( F ) Higher magnification of dendritic spines from neurons transfected with rescue RFP - SP ( magenta ) containing wild - type ( WT ) or mutated ( MUT ) 3 ' UTR and BFP - shRNA - SP ( grey ) and contacted by GFP - Syp + terminals ( green ) . Arrowheads indicate putative synaptic contacts . Scale bar : 5 μm . ( G ) Percentage of SP + spines in neurons expressing RFP - SP - 3 ' UTR - WT vs - MUT and depending on the apposition or not to a GFP - Syp + terminal ( SP - 3 ' UTR - WT : TetTx - , n = 220 , TetTx + , n = 26 ; SP - 3 ' UTR - MUT : TetTx - , n = 616 , TetTx + , n = 25 ; n indicates the number of spines from 5 - 7 pairs ) . ( H ) Spine head volume in same conditions as in ( G ) ( SP - 3 ' UTR - WT : TetTx - , n = 26 , TetTx + , n = 26 spines from 5 pairs , 4 slices ; SP - 3 ' UTR - MUT : TetTx - , n = 25 , TetTx + , n = 25 ; n indicates the number of spines from 7 pairs , 7 slices ) . * * * P < 0 . 001 , * P < 0 . 05 , ns , not significant ( Kruskall - Wallis test followed by Dunn ' s multiple test ) . ( I ) Cumulative probability distributions of spine head volumes for TetTx - and TetTx + spines from neurons expressing RFP - SP - 3 ' UTR - WT ( SP - WT , light gray and light green ) or RFP - SP - 3 ' UTR - MUT ( SP - MUT , dark gray and dark green ) . Data information : Data are represented as mean ± SEM .

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(F) Higher magnification of dendritic spines from neurons transfected with rescue RFP-SP (magenta) containing wild-type (WT) or mutated (MUT) 3'UTR and BFP-shRNA-SP (grey) and contacted by GFP-Syp+ terminals (green). Arrowheads indicate putative synaptic contacts. Scale bar: 5 μm. (G) Percentage of SP+ spines in neurons expressing RFP-SP-3'UTR-WT vs -MUT and depending on the apposition or not to a GFP-Syp+ terminal (SP-3'UTR-WT: TetTx-, n = 220, TetTx+, n = 26; SP-3'UTR-MUT: TetTx-, n = 616, TetTx+, n = 25; n indicates the number of spines from 5-7 pairs). (H) Spine head volume in same conditions as in (G) (SP-3'UTR-WT: TetTx-, n = 26, TetTx+, n = 26 spines from 5 pairs, 4 slices; SP-3'UTR-MUT: TetTx-, n = 25, TetTx+, n = 25; n indicates the number of spines from 7 pairs, 7 slices). ***P < 0.001, *P < 0.05, ns, not significant (Kruskall-Wallis test followed by Dunn's multiple test). (I) Cumulative probability distributions of spine head volumes for TetTx- and TetTx+ spines from neurons expressing RFP-SP-3'UTR-WT (SP-WT, light gray and light green) or RFP-SP-3'UTR-MUT (SP-MUT, dark gray and dark green). Data information: Data are represented as mean ± SEM.


( c ) Intravascular endothelial cell interactions and transmigration of neutrophils ( Ly - 6G + CX3CR - 1 - ; red ) and cMOs ( Ly - 6G - CX3CR - 1low ; green ) to the perivascular tissue as assessed 6 h after intrascrotal stimulation with recombinant murine uPA , PAI - 1 , or uPA - PAI - 1 in postcapillary venules of the cremaster of CX3CR - 1GFP / + mice by multi - channel in vivo microscopy . Representative still images ( scale bar : 50 : µm ) and quantitative data are shown ( mean ± SEM for n = 4 mice per group ; # p < 0 . 05 vs . saline ; * p < 0 . 05 vs . uPA ; § p < 0 . 05 vs . PAI - 1 ; One - way ANOVA ) .

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(c) Intravascular endothelial cell interactions and transmigration of neutrophils (Ly-6G+ CX3CR-1-; red) and cMOs (Ly-6G- CX3CR-1low; green) to the perivascular tissue as assessed 6 h after intrascrotal stimulation with recombinant murine uPA, PAI-1, or uPA-PAI-1 in postcapillary venules of the cremaster of CX3CR-1GFP/+ mice by multi-channel in vivo microscopy. Representative still images (scale bar: 50: µm) and quantitative data are shown (mean±SEM for n=4 mice per group; #p<0.05 vs. saline; *p<0.05 vs. uPA; §p<0.05 vs. PAI-1; One-way ANOVA).


( b ) Expression of PECAM - 1 / CD31 ( red ) and ICAM - 1 / CD54 or VCAM - 1 / CD106 ( green ) in the cremaster muscle of WT mice as assessed ex vivo by confocal laser scanning microscopy 6 h after intrascrotal injection of recombinant murine uPA - PAI - 1 or saline , representative images ( scale bar : 50 µm ) and quantitative data are shown ( mean ± SEM for n = 4 mice per group ; # p < 0 . 05 vs . saline ; t test ) .

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(b) Expression of PECAM-1/CD31 (red) and ICAM-1/CD54 or VCAM-1/CD106 (green) in the cremaster muscle of WT mice as assessed ex vivo by confocal laser scanning microscopy 6 h after intrascrotal injection of recombinant murine uPA-PAI-1 or saline, representative images (scale bar: 50 µm) and quantitative data are shown (mean±SEM for n=4 mice per group; #p<0.05 vs. saline; t test).


( g ) Quantitative analysis of LFA - 1 / CD11a clustering on the surface of mouse neutrophils under increasing flow conditions in vitro in the presence of either E - selectin / CD62E + ICAM - 1 / CD54 , E - selectin / CD62E + ICAM - 1 / CD54 + KC , or E - selectin / CD62E + ICAM - 1 / CD54 + KC + uPA - PAI - 1 as assessed by spinning disk confocal microscopy , representative images and quantitative data are shown ( scale bar : 10 μm ; mean ± SD ; n = 3 mice ; n = 10 - 13 flow chambers , n = 122 - 222 cells ) ; * p < 0 . 05 vs . E - selectin / CD62E + ICAM - 1 / CD54 ; One - way ANOVA ) .

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(g) Quantitative analysis of LFA-1/CD11a clustering on the surface of mouse neutrophils under increasing flow conditions in vitro in the presence of either E-selectin/CD62E + ICAM-1/CD54, E-selectin/CD62E + ICAM-1/CD54 + KC, or E-selectin/CD62E + ICAM-1/CD54 + KC + uPA-PAI-1 as assessed by spinning disk confocal microscopy, representative images and quantitative data are shown (scale bar: 10 μm; mean±SD; n=3 mice; n=10-13 flow chambers, n =122-222 cells); *p<0.05 vs. E-selectin/CD62E + ICAM-1/CD54; One-way ANOVA).


( b ) Correlation analyses of RNA expression levels of uPA ( PLAU gene ) , PAI - 1 ( SERPINE1 gene ) , and the neutrophil marker gene FPR1 , and overall survival of breast cancer patients ( stages 0 and 1 ) with high and low PLAU or SERPINE1 gene expression levels ( cut - off z ≥ 2 . 0 ) in the METABRIC breast cancer cohort .

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(b) Correlation analyses of RNA expression levels of uPA (PLAU gene), PAI-1 (SERPINE1 gene), and the neutrophil marker gene FPR1, and overall survival of breast cancer patients (stages 0 and 1) with high and low PLAU or SERPINE1 gene expression levels (cut-off z≥ 2.0) in the METABRIC breast cancer cohort.


( c ) Surface expression of MMP - 9 , VEGF , and NE as assessed on circulating neutrophils isolated from the peripheral blood of WT mice ( saline ) or from the peritoneal cavity of WT mice 6 h after intraperitoneal stimulation with uPA - PAI - 1 ( uPA - PAI - 1 ) by multi - channel flow cytometry , quantitative data are shown ( mean ± SEM for n = 4 - 6 mice per group ; # p < 0 . 05 vs . saline ; t test ) .

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(c) Surface expression of MMP-9, VEGF, and NE as assessed on circulating neutrophils isolated from the peripheral blood of WT mice (saline) or from the peritoneal cavity of WT mice 6 h after intraperitoneal stimulation with uPA-PAI-1 (uPA-PAI-1) by multi-channel flow cytometry, quantitative data are shown (mean±SEM for n=4-6 mice per group; #p<0.05 vs. saline; t test).


( f ) Formation of NETs ( histone H3 + ; green ) as assessed ex vivo by confocal microscopy in the cremaster muscle of WT mice 6 h after intra - scrotal injection of uPA - PAI - 1 , TNF , or saline , PECAM - 1 / CD31 + postcapillary venules ( blue ) and Ly - 6G + neutrophils ( red ) are depicted . Representative images are shown ( scale bar : 50 µm ) .

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(f) Formation of NETs (histone H3+; green) as assessed ex vivo by confocal microscopy in the cremaster muscle of WT mice 6 h after intra-scrotal injection of uPA-PAI-1, TNF, or saline, PECAM-1/CD31+ postcapillary venules (blue) and Ly-6G+ neutrophils (red) are depicted. Representative images are shown (scale bar: 50 µm).


( 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 ) .

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(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 ) .

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(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 ) .

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(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).


C 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 .

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C 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 .

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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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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.


A , 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 . ) .

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A, 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 .

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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.


A , 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 .

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A, 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 .

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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 .

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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.


A 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 .

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A 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 .

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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 .

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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.


B 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 .

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B 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 ) .

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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 .

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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.


C , 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 .

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C, 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 .

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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 .

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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.


B 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 ) .

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B 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 ) .

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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).


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 .

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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 ) .

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 .

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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 .

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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.


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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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.


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 .

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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.


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 .

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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.


J . As in ( I ) , but using RNF4 - depleted CSF - arrested extracts reconstituted with recombinant RNF4 WT or C130A from ( H ) .

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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 .

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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.


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 .

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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 .

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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 .

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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 .

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H. Immunoblot analysis of siRNA-treated HeLa cells that were exposed to indicated genotoxic agents and collected 1 h later.


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 .

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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 .

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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.


( 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 .

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(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 .

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(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.


( 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 .

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(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 .

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(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 .

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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 .

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(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 .

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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.


( 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 .

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(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.


( 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 .

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(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.


( 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 .

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(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 .

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(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 .

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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 .

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(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 .

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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 .

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(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.


( 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 .

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(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 .

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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 .

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(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.


Western 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 .

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Western 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 .

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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 .

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(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.


( 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 .

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(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 .

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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 .

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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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(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.


( 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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(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.


( 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 .

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(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 .

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.


( 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 .

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(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 .

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(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 .

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(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 .

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(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 .

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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.


( 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 .

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(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 .

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(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.


A . 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 .

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A. 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 .

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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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.


A , 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 .

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A, 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 .

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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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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.


A , 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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A, 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.


B . 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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B. 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.


D . 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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D. 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.


A - 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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A-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.


B . 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 .

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B. 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.


( 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 .

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(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 .

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(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.


( 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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(F) Samples were treated as in (D), and ferritin was immunoprecipitated and analyzed by Western blot analysis using an antibody to ubiquitin.