm . J . GFP - tagged DNMT1 from extracts of HeLa / GFP - DNMT1 cells treated or not with 5 - azadC was immobilized on GFP - Trap agarose , subjected to stringent washing to remove proteins non - covalently bound to GFP - DNMT1 and incubated with recombinant HA - ubiquitin , E1 and E2 ( UbcH5a ) enzymes and RNF4 proteins ( STUbL reaction ) at 37 ° C for 1 h . Samples were then subjected to immunoblotting to assay for RNF4 - dependent STUbL activity towards GFP - DNMT1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 A. HeLa cells released from double thymidine synchronization in early S phase were treated with 5-azadC for 30 min, washed and collected at the indicated times. Soluble and chromatin-enriched fractions were immunoblotted with indicated antibodies.  E. HeLa/GFP-DNMT1 cells transfected with indicated siRNAs were processed as in (D).  H. Representative images of U2OS cells transfected with indicated siRNAs followed by mCherry-RNF4 expression plasmid. Cells were treated with 5-azadC in the presence or absence of SUMOi, fixed 2 h later, pre-extracted and co-immunostained with DNMT1 and SUMO2/3 antibodies. Scale bar, 5 μm.  J. GFP-tagged DNMT1 from extracts of HeLa/GFP-DNMT1 cells treated or not with 5-azadC was immobilized on GFP-Trap agarose, subjected to stringent washing to remove proteins non-covalently bound to GFP-DNMT1 and incubated with recombinant HA-ubiquitin, E1 and E2 (UbcH5a) enzymes and RNF4 proteins (STUbL reaction) at 37 °C for 1 h. Samples were then subjected to immunoblotting to assay for RNF4-dependent STUbL activity towards GFP-DNMT1.


Figure 2 A . M . HpaII was crosslinked into a plasmid to generate p4xDPC and then incubated in nucleoplasmic egg extracts ( NPE ) in the presence or absence of 50 μM of SUMOi . DPC pull - down under stringent conditions was performed at the indicated time points and the recovered samples were immunoblotted for crosslinked M . HpaII ( Larsen et al , 2019 ) . Note that p4xDPC , which contains four M . HpaII DPCs , is sometimes used to increase sensitivity towards modified M . HpaII species . Identical results were obtained with plasmids containing one or four DPCs . B . p4xDPC or p4xDPCSUMO ( generated by crosslinking SUMO ∆ GG - M . HpaII ) were incubated in NPE and DPC pull - down performed as in ( A ) . Note that priming M . HpaII with SUMO stimulates rapid poly - SUMOylation of the DPC in NPE . Thus , this substrate was frequently used in subsequent experiments to stimulate DPC poly - SUMOylation . C . p4xDPCSUMO was incubated in NPE and plasmids analyzed as in ( A ) . Following plasmid pull - down , the samples were split and either left untreated or treated with the SUMO protease Ulp1 . I . Representative images of U2OS cells transfected with Myc - PIAS4 expression plasmid that were left untreated or exposed to 5 - azadC in the presence or absence of SUMOi , fixed 2 h later , pre - extracted and co - immunostained with DNMT1 and Myc antibodies . Scale bar , 5 μm . J . HeLa or HeLa / GFP - DNMT1 cells left untreated or exposed to 5 - azadC for 30 min were lysed and subjected to GFP IP under stringent conditions . After extensive washing , individual IPs were incubated with an equal amount ( 800 μg ) of whole cell lysate of HeLa cells transfected with Myc - PIAS4 expression construct ( Fig . EV2J ) , washed and immunoblotted with antibodies to Myc , SUMO2 / 3 and GFP . * , cross - reactive band . K . HeLa / GFP - DNMT1 cells transfected with previously validated siRNAs targeting established SUMO E3 ligases ( Fig . EV2L and Methods section ) were treated with 5 - azadC for 30 min , collected and subjected to GFP immunoprecipitation under denaturing conditions , and immunoblotted with antibodies to SUMO1 , SUMO2 / 3 , ubiquitin and GFP .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2 A. M.HpaII was crosslinked into a plasmid to generate p4xDPC and then incubated in nucleoplasmic egg extracts (NPE) in the presence or absence of 50 μM of SUMOi. DPC pull-down under stringent conditions was performed at the indicated time points and the recovered samples were immunoblotted for crosslinked M.HpaII (Larsen et al, 2019). Note that p4xDPC, which contains four M.HpaII DPCs, is sometimes used to increase sensitivity towards modified M.HpaII species. Identical results were obtained with plasmids containing one or four DPCs.  B. p4xDPC or p4xDPCSUMO (generated by crosslinking SUMO∆GG-M.HpaII) were incubated in NPE and DPC pull-down performed as in (A). Note that priming M.HpaII with SUMO stimulates rapid poly-SUMOylation of the DPC in NPE. Thus, this substrate was frequently used in subsequent experiments to stimulate DPC poly-SUMOylation.  C. p4xDPCSUMO was incubated in NPE and plasmids analyzed as in (A). Following plasmid pull-down, the samples were split and either left untreated or treated with the SUMO protease Ulp1.  I. Representative images of U2OS cells transfected with Myc-PIAS4 expression plasmid that were left untreated or exposed to 5-azadC in the presence or absence of SUMOi, fixed 2 h later, pre-extracted and co-immunostained with DNMT1 and Myc antibodies. Scale bar, 5 μm.  J. HeLa or HeLa/GFP-DNMT1 cells left untreated or exposed to 5-azadC for 30 min were lysed and subjected to GFP IP under stringent conditions. After extensive washing, individual IPs were incubated with an equal amount (800 μg) of whole cell lysate of HeLa cells transfected with Myc-PIAS4 expression construct (Fig. EV2J), washed and immunoblotted with antibodies to Myc, SUMO2/3 and GFP. *, cross-reactive band.  K. HeLa/GFP-DNMT1 cells transfected with previously validated siRNAs targeting established SUMO E3 ligases (Fig. EV2L and Methods section) were treated with 5-azadC for 30 min, collected and subjected to GFP immunoprecipitation under denaturing conditions, and immunoblotted with antibodies to SUMO1, SUMO2/3, ubiquitin and GFP.


Figure 3 B . p2xDPCLeads or p2xDPCSUMOLeads were replicated in egg extracts in the presence [ α - 32P ] dATP . Where indicated , 50 μM of SUMOi was added to the extracts . Reaction samples were analyzed by native agarose gel electrophoresis . RI , replication intermediates ; OC , open circular ; SC , supercoiled . Red arrowheads indicate OC molecules that have not yet undergone repair . C . Samples in ( B ) were recovered by stringent DPC pull - down and immunoblotted for crosslinked M . HpaII . J . As in ( I ) , but using RNF4 - depleted CSF - arrested extracts reconstituted with recombinant RNF4 WT or C130A from ( H ) . L . p4xDPCSUMO was subjected to sequential addition of NPE and CSF - arrested extract as in ( I ) . CSF - arrested extracts from ( K ) were supplemented with recombinant RNF4 , and the DPC plasmid was recovered by DPC pull - down at the indicated time points and immunoblotted against M . HpaII .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3 B. p2xDPCLeads or p2xDPCSUMOLeads were replicated in egg extracts in the presence [α-32P]dATP. Where indicated, 50 μM of SUMOi was added to the extracts. Reaction samples were analyzed by native agarose gel electrophoresis. RI, replication intermediates; OC, open circular; SC, supercoiled. Red arrowheads indicate OC molecules that have not yet undergone repair. C. Samples in (B) were recovered by stringent DPC pull-down and immunoblotted for crosslinked M.HpaII.  J. As in (I), but using RNF4-depleted CSF-arrested extracts reconstituted with recombinant RNF4 WT or C130A from (H).  L. p4xDPCSUMO was subjected to sequential addition of NPE and CSF-arrested extract as in (I). CSF-arrested extracts from (K) were supplemented with recombinant RNF4, and the DPC plasmid was recovered by DPC pull-down at the indicated time points and immunoblotted against M.HpaII.


Figure 4 D . HeLa cells transfected with indicated siRNAs and synchronized in early S phase by double thymidine block were pulse - labeled with 5 - azadC in late S phase as outlined in ( A ) . Cells were then collected at the indicated times after 5 - azadC withdrawal and analyzed by flow cytometry . Data are representative of three independent experiments . Proportion of cells with G2 / M DNA content is indicated . E . HeLa cells transfected with indicated siRNAs were treated with 5 - azadC and / or SUMOi in late S phase as outlined in ( A ) . Cells were then collected at the indicated times after 5 - azadC withdrawal and analyzed by flow cytometry . Data are representative of three independent experiments . Proportion of cells with G2 / M DNA content is indicated . F . HeLa cells transfected with indicated siRNAs were pulse - labeled or not with 5 - azadC for 30 min in late S phase according to the experimental setup in ( A ) . Following 5 - azadC withdrawal cells were incubated with nocodazole , collected at the indicated times and immunoblotted with indicated antibodies . H . Immunoblot analysis of siRNA - treated HeLa cells that were exposed to indicated genotoxic agents and collected 1 h later .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4 D. HeLa cells transfected with indicated siRNAs and synchronized in early S phase by double thymidine block were pulse-labeled with 5-azadC in late S phase as outlined in (A). Cells were then collected at the indicated times after 5-azadC withdrawal and analyzed by flow cytometry. Data are representative of three independent experiments. Proportion of cells with G2/M DNA content is indicated.  E. HeLa cells transfected with indicated siRNAs were treated with 5-azadC and/or SUMOi in late S phase as outlined in (A). Cells were then collected at the indicated times after 5-azadC withdrawal and analyzed by flow cytometry. Data are representative of three independent experiments. Proportion of cells with G2/M DNA content is indicated.  F. HeLa cells transfected with indicated siRNAs were pulse-labeled or not with 5-azadC for 30 min in late S phase according to the experimental setup in (A). Following 5-azadC withdrawal cells were incubated with nocodazole, collected at the indicated times and immunoblotted with indicated antibodies.  H. Immunoblot analysis of siRNA-treated HeLa cells that were exposed to indicated genotoxic agents and collected 1 h later.


Figure 5 B . Representative time - lapse sequences of mitotic progression in cells in ( A ) . NEBD corresponds to t = 0 . White arrowheads indicate cells undergoing division . Scale bar , 10 μm . E . HeLa cells transfected with RNF4 siRNA were synchronized in early S phase by double thymidine block . Six h after release from the block , cells were pulse - labeled with 5 - azadC for 30 min . Mitotic cells were isolated by shake - off 8 h later , washed and incubated or not with MPS1 inhibitor ( MPS1i ) and collected at the indicated times . Cells were then processed for immunoblotting with indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5 B. Representative time-lapse sequences of mitotic progression in cells in (A). NEBD corresponds to t=0. White arrowheads indicate cells undergoing division. Scale bar, 10 μm.  E. HeLa cells transfected with RNF4 siRNA were synchronized in early S phase by double thymidine block. Six h after release from the block, cells were pulse-labeled with 5-azadC for 30 min. Mitotic cells were isolated by shake-off 8 h later, washed and incubated or not with MPS1 inhibitor (MPS1i) and collected at the indicated times. Cells were then processed for immunoblotting with indicated antibodies.


Figure 1 ( A - B ) Spinal cord sections from 5 - month - old WT and Tmem106b - / - mice were stained with anti - neurofascin , anti - LAMP1 and anti - Cath D antibodies . Accumulation of LAMP1 and CathD positive vacuoles at the distal end of AIS was observed in Tmem106b - / - motor neurons . Representative images from three independent mice were shown . Scale bar = 10 µm . n = 3 . Data presented as mean ± SEM . Unpaired Student ' s t test . * * * * , p < 0 . 0001 . ( C - F ) Western blot analysis of p62 , ubiquitin ( Ub ) , TDP - 43 and p - TDP - 43 in RIPA - and urea - soluble fractions from brain ( C and E ) and spinal cord ( D and F ) of 16 - month - old WT and Tmem106b - / - mice . n = 5 . Data presented as mean ± SEM . Unpaired Student ' s t test . * , p < 0 . 05 , * * * , p < 0 . 001 , * * * * , p < 0 . 0001 .

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 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

Figure 1(A-B) Spinal cord sections from 5-month-old WT and Tmem106b-/- mice were stained with anti- neurofascin, anti- LAMP1 and anti-Cath D antibodies. Accumulation of LAMP1 and CathD positive vacuoles at the distal end of AIS was observed in Tmem106b-/- motor neurons. Representative images from three independent mice were shown. Scale bar = 10 µm. n=3. Data presented as mean ± SEM. Unpaired Student's t test. ****, p<0.0001.(C-F) Western blot analysis of p62, ubiquitin (Ub), TDP-43 and p-TDP-43 in RIPA- and urea-soluble fractions from brain (C and E) and spinal cord (D and F) of 16-month-old WT and Tmem106b-/- mice. n=5. Data presented as mean ± SEM. Unpaired Student's t test. *, p<0.05, ***, p<0.001, ****, p<0.0001.


Figure 2 ( A ) Tmem106b - / - Grn - / - mice do not show any changes in body weight . Body weight of 5 - month - old mice of indicated genotypes were measured and analyzed . n = 6 - 10 . ( B Tmem106b - / - Grn - / - mice show reduced activities in the open field test . 4 . 5 - month - old mice were used in the study . C ) Tmem106b - / - Grn - / - mice show reduced activities in the open field test . 4 . 5 - month - old mice were used in the study . Total movement was quantified . n = 8 - 10 . Data presented as mean ± SEM . One - way ANOVA tests with Bonferroni ' s multiple comparisons : * * * * , p < 0 . 0001 . ( D Tmem106b - / - Grn - / - mice show abnormal hindlimb clasping behavior . 5 - month - old mice were used in the study . E ) Tmem106b - / - Grn - / - mice show abnormal hindlimb clasping behavior . 5 - month - old mice were used in the study . Severity score was quantified . n = 8 - 10 . Data presented as mean ± SEM . One - way ANOVA tests with Bonferroni ' s multiple comparisons : * * * , p < 0 . 001 , * * * * , p < 0 . 0001 .

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 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 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 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 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

Figure 2(A) Tmem106b-/-Grn-/- mice do not show any changes in body weight. Body weight of 5-month-old mice of indicated genotypes were measured and analyzed. n=6-10.(B Tmem106b-/-Grn-/- mice show reduced activities in the open field test. 4.5-month-old mice were used in the study.C) Tmem106b-/-Grn-/- mice show reduced activities in the open field test. 4.5-month-old mice were used in the study. Total movement was quantified. n=8-10. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ****, p<0.0001.(D Tmem106b-/-Grn-/- mice show abnormal hindlimb clasping behavior. 5-month-old mice were used in the study.E) Tmem106b-/-Grn-/- mice show abnormal hindlimb clasping behavior. 5-month-old mice were used in the study. Severity score was quantified. n= 8-10. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ***, p<0.001, ****, p<0.0001.


Figure 3 ( A , B ) Spinal cord sections ( C1 - C4 ) from 5 - month - old mice of indicated genotypes were stained with indicated antibodies ( A ) and the numbers of NeuN , or CHAT positive cells , as well as IBA1 and GFAP intensities were quantified ( B ) . 3 - 5 sections / mouse were used for quantification . n = 3 . Data presented as mean ± SEM . One - way ANOVA tests with Bonferroni ' s multiple comparisons : * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 , * * * * , p < 0 . 0001 . Scale bar : 20 µm for CHAT staining , 200 µm for other images .

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

Figure 3(A,B) Spinal cord sections (C1-C4) from 5-month-old mice of indicated genotypes were stained with indicated antibodies (A) and the numbers of NeuN, or CHAT positive cells, as well as IBA1 and GFAP intensities were quantified (B). 3-5 sections/mouse were used for quantification. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: *, p<0.05, **, p<0.01, ***, p<0.001, ****, p<0.0001. Scale bar: 20 µm for CHAT staining, 200 µm for other images.


Figure 4 ( A - D ) Western blot analysis of NeuN , PSD95 , SYN , GFAP , TMEM106B , PGRN and GAPDH in spinal cord ( C5 - C8 ) ( A , B ) and brain ( C , D ) lysates from 5 - month - old WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - mice .

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

Figure 4(A-D) Western blot analysis of NeuN, PSD95, SYN, GFAP, TMEM106B, PGRN and GAPDH in spinal cord (C5-C8) (A, B) and brain (C, D) lysates from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice.


Figure 5 ( A , B ) Retinal sections from 5 - month - old mice were stained with indicated antibodies . Thickness of each layer , number of Brn3a positive cells ( retinal ganglion cells ) per section and length of outer segment as indicated by opsin staining are quantified . Data information : ONL : outer nuclear layer ; INL : Inner nuclear layer ; OPL : outer plexiform layer ; IPL : inner plexiform layer ; GCL : ganglion cell layer . ( C Increase in apoptosis in Grn - / - and Tmem106b - / - Grn - / - retina as indicated by TUNEL assay . TUNEL positive signals per section were quantified . Data information : ONL : outer nuclear layer ; INL : Inner nuclear layer ; OPL : outer plexiform layer ; IPL : inner plexiform layer ; GCL : ganglion cell layer . D ) Increase in apoptosis in Grn - / - and Tmem106b - / - Grn - / - retina as indicated by TUNEL assay . TUNEL positive signals per section were quantified . ( E Increase in Müller glial activation in Tmem106b - / - Grn - / - retina as indicated by GFAP staining . GFAP intensity per section was quantified and normalized to WT control . Data information : ONL : outer nuclear layer ; INL : Inner nuclear layer ; OPL : outer plexiform layer ; IPL : inner plexiform layer ; GCL : ganglion cell layer . F ) Increase in Müller glial activation in Tmem106b - / - Grn - / - retina as indicated by GFAP staining . GFAP intensity per section was quantified and normalized to WT control . ( G , H ) Increase in microglia activation in Grn - / - and Tmem106b - / - Grn - / - retina as indicated by IBA1 staining . The number of IBA1 positive cells per section was quantified . Data information : ONL : outer nuclear layer ; INL : Inner nuclear layer ; OPL : outer plexiform layer ; IPL : inner plexiform layer ; GCL : ganglion cell layer .

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 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 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 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

Figure 5(A, B) Retinal sections from 5-month-old mice were stained with indicated antibodies. Thickness of each layer, number of Brn3a positive cells (retinal ganglion cells) per section and length of outer segment as indicated by opsin staining are quantified. Data information: ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer.(C Increase in apoptosis in Grn-/- and Tmem106b-/-Grn-/- retina as indicated by TUNEL assay. TUNEL positive signals per section were quantified. Data information: ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer.D) Increase in apoptosis in Grn-/- and Tmem106b-/-Grn-/- retina as indicated by TUNEL assay. TUNEL positive signals per section were quantified.(E Increase in Müller glial activation in Tmem106b-/-Grn-/- retina as indicated by GFAP staining. GFAP intensity per section was quantified and normalized to WT control. Data information: ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer.F) Increase in Müller glial activation in Tmem106b-/-Grn-/- retina as indicated by GFAP staining. GFAP intensity per section was quantified and normalized to WT control.(G, H) Increase in microglia activation in Grn-/- and Tmem106b-/-Grn-/- retina as indicated by IBA1 staining. The number of IBA1 positive cells per section was quantified. Data information: ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer.


Figure 6 ( A Heatmap illustrating gene expression changes in WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - cortex samples isolated from 2 . 7 - month - old mice . n = 3 . Genes with FDR p ≤ 0 . 05 , LogFC ≥ 0 . 5 or ≤ 0 . 5 between WT and Tmem106b - / - Grn - / - groups are plotted based on hierarchical clustering analysis . Data information : Inflammation genes are highlighted in magenta and lysosomal genes are highlighted in blue . B ) Heatmap illustrating gene expression changes in WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - spinal cord samples isolated from 2 . 7 - month - old mice . n = 3 . Genes with FDR p ≤ 0 . 05 , LogFC ≥ 0 . 5 or ≤ 0 . 5 between WT and Tmem106b - / - Grn - / - groups are plotted based on hierarchical clustering analysis . Data information : Inflammation genes are highlighted in magenta and lysosomal genes are highlighted in blue . ( C ) Heatmap illustrating gene expression changes in the inflammation and lysosome pathways in WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - spinal cord samples . Data information : Inflammation genes are highlighted in magenta and lysosomal genes are highlighted in blue .

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 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

Figure 6(A Heatmap illustrating gene expression changes in WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- cortex samples isolated from 2.7-month-old mice. n=3. Genes with FDR p≤0.05, LogFC≥0.5 or ≤0.5 between WT and Tmem106b-/-Grn-/- groups are plotted based on hierarchical clustering analysis. Data information: Inflammation genes are highlighted in magenta and lysosomal genes are highlighted in blue.B) Heatmap illustrating gene expression changes in WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- spinal cord samples isolated from 2.7-month-old mice. n=3. Genes with FDR p≤0.05, LogFC≥0.5 or ≤0.5 between WT and Tmem106b-/-Grn-/- groups are plotted based on hierarchical clustering analysis. Data information: Inflammation genes are highlighted in magenta and lysosomal genes are highlighted in blue.(C) Heatmap illustrating gene expression changes in the inflammation and lysosome pathways in WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- spinal cord samples. Data information: Inflammation genes are highlighted in magenta and lysosomal genes are highlighted in blue.


Figure 7Western blot analysis of Galectin - 3 and GPNMB in the spinal cord lysates from 5 - month - old WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - mice . n = 3 . Data presented as mean ± SEM . One - way ANOVA tests with Bonferroni ' s multiple comparisons : * * * , p < 0 . 001 , * * * * , p < 0 . 0001 . Western blot analysis of Galectin - 3 and GPNMB in the brain lysates from 5 - month - old WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - mice . n = 3 . Data presented as mean ± SEM . One - way ANOVA tests with Bonferroni ' s multiple comparisons : * * * , p < 0 . 001 , * * * * , p < 0 . 0001 . ( E ) Immunostaining of Galectin - 3 , CathD and CD68 in spinal cord sections from 5 - month - old WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - mice . Scale bar = 10 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 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 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 7Western blot analysis of Galectin-3 and GPNMB in the spinal cord lysates from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ***, p<0.001, ****, p<0.0001.Western blot analysis of Galectin-3 and GPNMB in the brain lysates from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ***, p<0.001, ****, p<0.0001.(E) Immunostaining of Galectin-3, CathD and CD68 in spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Scale bar = 10 µm.


Figure 8 ( A - B ) Lipofuscin accumulation in the retina : the number of auto fluorescent puncta per section was quantified . ONL : outer nuclear layer ; INL : Inner nuclear layer ; OPL : outer plexiform layer ; IPL : inner plexiform layer ; GCL : ganglion cell layer . n = 3 - 6 . Scale bar = 50 µm . TMEM106B and PGRN deletion results in increased autofluorescence in the spinal cord of 5 - month - old mice . n = 3 . Scale bar = 10 µm . TMEM106B and PGRN deletion results in increased autofluorescence in the thalamus of 5 - month - old mice . n = 3 . Scale bar = 10 µm . ( G , H ) Western blot analysis of lysosomal proteins in the spinal cord lysates from 5 - month - old WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - mice . Asterisk indicates non - specific bands . n = 3 .

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Figure 8(A-B) Lipofuscin accumulation in the retina: the number of auto fluorescent puncta per section was quantified. ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer. n=3-6. Scale bar = 50 µm.TMEM106B and PGRN deletion results in increased autofluorescence in the spinal cord of 5-month-old mice. n=3. Scale bar = 10 µm.TMEM106B and PGRN deletion results in increased autofluorescence in the thalamus of 5-month-old mice. n=3. Scale bar = 10 µm.(G, H) Western blot analysis of lysosomal proteins in the spinal cord lysates from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Asterisk indicates non-specific bands. n=3.


Figure 9 ( A ) Spinal cord sections from 5 - month - old WT , Tmem106b - / - , Grn - / - and Tmem106b - / - Grn - / - mice were stained with anti - neurofascin , anti - LAMP1 and anti - CathD antibodies . Representative images from three independent mice were shown . Scale bar = 10 µm . ( B ) Quantification of LAMP1 positive vacuoles ( > 2 µm ) for experiments in ( A ) . 4 - 6 sections / spinal cord from 3 mice were analyzed for each genotype . Data presented as mean ± SEM . Unpaired Student ' s t test : * * * * , p < 0 . 0001 .

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Figure 9(A) Spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/- and Tmem106b-/-Grn-/- mice were stained with anti-neurofascin, anti-LAMP1 and anti-CathD antibodies. Representative images from three independent mice were shown. Scale bar = 10 µm. (B) Quantification of LAMP1 positive vacuoles (> 2 µm) for experiments in (A). 4-6 sections/spinal cord from 3 mice were analyzed for each genotype. Data presented as mean ± SEM. Unpaired Student's t test: ****, p<0.0001.


Figure 10 ( A Immunostaining of GFAP with LAMP1 and Cath D in the spinal cord sections from 5 - month - old WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - mice . Scale bar = 5 µm . B ) Immunostaining of IBA1 with LAMP1 and Cath D in the spinal cord sections from 5 - month - old WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - mice . Scale bar = 5 µm .

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Figure 10(A Immunostaining of GFAP with LAMP1 and Cath D in the spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Scale bar = 5 µm.B) Immunostaining of IBA1 with LAMP1 and Cath D in the spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Scale bar = 5 µm.


Figure 11 ( A ) Accumulation of autophagosomes and lysosomes in the spinal cord sections from a 5 - month - old Tmem106b - / - Grn - / - mouse as shown by transmission electron microscope analysis . Scale bar = 1µm . Arrows : autophagosome ; arrow heads : lysosomes ; ( a ) : axon . ( B ) Accumulation of myelin debris and amorphous inclusions in the microglia in the spinal cord sections from a 5 - month - old Tmem106b - / - Grn - / - mouse as shown by transmission electron microscope analysis . Scale bar = 1µm . Arrows : myelin debris ; arrow heads : dark amorphous inclusions ; ( m ) : microglia . ( C ) Western blot analysis of LC3 - I and LC3 - II in the spinal cord lysates from 5 - month - old WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - mice . n = 3 . Data presented as mean ± SEM . One - way ANOVA tests with Bonferroni ' s multiple comparisons : * * * * , p < 0 . 0001 . ( D Immunostaining of TFE3 and IBA1 in the spinal cord sections from 5 - month - old WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - mice . E ) Immunostaining of TFE3 and IBA1 in the spinal cord sections from 5 - month - old WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - mice . Nuclear signals of TFE3 in IBA1 positive microglia were quantified . n = 3 . Data presented as mean ± SEM . One - way ANOVA tests with Bonferroni ' s multiple comparisons : * * * , p < 0 . 001 , * * * * , p < 0 . 0001 . Scale bar = 10 µm .

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 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 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 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

Figure 11(A) Accumulation of autophagosomes and lysosomes in the spinal cord sections from a 5-month-old Tmem106b-/-Grn-/- mouse as shown by transmission electron microscope analysis. Scale bar = 1µm. Arrows: autophagosome; arrow heads: lysosomes; (a): axon.(B) Accumulation of myelin debris and amorphous inclusions in the microglia in the spinal cord sections from a 5-month-old Tmem106b-/-Grn-/- mouse as shown by transmission electron microscope analysis. Scale bar = 1µm. Arrows: myelin debris; arrow heads: dark amorphous inclusions; (m): microglia.(C) Western blot analysis of LC3-I and LC3-II in the spinal cord lysates from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ****, p<0.0001.(D Immunostaining of TFE3 and IBA1 in the spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice.E) Immunostaining of TFE3 and IBA1 in the spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Nuclear signals of TFE3 in IBA1 positive microglia were quantified. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ***, p<0.001, ****, p<0.0001. Scale bar = 10 µm.


Figure 12 ( A , B ) Western blot analysis of p62 , ubiquitin ( Ub ) , TDP - 43 and p - TDP - 43 in RIPA - and urea - soluble fractions from spinal cord ( C5 - C8 ) and brain of 5 - month - old WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - mice . Spinal cord : n = 3 ; brain : n = 4 . Data presented as mean ± SEM . One - way ANOVA tests with Bonferroni ' s multiple comparisons : * , p < 0 . 05 ; * * , p < 0 . 01 * * * , p < 0 . 001 , * * * * , p < 0 . 0001 . ( C , D ) Immunostaining of p62 and ubiquitin in the spinal cord sections from 5 - month - old WT , Tmem106b - / - , Grn - / - , and Tmem106b - / - Grn - / - mice . The number of p62 or Ub positive puncta was quantified . n = 3 . Data presented as mean ± SEM . One - way ANOVA tests with Bonferroni ' s multiple comparisons : * * * , p < 0 . 001 , * * * * , p < 0 . 0001 . Scale bar = 10 µm .

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

Figure 12(A, B) Western blot analysis of p62, ubiquitin (Ub), TDP-43 and p-TDP-43 in RIPA- and urea-soluble fractions from spinal cord (C5-C8) and brain of 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Spinal cord: n=3; brain: n=4. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: *, p<0.05; **, p<0.01***, p<0.001, ****, p<0.0001.(C, D) Immunostaining of p62 and ubiquitin in the spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. The number of p62 or Ub positive puncta was quantified. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ***, p<0.001, ****, p<0.0001. Scale bar = 10 µm.


Figure 1A . H3122 cells expressing endogenous EML4 - ALK V1 and HEK293 transfected with YFP - EML4 - ALK V1 WT and KD were stained for either anti - ALK or anti - GFP ( green ) , anti - α - tubulin ( red ) , and DAPI ( blue ) . Scale bars , 10 μm ; magnified views of a selected area are shown . B . Violin plots showing the number of EML4 - ALK V1 cytoplasmic foci per cell . Data represents 30 - 50 counts from three biological replicates . * * * P < 0 . 001 , * * * * P < 0 . 0001 in comparison to HEK293 YFP - EML4 - ALK - V1 KD by one - way ANOVA analysis . C . H2228 cells expressing endogenous EML4 - ALK V3 and HEK293 transfected with YFP - EML4 - ALK V3 WT and KD were stained for either anti - ALK or anti - GFP ( green ) , anti - α - tubulin ( red ) , and DAPI ( blue ) . Scale bars , 10 μm ; magnified views of a selected area are shown . D . Violin plots showing the number of EML4 - ALK V3 cytoplasmic foci per cell . Data represents 30 - 50 counts from four biological replicates . * * * P < 0 . 001 , * * * * P < 0 . 0001 in comparison to HEK293 YFP - EML4 - ALK - V3 KD by one - way ANOVA analysis . H . Whisker plot showing the calculated velocity of single events in the kymograph . Data represent 20 counts from 10 kymographs . n = 2 . Error bar represents SD of two biological replicates . * P < 0 . 05 in comparison to YFP EML4 - ALK V3 KD by unpaired t test .

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Figure 1A. H3122 cells expressing endogenous EML4-ALK V1 and HEK293 transfected with YFP-EML4-ALK V1 WT and KD were stained for either anti-ALK or anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. B. Violin plots showing the number of EML4-ALK V1 cytoplasmic foci per cell. Data represents 30-50 counts from three biological replicates. ***P<0.001, ****P<0.0001 in comparison to HEK293 YFP-EML4-ALK-V1 KD by one-way ANOVA analysis. C. H2228 cells expressing endogenous EML4-ALK V3 and HEK293 transfected with YFP-EML4-ALK V3 WT and KD were stained for either anti-ALK or anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. D. Violin plots showing the number of EML4-ALK V3 cytoplasmic foci per cell. Data represents 30-50 counts from four biological replicates. ***P<0.001, ****P<0.0001 in comparison to HEK293 YFP-EML4-ALK-V3 KD by one-way ANOVA analysis. H. Whisker plot showing the calculated velocity of single events in the kymograph. Data represent 20 counts from 10 kymographs. n=2. Error bar represents SD of two biological replicates. *P< 0.05 in comparison to YFP EML4-ALK V3 KD by unpaired t test.


Figure 2A , B . H3122 and H2228 cells were stained for anti - ALK ( green ) , anti - GRB2 ( red ) , anti - SOS1 ( red ) , anti - pC - KITY721 ( red ) , anti - PI3K p85β or anti - pPLCγ2Y759 and DAPI ( blue ) . Scale bars , 10 μm ; magnified views of a selected area are shown . C . HEK293 cells transfected with YFP - EM4L - ALK V1 WT or V3 WT for 48 hrs and treated with 5 % or 10 % 1 , 6 - hexanediol . Representative still images after 5 min ( taken from time - lapse movies ) are shown of cells treated with 10 % 1 , 6 - Hexanediol or DMSO . Scale bars , 10 μm ; the arrowheads indicate cytoplasmic foci . D . Box plot showing the number of cytoplasmic foci in DMSO and 5 % or 10 % Hexanediol ( 5 min ) . Data represent 5 - 10 counts from 3 independent experiments ( n = 3 ) . Error bar represents SD of three biological replicates . * * * * P < 0 . 0001 in comparison to YFP - EML4 - ALK V3 WT DMSO by one - way ANOVA analysis . E . Representative western blots of HEK293 transfected EML4 - ALK V1 WT and V3 WT for 48 hrs . Cells were treated with either 5 % or 10 % 1 , 6 - Hexanediol for 5 minutes . DMSO was used a control . Lysates were analysed for the phosphorylation and expression of the indicated antibodies . GAPDH was used as a loading control . Data representative of n = 2 experiments .

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Figure 2A, B. H3122 and H2228 cells were stained for anti-ALK (green), anti-GRB2 (red), anti-SOS1 (red), anti-pC-KITY721 (red), anti-PI3K p85β or anti-pPLCγ2Y759 and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown.C. HEK293 cells transfected with YFP-EM4L-ALK V1 WT or V3 WT for 48 hrs and treated with 5% or 10% 1,6-hexanediol. Representative still images after 5 min (taken from time-lapse movies) are shown of cells treated with 10% 1,6-Hexanediol or DMSO. Scale bars, 10 μm; the arrowheads indicate cytoplasmic foci. D. Box plot showing the number of cytoplasmic foci in DMSO and 5% or 10% Hexanediol (5 min). Data represent 5-10 counts from 3 independent experiments (n=3). Error bar represents SD of three biological replicates. ****P< 0.0001 in comparison to YFP-EML4-ALK V3 WT DMSO by one-way ANOVA analysis. E. Representative western blots of HEK293 transfected EML4-ALK V1 WT and V3 WT for 48 hrs. Cells were treated with either 5% or 10% 1,6-Hexanediol for 5 minutes. DMSO was used a control. Lysates were analysed for the phosphorylation and expression of the indicated antibodies. GAPDH was used as a loading control. Data representative of n=2 experiments.


Figure 3A , B , C , D . HEK293 cells were transfected with EML4 - ALK V1 WT or V3 WT or Kinase Dead ( KD ) for 48 hours . Cells were either untreated ( DMSO ) or treated with ALK inhibitors for 4 hours before fixation and staining with anti - GFP ( green ) , anti - α - tubulin ( red ) , and DAPI ( blue ) . Scale bars , 10 μm ; magnified views of a selected area are shown .

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Figure 3A, B, C, D. HEK293 cells were transfected with EML4-ALK V1 WT or V3 WT or Kinase Dead (KD) for 48 hours. Cells were either untreated (DMSO) or treated with ALK inhibitors for 4 hours before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown.


Figure 4B . HEK293 cells were transfected with EML4 - ALK V1 deletion 12N blade for 48 hours . Cells were either untreated ( DMSO ) or treated with ALK inhibitors for 4 hours before fixation and staining with anti - GFP ( green ) , anti - α - tubulin ( red ) , and DAPI ( blue ) . Scale bars , 10 μm ; magnified views of a selected area are shown .

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Figure 4B. HEK293 cells were transfected with EML4-ALK V1 deletion 12N blade for 48 hours. Cells were either untreated (DMSO) or treated with ALK inhibitors for 4 hours before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown.


Figure 5D . HEK293 cells were transfected with EML4 - ALK V3 ∆ JM 1058 - 1100 for 48 hours . Cells were either untreated ( DMSO ) or treated with ALK inhibitors for 4 hours before fixation and staining with anti - GFP ( green ) , anti - α - tubulin ( red ) , and DAPI ( blue ) . Scale bars , 10 μm ; magnified views of a selected area are shown . E . Violin plot representing the number of cytoplasmic foci counted per cell from D . Data represent counts from at least 30 cells , n = 3 . * * * * P < 0 . 0001 in comparison to DMSO by one - way ANOVA .

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Figure 5D. HEK293 cells were transfected with EML4-ALK V3 ∆JM 1058-1100 for 48 hours. Cells were either untreated (DMSO) or treated with ALK inhibitors for 4 hours before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. E. Violin plot representing the number of cytoplasmic foci counted per cell from D. Data represent counts from at least 30 cells, n=3. ****P< 0.0001 in comparison to DMSO by one-way ANOVA.


Figure 6A - F HEK293 cells were transfected with A . YFP - EML4 - ALK V3 K1150M , C . R1275Q or E . F1174L constructs and treated with ALK inhibitors or DMSO for 4 hours before fixation and staining with anti - GFP ( green ) , anti - α - tubulin ( red ) , and DAPI ( blue ) . Scale bars , 10 μm ; magnified views of a selected area are shown . B , D , F . Violin plots representing the number of cytoplasmic foci counted per cell from A , C and E . Data represent counts from at least 30 cells , n = 3 or 4 . * * * * P < 0 . 0001 in comparison to DMSO by one - way ANOVA .

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Figure 6A-F HEK293 cells were transfected with A. YFP-EML4-ALK V3 K1150M, C. R1275Q or E. F1174L constructs and treated with ALK inhibitors or DMSO for 4 hours before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. B, D, F. Violin plots representing the number of cytoplasmic foci counted per cell from A, C and E. Data represent counts from at least 30 cells, n=3 or 4. ****P< 0.0001 in comparison to DMSO by one-way ANOVA.


Figure 7B . HEK293 cells transfected with YFP - EML4 - GyrB and either untreated ( DMSO ) or treated with Coumermycin A1 to induce dimerization of GyrB before fixation and staining with anti - GFP ( green ) , anti - α - tubulin ( red ) , and DAPI ( blue ) . Scale bars , 10 μm ; magnified views of a selected area are shown . C . Violin plot showing the number of cytoplasmic foci per HEK293 cell transfected with YFP - EML4 - GyrB . Data represent counts from > 30 cells , n = 4 . * * * * P < 0 . 0001 in comparison to DMSO by unpaired t 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7B. HEK293 cells transfected with YFP-EML4-GyrB and either untreated (DMSO) or treated with Coumermycin A1 to induce dimerization of GyrB before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. C. Violin plot showing the number of cytoplasmic foci per HEK293 cell transfected with YFP-EML4-GyrB. Data represent counts from >30 cells, n= 4. ****P< 0.0001 in comparison to DMSO by unpaired t test.


Figure 1 ( A ) HEK293T ‐ Fpn cells were incubated with FAC ( 10 μM Fe ) for 24 h followed by incubation for 6 h in the absence or presence of 10 μM Ponasterone A . Cells were then incubated with and without 100 μM chloroquine or 10 μM leupeptin for 10 h and harvested . The ferritin content was determined by ELISA . Induction of Fpn resulted in decreased ferritin levels and this was not prevented by treatment with chloroquine or leupeptin . The data are presented as the standard deviation from three different experiments . ( B ) Cells treated as in ( A ) were incubated in the presence of 1 μg / ml EGF for 2 h . Cells were fixed and processed for immunofluorescence using mouse anti ‐ EGF receptor and Alexa 594 conjugated goat anti ‐ mouse IgG . Chloroquine and leupeptin effectively inhibit degradation of EGF receptor .

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 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

Figure 1(A) HEK293T‐Fpn cells were incubated with FAC (10 μM Fe) for 24 h followed by incubation for 6 h in the absence or presence of 10 μM Ponasterone A. Cells were then incubated with and without 100 μM chloroquine or 10 μM leupeptin for 10 h and harvested. The ferritin content was determined by ELISA. Induction of Fpn resulted in decreased ferritin levels and this was not prevented by treatment with chloroquine or leupeptin. The data are presented as the standard deviation from three different experiments.(B) Cells treated as in (A) were incubated in the presence of 1 μg/ml EGF for 2 h. Cells were fixed and processed for immunofluorescence using mouse anti‐EGF receptor and Alexa 594 conjugated goat anti‐mouse IgG. Chloroquine and leupeptin effectively inhibit degradation of EGF receptor.


Figure 2 ( A ) HEK293T ‐ Fpn cells were incubated with FAC ( 10 μM Fe ) for 24 h followed by incubation for 6 h in the absence or presence of 10 μM Ponasterone A . Cells were then treated with or without 10 μM MG132 or 10 μM lactacystin in the presence of Ponasterone A for 10 h and harvested . The ferritin content was determined by ELISA . Error bars represent the standard deviation from three different experiments in duplicate . ( B ) Samples treated as in ( A ) were immunoprecipitated using anti ‐ ferritin antibodies and the immunoprecipitate examined for the presence of ferritin or ubiquitin by Western blot analysis . The arrows indicate the migration of H and L chains . ( C ) Cells were incubated with 1 . 0 × 10 − 7 M Tf ( 59Fe ) 2 for 24 h followed by incubation for 6 h in the absence or presence of 10 μM Ponasterone A . Cells were then treated with or without 10 μM MG132 in the presence of Ponasterone A for 10 h and harvested and the amount of 59Fe in immunoprecipitatedferritin was determined . ( D ) Cells were treated as described in ( A ) and cell extracts were applied to size exclusion chromatography and the ferritin content , in selected fractions , was determined by ELISA . Error bars represent the standard deviation from three different experiments in duplicate . ( E ) Cells were treated as described in ( C ) , ferritin was then immunoprecipitated , eluted using 100 mM glycine , pH 2 . 5 and measured by ELISA . The amount of ferritin ‐ associated 59Fe was measured and the specific activity of ferritin determined . Error bars represent the standard deviation from three different experiments in duplicate . ( F ) Samples were treated as in ( D ) , and ferritin was immunoprecipitated and analyzed by Western blot analysis using an antibody to ubiquitin .

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 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 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 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

Figure 2(A) HEK293T‐Fpn cells were incubated with FAC (10 μM Fe) for 24 h followed by incubation for 6 h in the absence or presence of 10 μM Ponasterone A. Cells were then treated with or without 10 μM MG132 or 10 μM lactacystin in the presence of Ponasterone A for 10 h and harvested. The ferritin content was determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.(B) Samples treated as in (A) were immunoprecipitated using anti‐ferritin antibodies and the immunoprecipitate examined for the presence of ferritin or ubiquitin by Western blot analysis. The arrows indicate the migration of H and L chains.(C) Cells were incubated with 1.0 × 10−7 M Tf(59Fe)2 for 24 h followed by incubation for 6 h in the absence or presence of 10 μM Ponasterone A. Cells were then treated with or without 10 μM MG132 in the presence of Ponasterone A for 10 h and harvested and the amount of 59Fe in immunoprecipitatedferritin was determined.(D) Cells were treated as described in (A) and cell extracts were applied to size exclusion chromatography and the ferritin content, in selected fractions, was determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.(E) Cells were treated as described in (C), ferritin was then immunoprecipitated, eluted using 100 mM glycine, pH 2.5 and measured by ELISA. The amount of ferritin‐associated 59Fe was measured and the specific activity of ferritin determined. Error bars represent the standard deviation from three different experiments in duplicate.(F) Samples were treated as in (D), and ferritin was immunoprecipitated and analyzed by Western blot analysis using an antibody to ubiquitin.


Figure 3 ( A ) FM3A ( black ) and ts85 ( grey ) cells were transiently transfected with a plasmid containing CMV ‐ regulated Fpn ‐ GFP and incubated in the presence of FAC ( 10 μM Fe ) and 0 . 5 μM hepcidin for 24 h . Hepcidin was either maintained ( + ) or removed ( − ) to allow Fpn ‐ GFP localization at the plasma membrane . Cells were maintained at the permissive temperature ( 33 ° C ) or moved to the restrictive temperature ( 39 ° C ) and incubated for 6 h . Cells were harvested and ferritin content determined by ELISA . Error bars represent the standard deviation from three different experiments in duplicate . ( B ) FM3A and ts85 cells were treated as in ( A ) but FAC was replaced with 1 . 0 × 10 − 7 M Tf ( 59Fe ) 2 . Ferritin was immunoprecipitated and the specific activity of 59Fe ‐ ferritin determined . Error bars represent the standard deviation from three different experiments in duplicate . ( C ) ts85 cells were treated as in ( B ) and cell extracts applied to size exclusion chromatography . Ferritin content in selected fractions was measured by ELISA . Black bars represent assembled ferritin ( > 400 kDa ) , gray bars represent monomeric ( 50 kDa ) . ( D ) FM3A and ts85 cells were treated as in ( A ) but cells that had been incubated at the restrictive temperature ( 39 ° C ) were then returned to the permissive temperature ( 33 ° C ) and incubated in the presence of cycloheximide ( 75 μg / ml ) and 10 μM FAC for 1 h . Cells were then harvested and ferritin levels determined by ELISA . Error bars represent the standard deviation from three different experiments in duplicate .

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 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 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

Figure 3(A) FM3A (black) and ts85 (grey) cells were transiently transfected with a plasmid containing CMV‐regulated Fpn‐GFP and incubated in the presence of FAC (10 μM Fe) and 0.5 μM hepcidin for 24 h. Hepcidin was either maintained (+) or removed (−) to allow Fpn‐GFP localization at the plasma membrane. Cells were maintained at the permissive temperature (33°C) or moved to the restrictive temperature (39°C) and incubated for 6 h. Cells were harvested and ferritin content determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.(B) FM3A and ts85 cells were treated as in (A) but FAC was replaced with 1.0 × 10−7 M Tf(59Fe)2. Ferritin was immunoprecipitated and the specific activity of 59Fe‐ferritin determined. Error bars represent the standard deviation from three different experiments in duplicate.(C) ts85 cells were treated as in (B) and cell extracts applied to size exclusion chromatography. Ferritin content in selected fractions was measured by ELISA. Black bars represent assembled ferritin (>400 kDa), gray bars represent monomeric (50 kDa).(D) FM3A and ts85 cells were treated as in (A) but cells that had been incubated at the restrictive temperature (39°C) were then returned to the permissive temperature (33°C) and incubated in the presence of cycloheximide (75 μg/ml) and 10 μM FAC for 1 h. Cells were then harvested and ferritin levels determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.


Figure 4 ( A ) Cells were then incubated at the restrictive temperature ( 39 ° C ) for 6 h in the presence or absence of 100 μM DFO , with or without 100 μM chloroquine or 10 μM MG132 . Cells were then harvested and ferritin content determined by ELISA . Error bars represent the standard deviation from three different experiments in duplicate . ( B ) Cells treated as in ( A ) were incubated in the presence or absence of 5 mM 3 ‐ methyladenine .

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 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

Figure 4(A) Cells were then incubated at the restrictive temperature (39°C) for 6 h in the presence or absence of 100 μM DFO, with or without 100 μM chloroquine or 10 μM MG132. Cells were then harvested and ferritin content determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.(B) Cells treated as in (A) were incubated in the presence or absence of 5 mM 3‐methyladenine.


Figure 5 ( A ) Δccc1 / pGAL ‐ H + L cells were transformed with a plasmid containing a methionine regulated CCC1 ( pMET3CCC1 ) . Cells were grown in medium with galactose for 20 h , washed and incubated in galactose ( • ) , glucose with 10 × methionine ( ▵ ) , glucose ( ○ ) or glucose without methionine ( ▾ ) for 10 h . Cells were then harvested and ferritin levels determined by ELISA . Error bars represent the standard deviation from three different experiments in duplicate . The absence of methionine leads to expression of the vacuolar iron transporter Ccc1p . ( B ) erg6 ‐ 2 and erg6 ‐ 2 pGAL ‐ H + L strains were grown in medium with galactose and 250 μM FeSO4 for 24 h . Cells were then washed and incubated in galactose or glucose in the absence or presence of 50 μM MG132 for 7 h . ( C ) Cells were then harvested , ferritin levels determined by ELISA and immunoprecipitated using anti ‐ ferritin antibodies and the immunoprecipitate examined for the presence of ubiquitin by Western analysis . Cells with the erg6 ‐ 2 allele permit the entry of MG132 ( Lee and Goldberg , 1996 ) .

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 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 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 5(A) Δccc1/pGAL‐H+L cells were transformed with a plasmid containing a methionine regulated CCC1 (pMET3CCC1). Cells were grown in medium with galactose for 20 h, washed and incubated in galactose (•), glucose with 10 × methionine (▵), glucose (○) or glucose without methionine (▾) for 10 h. Cells were then harvested and ferritin levels determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate. The absence of methionine leads to expression of the vacuolar iron transporter Ccc1p.(B) erg6‐2 and erg6‐2 pGAL‐H+L strains were grown in medium with galactose and 250 μM FeSO4 for 24 h. Cells were then washed and incubated in galactose or glucose in the absence or presence of 50 μM MG132 for 7 h.(C) Cells were then harvested, ferritin levels determined by ELISA and immunoprecipitated using anti‐ferritin antibodies and the immunoprecipitate examined for the presence of ubiquitin by Western analysis. Cells with the erg6‐2 allele permit the entry of MG132 ( Lee and Goldberg, 1996).


Figure 1 ( A ) Mitochondrial extracts from 143B cells , COX1 ∆ and COX2 ∆ mutant cybrids and their respective isogenic controls ( CON - 1 and CON - 2 ) were analyzed by SDS - PAGE and immunoblotting with the indicated antibodies . The graphs ( right ) show densitometric quantifications of the signals normalized to CII subunit SDHA . The mean of three independent controls ( CON ) was set to 100 and all measurements were referenced to that value . The values represent mean ± SD ( n = 3 - 8 ) . Mann - Whitney U test was applied for statistical analyses . Variations between controls and mutants : α , p < 0 . 05 ; β , p < 0 . 01 ; γ , p < 0 . 001 . Specific variations between COX1 ∆ and COX2 ∆ mutants : ∗ p < 0 . 05 ; ∗ ∗ p < 0 . 01 . ( B ) Digitonized mitochondria ( digitonin - to - protein ratio of 2 : 1 ) were analyzed by BN - PAGE , followed by CI in gel activity ( IGA ) assays and Coomassie staining . The SC I + III2 + IV1 band ( b1 ) was excised from the control ( 143B ) lane and the SC I + III2 bands ( b2 to b4 ) were excised from the COX1 ∆ and COX2 ∆ lanes . Their protein compositions were subsequently analyzed by nano - LC / ESI - MS ( see also Figure EV2 ) . The relative position of the molecular weight marker is indicated on the left . ( C ) BN - PAGE was followed by CI - IGA assay and immunoblotting with the indicated antibodies ( in brackets ) . The identity of the MRC complexes and SCs is : I + III2 + IV1 , SC containing CI , CIII2 , and CIV ; I + III2 , SC containing CI and CIII2 ; I + III2plus , SC containing CI , CIII2 and a COX1 - submodule ; III2 + IV , SC containing CIII2 and CIV ; I , complex I ; III2 , CIII dimer ; IV2 , CIV dimer ; subCOX1 , COX1 - containing subcomplexes .

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 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 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

Figure 1(A) Mitochondrial extracts from 143B cells, COX1∆ and COX2∆ mutant cybrids and their respective isogenic controls (CON-1 and CON-2) were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. The graphs (right) show densitometric quantifications of the signals normalized to CII subunit SDHA. The mean of three independent controls (CON) was set to 100 and all measurements were referenced to that value. The values represent mean ± SD (n=3-8). Mann-Whitney U test was applied for statistical analyses. Variations between controls and mutants: α, p<0.05; β, p<0.01; γ, p<0.001. Specific variations between COX1∆ and COX2∆ mutants: ∗p<0.05; ∗∗p<0.01.(B) Digitonized mitochondria (digitonin-to-protein ratio of 2:1) were analyzed by BN-PAGE, followed by CI in gel activity (IGA) assays and Coomassie staining. The SC I+III2+IV1 band (b1) was excised from the control (143B) lane and the SC I+III2 bands (b2 to b4) were excised from the COX1∆ and COX2∆ lanes. Their protein compositions were subsequently analyzed by nano-LC/ESI-MS (see also Figure EV2). The relative position of the molecular weight marker is indicated on the left.(C) BN-PAGE was followed by CI-IGA assay and immunoblotting with the indicated antibodies (in brackets). The identity of the MRC complexes and SCs is: I+III2+IV1, SC containing CI, CIII2, and CIV; I+III2, SC containing CI and CIII2; I+III2plus, SC containing CI, CIII2 and a COX1-submodule; III2+IV, SC containing CIII2 and CIV; I, complex I; III2, CIII dimer; IV2, CIV dimer; subCOX1, COX1-containing subcomplexes.


Figure 2 ( A ) Control 143B cells ( left panels ) , COX1 ∆ cybrids ( middle panels ) , and COX2 ∆ cybrids ( right panels ) . The right lane highlights structural subunits of CI ( yellow ) , CIII ( light green ) , and CIV ( orange brown ) , and assembly factors ( orange ) . Intensity - based absolute quantification ( iBAQ ) values were normalized to the sum of all control values . Top lanes indicate gel slice numbers and their relative native masses . ( B ) Intensity profiles showing abundance and relative distribution of structural subunits from CI ( upper graphs ) , CIII ( middle graphs ) , and CIV ( bottom graphs ) . The identities of MRC complexes and SCs are as in Figure 1 .

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 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

Figure 2(A) Control 143B cells (left panels), COX1∆ cybrids (middle panels), and COX2∆ cybrids (right panels). The right lane highlights structural subunits of CI (yellow), CIII (light green), and CIV (orange brown), and assembly factors (orange). Intensity-based absolute quantification (iBAQ) values were normalized to the sum of all control values. Top lanes indicate gel slice numbers and their relative native masses.(B) Intensity profiles showing abundance and relative distribution of structural subunits from CI (upper graphs), CIII (middle graphs), and CIV (bottom graphs). The identities of MRC complexes and SCs are as in Figure 1.


Figure 3 ( A ) Excerpts of complexome profiling data showing the distribution of CIV subunits and assembly factors HIGD1A , HIGD2A , CMC1 and COA3 in control ( 143B ) , COX1 ∆ and COX2 ∆ cybrids . The relative abundance and distribution of detected proteins is shown in the lower graphs . The localization of SC I + III2plus in COX2Δ cybrids is highlighted with a red line . The approximate molecular weights of CIV - containing structures are indicated on the top ( in kDa ) . ( B ) 2D - BN / SDS - PAGE analysis from control ( 143B ) and COX2Δ cybrids followed by immunoblotting with the indicated antibodies . I + III2 + IV1 , SC containing CI , CIII , and CIV ; I + III2plus , SC containing CI , CIII and a COX1 - submodule ; III2 + IV , SC containing CIII and CIV ; IV2 , CIV dimer ; IV , CIV monomer . The presence of a COX1 - CMC1 - COA3 - containing subcomplex is indicated .

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 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

Figure 3(A) Excerpts of complexome profiling data showing the distribution of CIV subunits and assembly factors HIGD1A, HIGD2A, CMC1 and COA3 in control (143B), COX1∆ and COX2∆ cybrids. The relative abundance and distribution of detected proteins is shown in the lower graphs. The localization of SC I+III2plus in COX2Δ cybrids is highlighted with a red line. The approximate molecular weights of CIV-containing structures are indicated on the top (in kDa).(B) 2D-BN/SDS-PAGE analysis from control (143B) and COX2Δ cybrids followed by immunoblotting with the indicated antibodies. I+III2+IV1, SC containing CI, CIII, and CIV; I+III2plus, SC containing CI, CIII and a COX1-submodule; III2+IV, SC containing CIII and CIV; IV2, CIV dimer; IV, CIV monomer. The presence of a COX1-CMC1-COA3-containing subcomplex is indicated.