Figure 4HeLa CALCOCO1 KO cells stably expressing EGFP - CALCOCO1 were starved for 4 hours and then immunostained with anti - ATG13 and anti - WIPI2 antibodies . Scale bars in A are 5 μm for the confocal microscopy images and 2 μm for the airyscans . In B , the error bars represent mean ± sd of puncta per cell from three independent experiments and100 - 200 cells per each experiment . Immunoblot analysis of indicated cell lines treated as indicated . Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control ( GAPDH In D the bars represent the mean ± sd of band intensities relative to the GAPDH loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant . Immunoblot analysis of indicated cell lines treated as indicated . Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control ( GAPDH In , the bars represent the mean ± sd of band intensities relative to the GAPDH loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant . Immunoblot analysis of indicated cell lines treated as indicated . Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control actin ) . In , the bars represent the mean ± sd of band intensities relative to the actin loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant . , I the bars represent the mean ± sd of band intensities relative to the actin loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant . Immunoblot analysis of indicated cell lines treated as indicated . Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control ( GAPDHImmunoblot analysis of indicated cell lines treated as indicated . Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control ( GAPDH The asterisk in K indicates that endogenous CALCOCO1 is detected in WT and KO cell extracts and EGFP - CALCOCO1 in cells extracts from the rescued cells . In the bars represent the mean ± sd of band intensities relative to the actin or GAPDH loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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

Figure 4HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 were starved for 4 hours and then immunostained with anti-ATG13 and anti-WIPI2 antibodies. Scale bars in A are 5 μm for the confocal microscopy images and 2 μm for the airyscans.In B, the error bars represent mean±sd of puncta per cell from three independent experiments and100-200 cells per each experiment.Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control (GAPDH In D the bars represent the mean±sd of band intensities relative to the GAPDH loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control (GAPDH In , the bars represent the mean±sd of band intensities relative to the GAPDH loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control actin). In , the bars represent the mean±sd of band intensities relative to the actin loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant., I the bars represent the mean±sd of band intensities relative to the actin loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control (GAPDHImmunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control (GAPDH The asterisk in K indicates that endogenous CALCOCO1 is detected in WT and KO cell extracts and EGFP-CALCOCO1 in cells extracts from the rescued cells. In the bars represent the mean±sd of band intensities relative to the actin or GAPDH loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


Figure 5B Co - IP of Myc - VAPA or Myc - VAPB with EGFP - CALCOCO1 from transiently transfected HEK293 cells . The asterisks ( * ) indicate Myc - VAP previously detected on the same membraneC , D GST pulldown assays of in vitro transcribed / translated 35S - Myc - CALCOCO1 constructs with indicated recombinant GST - VAPA or GST - VAPB constructs . The scheme is a representation of CALCOCO1 domains and motifs . E HeLa cells transiently co - transfected with EGFP - CALCOCO1 and Myc - VAPA or - VAPB were immunostained with anti - Myc antibody . Scale bars , 5 μm .

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Figure 5B Co- IP of Myc-VAPA or Myc-VAPB with EGFP-CALCOCO1 from transiently transfected HEK293 cells. The asterisks (*) indicate Myc-VAP previously detected on the same membraneC, D GST pulldown assays of in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-VAPA or GST-VAPB constructs.The scheme is a representation of CALCOCO1 domains and motifs.E HeLa cells transiently co-transfected with EGFP-CALCOCO1 and Myc-VAPA or -VAPB were immunostained with anti-Myc antibody. Scale bars, 5 μm.


Figure 6A , B Immunoblot analysis of indicated cell lines , starved for 6 hours ( HBSS ) as indicated , and treated with MG132 or Baf A1 as indicated . Numbers below the blots in A represent relative intensity of the bands in the shown blots normalized against GAPDH loading control . In B , the bars represent the mean ± sd of band intensities of three independent experiments as quantified using ImageJ . Statistical comparison was analyzed by one - way ANOVA and significance displayed as * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant . C Immunoblot analysis of HeLa cells transfected with the indicated siRNAs . D , E Immunoblot analysis of HeLa cells transfected with the indicated siRNAs and treated with Baf A1 as indicated . In D , the panels are from more than one western blot experiment but only a single GAPDH loading control is shown . In E , the bars represent the mean ± sd of band intensities of three independent experiments as quantified using ImageJ . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison test and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 ; ns is not significant . F HeLa KO CALCOCO1 cells were transiently co - transfected with EGFP - CALCOCO1 and Myc - VAPA or Myc - VAPB , and then immunostained with anti - Myc and anti - LC3B antibodies . Arrows indicate dots of co - localization of all three proteins . Scale bars , 5 μm for large merged images and 1 μm for zoomed images .

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Figure 6A, B Immunoblot analysis of indicated cell lines, starved for 6 hours (HBSS) as indicated, and treated with MG132 or Baf A1 as indicated. Numbers below the blots in A represent relative intensity of the bands in the shown blots normalized against GAPDH loading control. In B, the bars represent the mean±sd of band intensities of three independent experiments as quantified using ImageJ. Statistical comparison was analyzed by one-way ANOVA and significance displayed as **p ˂ 0.005, *p ˂ 0.01; ns is not significant.C Immunoblot analysis of HeLa cells transfected with the indicated siRNAs.D, E Immunoblot analysis of HeLa cells transfected with the indicated siRNAs and treated with Baf A1 as indicated. In D, the panels are from more than one western blot experiment but only a single GAPDH loading control is shown. In E, the bars represent the mean±sd of band intensities of three independent experiments as quantified using ImageJ. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001, **p ˂ 0.005; ns is not significant.F HeLa KO CALCOCO1 cells were transiently co-transfected with EGFP-CALCOCO1 and Myc-VAPA or Myc-VAPB, and then immunostained with anti-Myc and anti-LC3B antibodies. Arrows indicate dots of co-localization of all three proteins. Scale bars, 5 μm for large merged images and 1 μm for zoomed images.


Figure 7A , B Immunoblot analysis of HeLa WT and HeLa CALCOCO1 KO cells . The cells were starved for 6 hours ( HBSS ) as indicated and treated with Baf A1 as indicated . Numbers below the blots in A represent relative intensity of the bands in the shown blots normalized against GAPDH loading control . In A , the panels are collected from more than one western blot experiment but for clarity , only a single GAPDH loading control is shown . In B , the bars represent the mean ± sd of band intensities of three independent experiments as quantified using ImageJ . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison test and significance displayed as * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant . C , D Immunoblot analysis of HeLa CALCOCO1 KO cell lines reconstituted with EGFP - CALCOCO1 . Expression of EGFP - CALCOCO1 was induced or not with tetracycline and the cells were treated with MG132 or Baf A1 as indicated . Numbers below the blots in C represent relative intensity of the bands in the shown blots normalized against actin loading control . In C , the panels are collected from more than one western blot experiment but only a single actin loading control is shown . In D , the bars represent the mean ± sd of band intensities of three independent experiments as quantified using ImageJ . Statistical comparison was analyzed as in B and significance displayed as * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant . E HeLa CALCOCO1 KO cell lines reconstituted with EGFP - CALCOCO1 were treated with tetracycline or not to induce expression of EGFP - CALCOCO1 . Abundance of the ER was quantified from widefield fluorescence images of endogenous RTN3 staining ( see Materials and methods ) . Data are presented as mean ± sd of three independent experiments . Statistical comparison was analyzed as in B and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant . F , G Immunoblot analysis of cells analyzed in C . The bars in G represent the mean ± sd of band intensities relative to the loading control from three independent experiments quantified using ImageJ . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison test . Significance is displayed as * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant . H , I Immunoblot analysis of HeLa CALCOCO1 KO cells stably expressing EGFP - CALCOCO1 in fed or starved conditions and transfected with the indicated VAPA / B siRNAs .

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 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 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 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 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

Figure 7A, B Immunoblot analysis of HeLa WT and HeLa CALCOCO1 KO cells. The cells were starved for 6 hours (HBSS) as indicated and treated with Baf A1 as indicated. Numbers below the blots in A represent relative intensity of the bands in the shown blots normalized against GAPDH loading control. In A, the panels are collected from more than one western blot experiment but for clarity, only a single GAPDH loading control is shown. In B, the bars represent the mean±sd of band intensities of three independent experiments as quantified using ImageJ. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as **p ˂ 0.005, *p ˂ 0.01; ns is not significant.C, D Immunoblot analysis of HeLa CALCOCO1 KO cell lines reconstituted with EGFP-CALCOCO1. Expression of EGFP-CALCOCO1 was induced or not with tetracycline and the cells were treated with MG132 or Baf A1 as indicated. Numbers below the blots in C represent relative intensity of the bands in the shown blots normalized against actin loading control. In C, the panels are collected from more than one western blot experiment but only a single actin loading control is shown. In D, the bars represent the mean±sd of band intensities of three independent experiments as quantified using ImageJ. Statistical comparison was analyzed as in B and significance displayed as **p ˂ 0.005, *p ˂ 0.01; ns is not significant.E HeLa CALCOCO1 KO cell lines reconstituted with EGFP-CALCOCO1 were treated with tetracycline or not to induce expression of EGFP-CALCOCO1. Abundance of the ER was quantified from widefield fluorescence images of endogenous RTN3 staining (see Materials and methods). Data are presented as mean ± sd of three independent experiments. Statistical comparison was analyzed as in B and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.F, G Immunoblot analysis of cells analyzed in C. The bars in G represent the mean±sd of band intensities relative to the loading control from three independent experiments quantified using ImageJ. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test. Significance is displayed as **p ˂ 0.005, *p ˂ 0.01; ns is not significant.H, I Immunoblot analysis of HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 in fed or starved conditions and transfected with the indicated VAPA/B siRNAs.


Figure 8A Immunoblot analysis of HeLa CALCOCO1 KO cells stably expressing EGFP - CALCOCO1 in fed or starved conditions and treated as indicated with Baf A1 or PI3KC3 inhibitor SAR405 . B Immunoblot analysis of HeLa CALCOCO1 KO cells stably expressing EGFP - CALCOCO1 mLIR + ∆ 623 - 691 for 24 hours or not , and then treated as indicated . C , Representative confocal images of HeLa CALCOCO1 KO cells transiently co - transfected with mCherry - CALCOCO1 , Myc - VAPA and EGFP - LAMP1 , and then treated as indicated before immunostaining with anti - Myc anti - body . Arrows indicate co - localization . Scale bars in C , 5 μm for large merged images and 1 μm for zoomed images . D , data are presented as mean ± sd . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison test and significance displayed as * * * p ˂ 0 . 001 ; ns is not significant , n = 3 .

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Figure 8A Immunoblot analysis of HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 in fed or starved conditions and treated as indicated with Baf A1 or PI3KC3 inhibitor SAR405.B Immunoblot analysis of HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 mLIR+∆623-691 for 24 hours or not, and then treated as indicated.C, Representative confocal images of HeLa CALCOCO1 KO cells transiently co-transfected with mCherry-CALCOCO1, Myc-VAPA and EGFP-LAMP1, and then treated as indicated before immunostaining with anti-Myc anti-body. Arrows indicate co-localization. Scale bars in C, 5 μm for large merged images and 1 μm for zoomed images.D, data are presented as mean ± sd. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001; ns is not significant, n=3.


Figure 1Schematic , western blot , and immunofluorescence analysis of the 3 × FLAG - UBXD8 knockin U2OS cell line . Scale bar , 5 μm . Data information : In C , mitochondria were visualized by anti - Tom20 immunostaining ; ER was labeled by anti - PDI immunostaining .

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Figure 1Schematic, western blot, and immunofluorescence analysis of the 3×FLAG-UBXD8 knockin U2OS cell line. Scale bar, 5 μm. Data information: In C, mitochondria were visualized by anti-Tom20 immunostaining; ER was labeled by anti-PDI immunostaining.


Figure 2Blue native gel analysis of UBXD8 in mitochondrial fractions purified from WT and ∆ UBXD8 HeLa cells . Immunoprecipitation analysis of UBXD8 interaction with mitochondrial ( MARCH5 and MUL1 ) and ER ( RNF185 ) ubiquitin E3 ligases . HeLa cells were infected with lentivirus to stably express HA - tagged ubiquitin E3 ligases . Whole cell lysates were prepared for anti - HA immunoprecipitation . Immunoprecipitation analysis of UBXD8 interaction with MARCH5 and RNF185 . Whole cell lysates from 3 × FLAG - UBXD8 knockin HEK293T cells were prepared for anti - FLAG immunoprecipitation . MARCH5 and RNF185 : short exposure for whole cell lysate blots , long exposure for FLAG - IP blots . Fractionation analysis of UBXD8 ' s effect on VCP recruitment to mitochondria and the ER . Whole cell lysate , mitochondria , and ER fractions were purified from WT , ∆ UBXD8 , and ∆ UBXD8 HeLa cells rescued with UBXD8 or the UBX * mutant . UBX * - UBXD8 carries four point mutations ( K367A , F407A , P408G , R409A ) in the UBX domain to disrupt the UBXD8 - VCP interaction . For each sample , 5 μg proteins were loaded for western blot analysis . Quantitative analysis of VCP levels in the mitochondria and ER fractions from the indicated HeLa cells . Data are shown as mean ± SE from three biological repeats . Statistics : one - way ANOVA ; * * P < 0 . 01 , * * * P < 0 . 001 .

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

Figure 2Blue native gel analysis of UBXD8 in mitochondrial fractions purified from WT and ∆UBXD8 HeLa cells.Immunoprecipitation analysis of UBXD8 interaction with mitochondrial (MARCH5 and MUL1) and ER (RNF185) ubiquitin E3 ligases. HeLa cells were infected with lentivirus to stably express HA-tagged ubiquitin E3 ligases. Whole cell lysates were prepared for anti-HA immunoprecipitation.Immunoprecipitation analysis of UBXD8 interaction with MARCH5 and RNF185. Whole cell lysates from 3×FLAG-UBXD8 knockin HEK293T cells were prepared for anti-FLAG immunoprecipitation. MARCH5 and RNF185: short exposure for whole cell lysate blots, long exposure for FLAG-IP blots.Fractionation analysis of UBXD8's effect on VCP recruitment to mitochondria and the ER. Whole cell lysate, mitochondria, and ER fractions were purified from WT, ∆UBXD8, and ∆UBXD8 HeLa cells rescued with UBXD8 or the UBX* mutant. UBX*-UBXD8 carries four point mutations (K367A, F407A, P408G, R409A) in the UBX domain to disrupt the UBXD8-VCP interaction. For each sample, 5 μg proteins were loaded for western blot analysis.Quantitative analysis of VCP levels in the mitochondria and ER fractions from the indicated HeLa cells. Data are shown as mean ± SE from three biological repeats. Statistics: one-way ANOVA; **P < 0.01, ***P < 0.001.


Figure 3A , B . Western blot ( A ) and quantitative ( B ) analyses of MiD49 and Mcl1 degradation in WT and ∆ MARCH5 HeLa cells . Cycloheximide ( CHX : 200 μg / ml ; protein synthesis inhibitor ) ; MG132 : 20 μM , proteasome inhibitor . Data information : Data are shown as mean ± SE from three biological repeats ( B , Statistics : two - tailed unpaired Student ' s t - test ( B , ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant . C , D . Western blot ( C ) and quantitative ( D ) analyses of Mcl1 degradation in the indicated HeLa cells . Data information : Data are shown as mean ± SE from three biological repeats D , Statistics : two - tailed unpaired Student ' s t - test D , ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant . E , F . Western blot ( E ) and quantitative ( F ) analyses of MiD49 degradation in the indicated HeLa cells . Data information : Data are shown as mean ± SE from three biological repeats F ) . Statistics : two - tailed unpaired Student ' s t - test F ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant .

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

Figure 3A, B. Western blot (A) and quantitative (B) analyses of MiD49 and Mcl1 degradation in WT and ∆MARCH5 HeLa cells. Cycloheximide (CHX: 200 μg/ml; protein synthesis inhibitor); MG132: 20 μM, proteasome inhibitor. Data information: Data are shown as mean ± SE from three biological repeats (B, Statistics: two-tailed unpaired Student's t-test (B, ; *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.C, D. Western blot (C) and quantitative (D) analyses of Mcl1 degradation in the indicated HeLa cells. Data information: Data are shown as mean ± SE from three biological repeats D, Statistics: two-tailed unpaired Student's t-test D, ; *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.E, F. Western blot (E) and quantitative (F) analyses of MiD49 degradation in the indicated HeLa cells. Data information: Data are shown as mean ± SE from three biological repeats F). Statistics: two-tailed unpaired Student's t-test F); *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.


Figure 4Fractionation analysis of the subcellular localization of mito - UBXD8 and ER - UBXD8 . Whole cell lysate , mitochondria , and ER fractions were purified from the indicated HeLa cells . Exogenously - expressed mito - UBXD8 has similar expression level with endogenous UBXD8 in WT cells ; exogenously - expressed WT UBXD8 and ER - UBXD8 have similar expression levels . D , E . Western blot ( D ) and quantitative ( E ) analyses of Mcl1 degradation in the indicated HeLa cells . ∆ UBXD8 HeLa cells were rescued with vector , WT - , mito - , and ER - UBXD8 . Data are shown as mean ± SE from three biological repeats ( E ) . Statistics : two - tailed unpaired Student ' s t - test ( E ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4Fractionation analysis of the subcellular localization of mito-UBXD8 and ER-UBXD8. Whole cell lysate, mitochondria, and ER fractions were purified from the indicated HeLa cells. Exogenously-expressed mito-UBXD8 has similar expression level with endogenous UBXD8 in WT cells; exogenously-expressed WT UBXD8 and ER-UBXD8 have similar expression levels.D, E. Western blot (D) and quantitative (E) analyses of Mcl1 degradation in the indicated HeLa cells. ∆UBXD8 HeLa cells were rescued with vector, WT-, mito-, and ER-UBXD8. Data are shown as mean ± SE from three biological repeats (E). Statistics: two-tailed unpaired Student's t-test (E); *P < 0.05, **P < 0.01, ***P < 0.001.


Figure 5Cell survival analysis of the indicated HeLa cells treated with Actinomycin D ( ActD , 1 μM ) or Doxorubicin ( Doxo , 10 μM ) . Representative images are shown . Cell viability was measured by Cell Titer - Glo . Scale bar , 50 μm . Data information : Data are shown as mean ± SE from three biological repeats D , Statistics : two - tailed unpaired Student ' s t - test D , ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Western blot analysis of apoptosis activation ( caspase cleavage and PARP cleavage ) in the indicated HeLa cells treated similarly as in ( D ) . Cell survival analysis of the indicated HeLa cells . 7 × 105 cells in one well of 6 - well plate were treated with ActD ( 200 nM ) for 4 hours or Doxo ( 50 nM ) for 2 hours and subsequently cultured in normal medium for 21 days . Surviving colonies were stained by 0 . 1 % ( W / V ) methylene blue . Representative images and quantifications of the surviving colony numbers are shown . Data information : Data are shown as mean ± SE from three biological repeats G ) . Statistics : one - way ANOVA ( G ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

Figure 5Cell survival analysis of the indicated HeLa cells treated with Actinomycin D (ActD, 1 μM) or Doxorubicin (Doxo, 10 μM). Representative images are shown. Cell viability was measured by Cell Titer-Glo. Scale bar, 50 μm. Data information: Data are shown as mean ± SE from three biological repeats D, Statistics: two-tailed unpaired Student's t-test D, ; *P < 0.05, **P < 0.01, ***P < 0.001.Western blot analysis of apoptosis activation (caspase cleavage and PARP cleavage) in the indicated HeLa cells treated similarly as in (D).Cell survival analysis of the indicated HeLa cells. 7 × 105 cells in one well of 6-well plate were treated with ActD (200 nM) for 4 hours or Doxo (50 nM) for 2 hours and subsequently cultured in normal medium for 21 days. Surviving colonies were stained by 0.1% (W/V) methylene blue. Representative images and quantifications of the surviving colony numbers are shown. Data information: Data are shown as mean ± SE from three biological repeats G). Statistics: one-way ANOVA (G); *P < 0.05, **P < 0.01, ***P < 0.001.


Figure 6Western blot analysis of BH3 - domain containing proteins in the indicated HeLa cells treated with Doxo ( 10 μM ) . Three proteins with enhanced level in ∆ UBXD8 cells ( Noxa , Bik , and Bnip3 ) are highlighted in red . Western blot and quantitative analysis of Noxa degradation in the indicated HeLa cells . Data information : Data are shown as mean ± SE from three biological repeats ( B , Statistics : two - tailed unpaired Student ' s t - test ( B , ; * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 001 . Western blot and quantitative analysis of Bik and Bnip3 degradation in the indicated HeLa cells . Data information : Data are shown as mean ± SE from three biological repeats C , Statistics : two - tailed unpaired Student ' s t - test C , ; * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 001 . Western blot ( D , analysis of the indicated HeLa cells . Western blot F ) analysis of the indicated HeLa cells .

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

Figure 6Western blot analysis of BH3-domain containing proteins in the indicated HeLa cells treated with Doxo (10 μM). Three proteins with enhanced level in ∆UBXD8 cells (Noxa, Bik, and Bnip3) are highlighted in red.Western blot and quantitative analysis of Noxa degradation in the indicated HeLa cells. Data information: Data are shown as mean ± SE from three biological repeats (B, Statistics: two-tailed unpaired Student's t-test (B, ; *P < 0.05, **P < 0.001, ***P < 0.001.Western blot and quantitative analysis of Bik and Bnip3 degradation in the indicated HeLa cells. Data information: Data are shown as mean ± SE from three biological repeats C, Statistics: two-tailed unpaired Student's t-test C, ; *P < 0.05, **P < 0.001, ***P < 0.001.Western blot (D, analysis of the indicated HeLa cells.Western blot F) analysis of the indicated HeLa cells.


Figure 7Representative images of the mitophagy events in the indicated HeLa cells . mtKeima emission excited by 405 nm laser is shown as green ; mtKeima emission excited by 561 nm laser is shown as red . Mitolysosomes are pointed by white arrows . Scale bar , 5 μm . C - F . Quantitative analysis of mitophagy events in the indicated HeLa cells . Only cells with mitophagy were selected to quantify mitolysosome numbers ( D , F ) . Data information : Data are shown as mean ± SE from three biological repeats ( C - F , Statistics : two - tailed unpaired Student ' s t - test ( C - F , ; * * P < 0 . 01 , * * * P < 0 . 001 . I - K , Representative images ( I ) and quantitative analysis of mitophagy events ( J , K ) in the indicated HeLa cells . White arrows point to mitolysosomes . Scale bar , 5 μm . Data information : Data are shown as mean ± SE from three biological repeats J , K , Statistics : two - tailed unpaired Student ' s t - test J , K ) ; * * P < 0 . 01 , * * * P < 0 . 001 .

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 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

Figure 7Representative images of the mitophagy events in the indicated HeLa cells. mtKeima emission excited by 405 nm laser is shown as green; mtKeima emission excited by 561 nm laser is shown as red. Mitolysosomes are pointed by white arrows. Scale bar, 5 μm.C-F. Quantitative analysis of mitophagy events in the indicated HeLa cells. Only cells with mitophagy were selected to quantify mitolysosome numbers (D, F). Data information: Data are shown as mean ± SE from three biological repeats (C-F, Statistics: two-tailed unpaired Student's t-test (C-F, ; **P < 0.01, ***P < 0.001.I-K, Representative images (I) and quantitative analysis of mitophagy events (J, K) in the indicated HeLa cells. White arrows point to mitolysosomes. Scale bar, 5 μm. Data information: Data are shown as mean ± SE from three biological repeats J, K, Statistics: two-tailed unpaired Student's t-test J, K); **P < 0.01, ***P < 0.001.


Figure 1B . Carfilzomib , Bortezomib and MLN9708 activate luciferase activity in M2 macrophages derived from Il - 1β - luciferase transgenic mice . BMDMs from IL - 1β - luciferase transgenic mice were treated by IL - 4 ( 20 ng / mL ) for 24 hours to differentiate into mature M2 macrophages , and then used for screening of FDA compounds ( 5 μM ) . The X axis stands for the code of the drug . F , G . Carfilzomib , Bortezomib and MLN9708 promote the mRNA expression of Il - 1β in M2 macrophages . BMDMs ( F ) or Raw264 . 7 cells ( G ) were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then treated by DMSO , Carfilzomib ( 1 μM ) , Bortezomib ( 1 μM ) , or MLN9708 ( 2 μM ) . RNA was extracted from BMDMs or Raw264 . 7 cells 6 hours after stimulation and the expression of Il - 1β were quantified through RT - qPCR . Data from three experiments are presented as the mean ± SD . T test was used for statistical analysis of differences between groups . * * * p < 0 . 001 ( student ' s t - test ) . H , I . Carfilzomib , Bortezomib and MLN9708 promote the secretion of inflammatory cytokine IL - 1β in M2 macrophages . BMDMs ( H ) or Raw264 . 7 cells ( I ) were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then stimulated by DMSO , Carfilzomib ( 500 nM ) , Bortezomib ( 500 nM ) or MLN9708 ( 500 nM ) . Secretion of IL - 1β were measured through ELISA 24 hours later . Data from three experiments are presented as the mean ± SD . T test was used for statistical analysis of differences between groups . * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1B. Carfilzomib, Bortezomib and MLN9708 activate luciferase activity in M2 macrophages derived from Il-1β-luciferase transgenic mice. BMDMs from IL-1β-luciferase transgenic mice were treated by IL-4 (20 ng/mL) for 24 hours to differentiate into mature M2 macrophages, and then used for screening of FDA compounds (5 μM). The X axis stands for the code of the drug.F, G. Carfilzomib, Bortezomib and MLN9708 promote the mRNA expression of Il-1β in M2 macrophages. BMDMs (F) or Raw264.7 cells (G) were pretreated by IL-4 (20 ng/mL) for 24 hours, then treated by DMSO, Carfilzomib (1 μM), Bortezomib (1 μM), or MLN9708 (2 μM). RNA was extracted from BMDMs or Raw264.7 cells 6 hours after stimulation and the expression of Il-1β were quantified through RT-qPCR. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. ***p < 0.001 (student's t-test). H, I. Carfilzomib, Bortezomib and MLN9708 promote the secretion of inflammatory cytokine IL-1β in M2 macrophages. BMDMs (H) or Raw264.7 cells (I) were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO, Carfilzomib (500 nM), Bortezomib (500 nM) or MLN9708 (500 nM). Secretion of IL-1β were measured through ELISA 24 hours later. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. **p < 0.01, ***p < 0.001 (student's t-test).


Figure 2A . The most effective one among the candidate compounds , Carfilzomib , significantly promotes the expression of M1 macrophage markers , as well as reduces the expression of M2 macrophage markers . BMDMs were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then stimulated by DMSO or Carfilzomib ( 1 μM ) . RNA was extracted from cells 6 hours after stimulation and the expression of M1 ( Il - 6 / Inos ) or M2 ( Cd206 / Arg1 ) macrophage markers were quantified through RT - qPCR . Data from three experiments are presented as the mean ± SD . T test was used for statistical analysis of differences between groups . * * * p < 0 . 001 ( student ' s t - test ) . D . Statistics for proportion of CD86 , CD80 , MHC - II or CD206 positive cells in BMDMs . Data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( student ' s t - test ) . E . Carfilzomib promotes the phagocytosis of macrophages . BMDMs were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then stimulated by DMSO or Carfilzomib ( 500 nM ) for 12 hours . After starving for 2 hours and stained with Red membrane dye , BMDMs ( red ) were incubated with L1210 - GFP cells ( green ) in serum - free medium for another 2 hours . Phagocytosis effect was observed and photographed under fluorescence microscope . Scale bars , 50 μm ( up ) , 20 μm ( down ) . The yellow arrows indicated L1210 that is phagocytosed by macrophages . F . Statistics represent the number of phagocytosis L1210 in 100 macrophages . Data are means ± SD of three independent experiments . * * p < 0 . 01 ( student ' s t - test ) . M . Carfilzomib promotes the M1 - polarization of Tumor associated macrophages in vitro . BMDMs were cultured with tumor culture supernatant ( TSN ) produced by L1210 cells , followed by activation with DMSO or Carfilzomib ( 1 μM ) . RNA was extracted from cells and the expression of Il - 1β , Il - 6 , Inos were quantified through RT - qPCR 6 hours after stimulation . The data are means ± SD of three independent experiments . * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 2A. The most effective one among the candidate compounds, Carfilzomib, significantly promotes the expression of M1 macrophage markers, as well as reduces the expression of M2 macrophage markers. BMDMs were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO or Carfilzomib (1 μM). RNA was extracted from cells 6 hours after stimulation and the expression of M1 (Il-6/Inos) or M2 (Cd206/Arg1) macrophage markers were quantified through RT-qPCR. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. ***p < 0.001 (student's t-test).D. Statistics for proportion of CD86, CD80, MHC-II or CD206 positive cells in BMDMs. Data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01 (student's t-test).E. Carfilzomib promotes the phagocytosis of macrophages. BMDMs were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO or Carfilzomib (500 nM) for 12 hours. After starving for 2 hours and stained with Red membrane dye, BMDMs (red) were incubated with L1210-GFP cells (green) in serum-free medium for another 2 hours. Phagocytosis effect was observed and photographed under fluorescence microscope. Scale bars, 50 μm (up), 20 μm (down). The yellow arrows indicated L1210 that is phagocytosed by macrophages. F. Statistics represent the number of phagocytosis L1210 in 100 macrophages. Data are means ± SD of three independent experiments. **p < 0.01 (student's t-test).M. Carfilzomib promotes the M1-polarization of Tumor associated macrophages in vitro. BMDMs were cultured with tumor culture supernatant (TSN) produced by L1210 cells, followed by activation with DMSO or Carfilzomib (1 μM). RNA was extracted from cells and the expression of Il-1β, Il-6, Inos were quantified through RT-qPCR 6 hours after stimulation. The data are means ± SD of three independent experiments. **p < 0.01, ***p < 0.001 (student's t-test).


Figure 3A , B . Carfilzomib and proteasome inhibitor MG132 activate Il - 1β luciferase in macrophages . ( A ) BMDMs were treated with IL - 4 ( 20 ng / mL ) for 24 hours and then stimulated by DMSO , Carfilzomib ( 1 μM ) , or MG132 ( 5 μM ) . ( B ) BMDMs were stimulated by DMSO , Carfilzomib ( 1 μM ) or MG132 ( 5 μM ) ) for 1 hour , then induced with IL - 4 ( 20 ng / mL ) . Luciferase assays were monitored 12 hours after stimulation . The data are means ± SD of three independent experiments . * * * p < 0 . 001 ( student ' s t - test ) . E . Carfilzomib , Bortezomib , MLN9708 and MG132 induce ER stress response . BMDMs were pretreated with IL - 4 ( 20 ng / mL ) for 24 hours and then stimulated by DMSO or Carfilzomib ( 1 μM ) , Bortezomib ( 1 μM ) , MLN9708 ( 2 μM ) , MG132 ( 5 μM ) . RNA was extracted from cells 3 hours or 6 hours after stimulation and the expression of ER stress related genes ( Bip , Chop ) were detected through RT - qPCR . The data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - test ) . F . ER stress agonists , TUN and TG , promote the expression of M1 macrophage markers , as well as reduce the expression of M2 macrophage markers . BMDMs were pretreated with IL - 4 ( 20 ng / mL ) for 24 hours and then stimulated by DMSO , TUN ( 500 nM ) or TG ( 500 nM ) . RNA was extracted from cells 6 hours after stimulation and indicated genes were quantified through RT - qPCR . The data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - test ) . G . Inhibition of ER stress impairs the ability of Carfilzomib , Bortezomib , MLN9708 and MG132 to reprogram M2 towards M1 - like macrophages . BMDMs were induced by IL - 4 ( 20 ng / mL ) for 24 hours , then pretreated with 4 - PBA ( 5 mM ) for 1 hour and stimulated by Carfilzomib ( 1μM ) , Bortezomib ( 1μM ) , MLN9708 ( 2 μM ) , MG132 ( 5 μM ) . RNA was extracted from cells 6 hours after stimulation and the expression of Il - 1β , Il - 6 and Inos were detected through RT - qPCR . The data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B. Carfilzomib and proteasome inhibitor MG132 activate Il-1β luciferase in macrophages. (A) BMDMs were treated with IL-4 (20 ng/mL) for 24 hours and then stimulated by DMSO, Carfilzomib (1 μM), or MG132 (5 μM). (B) BMDMs were stimulated by DMSO, Carfilzomib (1 μM) or MG132 (5 μM)) for 1 hour, then induced with IL-4 (20 ng/mL). Luciferase assays were monitored 12 hours after stimulation. The data are means ± SD of three independent experiments. ***p < 0.001 (student's t-test).E. Carfilzomib, Bortezomib, MLN9708 and MG132 induce ER stress response. BMDMs were pretreated with IL-4 (20 ng/mL) for 24 hours and then stimulated by DMSO or Carfilzomib (1 μM), Bortezomib (1 μM), MLN9708 (2 μM), MG132 (5 μM). RNA was extracted from cells 3 hours or 6 hours after stimulation and the expression of ER stress related genes (Bip, Chop) were detected through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).F. ER stress agonists, TUN and TG, promote the expression of M1 macrophage markers, as well as reduce the expression of M2 macrophage markers. BMDMs were pretreated with IL-4 (20 ng/mL) for 24 hours and then stimulated by DMSO, TUN (500 nM) or TG (500 nM). RNA was extracted from cells 6 hours after stimulation and indicated genes were quantified through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).G. Inhibition of ER stress impairs the ability of Carfilzomib, Bortezomib, MLN9708 and MG132 to reprogram M2 towards M1-like macrophages. BMDMs were induced by IL-4 (20 ng/mL) for 24 hours, then pretreated with 4-PBA (5 mM) for 1 hour and stimulated by Carfilzomib (1μM), Bortezomib (1μM), MLN9708 (2 μM), MG132 (5 μM). RNA was extracted from cells 6 hours after stimulation and the expression of Il-1β, Il-6 and Inos were detected through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).


Figure 4A , B Deficiency of Ern1 reduces the expression of inflammatory related genes . Wild - type and Ern1 - / - Raw264 . 7 cells ( A ) or BMDMs ( B ) were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then stimulated by DMSO or Carfilzomib ( 1 μM ) . The mRNA of Il - 1β and Il - 6 were quantified through RT - qPCR 6 hours after stimulation . The data are means ± SD of three independent experiments . * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - test ) . E . Kira6 inhibits the expression of ER stress related genes and inflammatory related genes . BMDMs was treated by IL - 4 ( 20 ng / mL ) for 24 hours , treated with Kira6 ( 100 nM ) for 1 hour , and then stimulated by Carfilzomib ( 1 μM ) for 6 hours . RNA was extracted from cells and the expression of Bip , Chop , Il - 1β and Il - 6 were quantified through RT - qPCR . The data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( student ' s t - test ) . F , G . Carfilzomib has no effect on splicing of XBP1 in M2 macrophages . BMDMs ( F ) or Raw264 . 7 cells ( G ) were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then stimulated with DMSO , Carfilzomib ( 1 μM ) or TUN ( 500 nM ) . The mRNA expression of sXBP1 and unXBP1 were detected through RT - qPCR 6 hours after stimulation . The data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - test ) . J . Carfilzomib promotes the association between IRE1α and TRAF2 in M2 macrophages . Raw264 . 7 cells were pretreated by IL - 4 ( 20ng / mL ) for 24 hours , then stimulated by Carfilzomib ( 1 μM ) for 2 or 4 hours . Coimmunoprecipitation ( Co - IP ) and IB were performed with indicated antibodies . L . Carfilzomib activates NF - κB signaling pathway through IRE1α in M2 macrophages . Wild - type and Ern1 - / - Raw264 . 7 cells were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then stimulated by Carfilzomib ( 1 μM ) for indicated time points . WCL were analyzed by IB with indicated antibodies .

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

Figure 4A, B Deficiency of Ern1 reduces the expression of inflammatory related genes. Wild-type and Ern1-/- Raw264.7 cells (A) or BMDMs (B) were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO or Carfilzomib (1 μM). The mRNA of Il-1β and Il-6 were quantified through RT-qPCR 6 hours after stimulation. The data are means ± SD of three independent experiments. **p < 0.01, ***p < 0.001 (student's t-test).E. Kira6 inhibits the expression of ER stress related genes and inflammatory related genes. BMDMs was treated by IL-4 (20 ng/mL) for 24 hours, treated with Kira6 (100 nM) for 1 hour, and then stimulated by Carfilzomib (1 μM) for 6 hours. RNA was extracted from cells and the expression of Bip, Chop, Il-1β and Il-6 were quantified through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01 (student's t-test). F, G. Carfilzomib has no effect on splicing of XBP1 in M2 macrophages. BMDMs (F) or Raw264.7 cells (G) were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated with DMSO, Carfilzomib (1 μM) or TUN (500 nM). The mRNA expression of sXBP1 and unXBP1 were detected through RT-qPCR 6 hours after stimulation. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).J. Carfilzomib promotes the association between IRE1α and TRAF2 in M2 macrophages. Raw264.7 cells were pretreated by IL-4 (20ng/mL) for 24 hours, then stimulated by Carfilzomib (1 μM) for 2 or 4 hours. Coimmunoprecipitation (Co-IP) and IB were performed with indicated antibodies.L. Carfilzomib activates NF-κB signaling pathway through IRE1α in M2 macrophages. Wild-type and Ern1-/- Raw264.7 cells were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by Carfilzomib (1 μM) for indicated time points. WCL were analyzed by IB with indicated antibodies.


Figure 5C . Representative images of Hematoxylin and eosin ( H & E ) staining of the lung tissue obtained from different treatment groups . Expression of Ki - 67 in lung tissues was detected by immunohistochemistry method . D . Statistics of the tumor area and the number of Ki67 positive cells in sections of lung tissue . Data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - test ) . E . Carfilzomib treatment promotes the infiltration of M1 macrophages . Tumor bearing TD mice were treated as indicated in ( A ) and euthanized . Lung tissues were dissected for evaluating M1 / M2 populations through analyze the expression of CD80 and CD206 by flow cytometry . F . Statistics represent the proportion of CD80 or CD206 positive cells in lung tissue . Data are means ± SD of three independent experiments . * * p < 0 . 01 ( student ' s t - test ) . G . Carfilzomib treatment promotes the infiltration and activation of CD8 + T cells . Lung tissues of ( E ) were used for evaluating the infiltration of CD8 + T cells and their expression of CD69 by flow cytometry . H . Statistics about the proportion of CD8 + or CD69 + positive cells in lung tissue . Data are means ± SD of three independent experiments . * * p < 0 . 01 ( student ' s t - test ) . K , L . Carfilzomib shrinks tumor through modulating tumor microenvironment . EG7 cells were inoculated in wild - type or LyzM - cre ; lsl - dTA to allow tumor xenograft to reach a volume of around 80 mm3 . Mice were randomized for treatment ( n = 5 ) by saline or Carfilzomib for two weeks . The xenografts were dissected to photograph ( K ) or weigh ( L ) after treatment . Data are shown as mean ± SD and n indicates the number of biological replicates . * p < 0 . 05 , * * * p < 0 . 001 ( student ' s 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 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 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

Figure 5C. Representative images of Hematoxylin and eosin (H&E) staining of the lung tissue obtained from different treatment groups. Expression of Ki-67 in lung tissues was detected by immunohistochemistry method. D. Statistics of the tumor area and the number of Ki67 positive cells in sections of lung tissue. Data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).E. Carfilzomib treatment promotes the infiltration of M1 macrophages. Tumor bearing TD mice were treated as indicated in (A) and euthanized. Lung tissues were dissected for evaluating M1/M2 populations through analyze the expression of CD80 and CD206 by flow cytometry. F. Statistics represent the proportion of CD80 or CD206 positive cells in lung tissue. Data are means ± SD of three independent experiments. **p < 0.01 (student's t-test). G. Carfilzomib treatment promotes the infiltration and activation of CD8+ T cells. Lung tissues of (E) were used for evaluating the infiltration of CD8+ T cells and their expression of CD69 by flow cytometry. H. Statistics about the proportion of CD8+ or CD69+ positive cells in lung tissue. Data are means ± SD of three independent experiments. **p < 0.01 (student's t-test).K, L. Carfilzomib shrinks tumor through modulating tumor microenvironment. EG7 cells were inoculated in wild-type or LyzM-cre;lsl-dTA to allow tumor xenograft to reach a volume of around 80 mm3. Mice were randomized for treatment (n=5) by saline or Carfilzomib for two weeks. The xenografts were dissected to photograph (K) or weigh (L) after treatment. Data are shown as mean ±SD and n indicates the number of biological replicates. *p < 0.05, ***p < 0.001 (student's t-test).


Figure 6D . CD4 + and CD8 + T cells play a role in mediating cancer - shrinking effect of Carfilzomib . Lung - cancer - bearing TD mice were treated by saline , Carfilzomib , anti - mouse CD4 antibody ( ip . ) , anti - mouse CD8 antibody ( ip . ) , or Carfilzomib plus anti - mouse CD4 / 8 antibody . CT scanning was performed after two - week treatment . E . Representative images of H & E staining of the treated lung tissues . Expression of Ki - 67 in lung tissues was detected by immunohistochemistry method . F . Carfilzomib synergized with PD1 antibody in shrinking lung cancers . Lung cancer bearing TD mice were treated by saline , anti - PD1 antibody ( ip ) , Carfilzomib , or Carfilzomib plus anti - PD1 ( ip ) . CT scanning was performed after two - week treatment . G . Representative images of H & E staining of the lung tissues . Expression of Ki - 67 in lung tissue was detected by immunohistochemistry method .

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

Figure 6D. CD4+ and CD8+ T cells play a role in mediating cancer-shrinking effect of Carfilzomib. Lung-cancer-bearing TD mice were treated by saline, Carfilzomib, anti-mouse CD4 antibody (ip.), anti-mouse CD8 antibody (ip.), or Carfilzomib plus anti-mouse CD4/8 antibody. CT scanning was performed after two-week treatment. E. Representative images of H&E staining of the treated lung tissues. Expression of Ki-67 in lung tissues was detected by immunohistochemistry method. F. Carfilzomib synergized with PD1 antibody in shrinking lung cancers. Lung cancer bearing TD mice were treated by saline, anti-PD1 antibody (ip), Carfilzomib, or Carfilzomib plus anti-PD1 (ip). CT scanning was performed after two-week treatment. G. Representative images of H&E staining of the lung tissues. Expression of Ki-67 in lung tissue was detected by immunohistochemistry method.


Figure 7A . Carfilzomib , Bortezomib and MLN9708 promote the expression of M1 macrophage markers ( Il - 1β , Il - 6 , Tnfα ) and reduce the expression of M2 macrophage markers ( Il - 10 , Tgfβ ) in macrophages derived from PBMCs . After induced by IL - 4 ( 20 ng / ml ) for 24 hours to differentiate into mature M2 macrophages , PBMCs derived macrophages were stimulated by Carfilzomib ( 1 μM ) , Bortezomib ( 1 μM ) or MLN9708 ( 2 μM ) . RNA was extracted from cells 6 hours after stimulation and the expression of mRNA were quantified through RT - qPCR . Data from three experiments are presented as the mean ± SD . T test was used for statistical analysis of differences between groups . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - test ) . C - F . Carfilzomib , Bortezomib and MLN9708 increase the expression of CD80 ( C ) and reduce the expression of CD206 ( E ) in M2 macrophages derived from PBMCs . After activated by IL - 4 ( 20 ng / ml ) for 24 hours to differentiate into mature M2 macrophages , PBMCs derived macrophages were stimulated by Carfilzomib ( 500 nM ) , Bortezomib ( 500 nM ) or MLN9708 ( 500 nM ) . The representative histogram of CD80 and CD206 expression was analyzed by flow cytometry 12 hours after stimulation . ( D , F ) Statistics represent the proportion of CD80 ( D ) or CD206 ( F ) positive cells in macrophages derived from PBMCs . Data from three experiments are presented as the mean ± SD . T test was used for statistical analysis of differences between groups . * p < 0 . 05 , * * p < 0 . 01 ( student ' s 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 7A. Carfilzomib, Bortezomib and MLN9708 promote the expression of M1 macrophage markers (Il-1β, Il-6, Tnfα) and reduce the expression of M2 macrophage markers (Il-10, Tgfβ) in macrophages derived from PBMCs. After induced by IL-4 (20 ng/ml) for 24 hours to differentiate into mature M2 macrophages, PBMCs derived macrophages were stimulated by Carfilzomib (1 μM), Bortezomib (1 μM) or MLN9708 (2 μM). RNA was extracted from cells 6 hours after stimulation and the expression of mRNA were quantified through RT-qPCR. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).C-F. Carfilzomib, Bortezomib and MLN9708 increase the expression of CD80 (C) and reduce the expression of CD206 (E) in M2 macrophages derived from PBMCs. After activated by IL-4 (20 ng/ml) for 24 hours to differentiate into mature M2 macrophages, PBMCs derived macrophages were stimulated by Carfilzomib (500 nM), Bortezomib (500 nM) or MLN9708 (500 nM). The representative histogram of CD80 and CD206 expression was analyzed by flow cytometry 12 hours after stimulation. (D, F) Statistics represent the proportion of CD80 (D) or CD206 (F) positive cells in macrophages derived from PBMCs. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. *p < 0.05, **p < 0.01 (student's t-test).


Figure 2C , AltFUS ( arrow ) endogenous expression in Human tissues ( brain , muscles and kidney - 100 μg ) , in HEK293 and HeLa cultured cells ( 100 μg ) and using the over - expression of altFUS CDS in HEK293 cells ( 50 μg ) as positive control ( representative image from n = 3 ) . The asterisk indicates a protein species detected with the anti - altFUS antibody specifically in the brain . AltFUS ( arrow ) endogenous expression in the motor cortex of three C9orf72 and three sporadic ALS patients ( D ) ( representative image from n = 3 ) . The asterisk indicates a protein species detected with the anti - altFUS antibody specifically in the brain . AltFUS ( arrow ) endogenous expression in iPSC - derived motor neurons of three lines from controls and from ALS patients ( E ) ( representative image from n = 3 ) . The asterisk indicates a protein species detected with the anti - altFUS antibody specifically in the brain . I , Endogenous altFUS mitochondrial expression in HEK293 cells following fractionation ( representative image from n = 3 ) , with Tubulin as a marker of the cytosolic fraction and VDAC as a marker of the mitochondrial fraction . We used siFUS transfected cells as a negative control and altFUS transfected cells as a positive control for altFUS expression ( WCL = Whole Cell Lysate , Cyto = Cytosol fraction , Mito = mitochondrial fraction ) . J , Representative images of the mitochondrial network ( Tomm20 in red ) in mock and altFUS - Flag ( green ) transfected HeLa cells ( n = 3 ) . The white scale bar corresponds to 10μm . K , Proportion of tubules and globules in the mitochondrial network of mock HeLa cells and HeLa cells transfected with altFUS - Flag Quantification was done over a minimum of 100 cells across a technical duplicate per independent experiments ( n = 3 , i . e . a minimum of 300 cells per biological conditions , p - value < 0 . 001 , Mann - Whitney U test ) . The boxes extend to the 25th and 75th percentiles , with the median marked . The whiskers correspond to the 5th and 95th percentiles .

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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2C, AltFUS (arrow) endogenous expression in Human tissues (brain, muscles and kidney - 100 μg), in HEK293 and HeLa cultured cells (100 μg) and using the over-expression of altFUS CDS in HEK293 cells (50 μg) as positive control (representative image from n=3). The asterisk indicates a protein species detected with the anti-altFUS antibody specifically in the brain.AltFUS (arrow) endogenous expression in the motor cortex of three C9orf72 and three sporadic ALS patients (D) (representative image from n=3). The asterisk indicates a protein species detected with the anti-altFUS antibody specifically in the brain.AltFUS (arrow) endogenous expression in iPSC-derived motor neurons of three lines from controls and from ALS patients (E) (representative image from n=3). The asterisk indicates a protein species detected with the anti-altFUS antibody specifically in the brain.I, Endogenous altFUS mitochondrial expression in HEK293 cells following fractionation (representative image from n=3), with Tubulin as a marker of the cytosolic fraction and VDAC as a marker of the mitochondrial fraction. We used siFUS transfected cells as a negative control and altFUS transfected cells as a positive control for altFUS expression (WCL = Whole Cell Lysate, Cyto = Cytosol fraction, Mito = mitochondrial fraction).J, Representative images of the mitochondrial network (Tomm20 in red) in mock and altFUS-Flag (green) transfected HeLa cells (n=3). The white scale bar corresponds to 10μm. K, Proportion of tubules and globules in the mitochondrial network of mock HeLa cells and HeLa cells transfected with altFUS-Flag Quantification was done over a minimum of 100 cells across a technical duplicate per independent experiments (n=3, i.e. a minimum of 300 cells per biological conditions, p-value < 0.001, Mann-Whitney U test). The boxes extend to the 25th and 75th percentiles, with the median marked. The whiskers correspond to the 5th and 95th percentiles.


Figure 3E , Scatter plot of the proteins identified by AP - MS indicating their enrichment ( Fold - change over control ) and their SAINT probability score . Proteins above the 0 . 8 threshold ( grey line ) are indicated in black , others in grey . AltFUS is indicated in red ( bait ) and preys known to regulate the autophagy or the cellular stress response are indicated in green .

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

Figure 3E, Scatter plot of the proteins identified by AP-MS indicating their enrichment (Fold-change over control) and their SAINT probability score. Proteins above the 0.8 threshold (grey line) are indicated in black, others in grey. AltFUS is indicated in red (bait) and preys known to regulate the autophagy or the cellular stress response are indicated in green.


Figure 4B , LC3 - II accumulation after bafilomycin treatment from mock , altFUS , FUS , FUS ( Ø ) , FUS - R495x , FUS ( Ø ) - R495x transfected cells and FUS ( Ø ) - R495x and altFUS co - transfected cells across 3 independent experiments ( n = 3 , mean ± SD ) . The quantification corresponds to the treated / untreated ratio of LC3 - II abundance Statistical significance is relative to the mock condition unless otherwise indicated ( * * * * = p value < 0 . 0001 , * * * = p value < 0 . 001 , * * = p value < 0 . 01 , n . s . = non - significant , two - way ANOVA with Tukey ' s multiple comparison correction ) . D - E , Quantification of FUS cytoplasmic granules , number ( D ) and area ( μm2 ) ( E ) in cells over - expressing the bicistronic ( + ) or monocistronic ( - ) construct for FUS , FUS - G156E , FUS - R495x , FUS - K510E , FUS - Q519x , FUS - Q519I - fs527x , FUS - R521C , and FUS - P525L . Statistical comparisons are made between bicistronic and monocistronic versions of each construct ( n = 3 - biological replicates , mean ± SD , * * * * = p value < 0 . 0001 , * * * = p value < 0 . 001 , * * = p value < 0 . 01 , * = p value < 0 . 05 , n . s . = non - significant , one - way ANOVA test with Sidak ' s multiple comparison ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4B, LC3-II accumulation after bafilomycin treatment from mock, altFUS, FUS, FUS(Ø), FUS-R495x, FUS(Ø)-R495x transfected cells and FUS(Ø)-R495x and altFUS co-transfected cells across 3 independent experiments (n=3, mean ± SD). The quantification corresponds to the treated/untreated ratio of LC3-II abundance Statistical significance is relative to the mock condition unless otherwise indicated (**** = p value < 0.0001, *** = p value < 0.001, ** = p value < 0.01, n.s. = non-significant, two-way ANOVA with Tukey's multiple comparison correction).D-E, Quantification of FUS cytoplasmic granules, number (D) and area (μm2) (E) in cells over-expressing the bicistronic (+) or monocistronic (-) construct for FUS, FUS-G156E, FUS-R495x, FUS-K510E, FUS-Q519x, FUS-Q519I-fs527x, FUS-R521C, and FUS-P525L. Statistical comparisons are made between bicistronic and monocistronic versions of each construct (n=3 - biological replicates, mean ± SD, **** = p value < 0.0001, *** = p value < 0.001, ** = p value < 0.01, * = p value < 0.05, n.s. = non-significant, one-way ANOVA test with Sidak's multiple comparison).


Figure 5C - E , Locomotion assay represented by the percentage of climbing success in control and RU - 486 - treated transgenic Drosophila expressing mCherry or altFUS ( C ) , the bicistronic FUS or the monocistronic FUS ( Ø ) ( D ) , and the bicistronic FUS - R495x or the monocistronic FUS ( Ø ) - R495x ( E ) at day 1 , 10 and 20 post - induction . Statistical comparisons were made between each population ( n = 4 - biological replicates ) . Indicated significance is between the monocistronic and the bicistronic transgenic flies of the RU - 486 - treated population ( mean ± SD , n . s . = non - significant , * = p value < 0 . 05 , * * * = p value < 0 . 001 , two - way ANOVA with Tukey ' s multiple comparison correction ) .

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Figure 5C-E, Locomotion assay represented by the percentage of climbing success in control and RU-486-treated transgenic Drosophila expressing mCherry or altFUS (C), the bicistronic FUS or the monocistronic FUS(Ø) (D), and the bicistronic FUS-R495x or the monocistronic FUS(Ø)-R495x (E) at day 1, 10 and 20 post-induction. Statistical comparisons were made between each population (n=4 - biological replicates). Indicated significance is between the monocistronic and the bicistronic transgenic flies of the RU-486-treated population (mean ± SD, n.s. = non-significant, * = p value < 0.05, *** = p value < 0.001, two-way ANOVA with Tukey's multiple comparison correction).


Figure 6A , Graphical representation of FUS synonymous mutations ( yellow ) found in ALS patients . The canonical FUS mRNA is represented in dark blue ( ENST00000254108 or NM _ 004960 ) . The FUS protein coding sequence is indicated in light blue , and the altFUS protein coding sequence is indicated in green . B , Images by confocal microscopy of TDP - 43 ( white ) , FUS ( GFP tagged - green ) and altFUS ( Flag tagged - red ) in HeLa cells over - expressing GFP - FUS ( Flag ) , GFP - FUS ( P31L - Flag ) - S44 = , GFP - FUS ( A38V - Flag ) - G51 = , GFP - FUS ( A46V - Flag ) - G59 = , GFP - FUS ( R64P - Flag ) - S77 = ( representative images from n = 3 ) . White arrows indicate some TDP - 43 aggregates . The white scale bar corresponds to 10 μm . C , Quantification of cells with TDP - 43 aggregates in HeLa cells ( see panel B ) . The data are represented as the fold - change compared to the GFP - FUS ( Flag ) expressing cells . Statistical significance is indicated above the bars ( n = 3 - biological replicates , mean ± SD , * * * * = p value < 0 . 0001 , two - way ANOVA with Tukey ' s multiple comparison correction ) .

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

Figure 6A, Graphical representation of FUS synonymous mutations (yellow) found in ALS patients. The canonical FUS mRNA is represented in dark blue (ENST00000254108 or NM_004960). The FUS protein coding sequence is indicated in light blue, and the altFUS protein coding sequence is indicated in green. B, Images by confocal microscopy of TDP-43 (white), FUS (GFP tagged - green) and altFUS (Flag tagged - red) in HeLa cells over-expressing GFP-FUS(Flag), GFP-FUS(P31L-Flag)-S44=, GFP-FUS(A38V-Flag)-G51=, GFP-FUS(A46V-Flag)-G59=, GFP-FUS(R64P-Flag)-S77= (representative images from n=3). White arrows indicate some TDP-43 aggregates. The white scale bar corresponds to 10 μm. C, Quantification of cells with TDP-43 aggregates in HeLa cells (see panel B). The data are represented as the fold-change compared to the GFP-FUS(Flag) expressing cells. Statistical significance is indicated above the bars (n=3 - biological replicates, mean ± SD, **** = p value < 0.0001, two-way ANOVA with Tukey's multiple comparison correction).


Figure 2Comparison of SWATH - MS - based quantification to DDA - MS - based strategies ( MS1 XIC and spectral counting ) with regard to consistency ( as judged based upon protein - level SEC chromatogram robustness towards increasing requirements on the number of consecutive detections ) and precision ( judged based on the correlation between sibling peptide SEC chromatograms ) of quantification and overall performance in error - controlled complex - centric query of CORUM complexes in the respective protein level chromatogram sets ( also see Appendix Figure S3C ) . TP , true positive assignments according to error model , P , all positives .

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Figure 2Comparison of SWATH-MS-based quantification to DDA-MS-based strategies (MS1 XIC and spectral counting) with regard to consistency (as judged based upon protein-level SEC chromatogram robustness towards increasing requirements on the number of consecutive detections) and precision (judged based on the correlation between sibling peptide SEC chromatograms) of quantification and overall performance in error-controlled complex-centric query of CORUM complexes in the respective protein level chromatogram sets (also see Appendix Figure S3C). TP, true positive assignments according to error model, P, all positives.


Figure 3Peptide - level SEC chromatograms are grouped by UniprotKB identifier to detect co - elution signals indicative of protein elution ranges / peaks . Based on external size calibration of the apparent analyte molecular weight per SEC fraction , signals can be attributed to likely assembled or monomeric state ( also see Appendix Figure S4 ) . For TCPE , three distinct elution signals , numbered 1 - 3 , are detected , two in the assembled and one in the monomer elution range . Proteins are observed eluting in 1 - 6 distinct peaks and with a wide range of apparent vs . monomeric molecular weight ratios ( distributions , left panels ) . The molecular weight ratios of the three peaks detected for TCPE ( displayed in A ) are indicated . Many of the proteins eluting in a single peak ( top panel and bar ) appear assembled . Proteins eluting multiple times ( 27 % of the proteins ) do so preferentially in the assembled range , suggesting frequent participation in multiple differently sized macromolecular assemblies ( lower panels and pie chart ) . For a list of all detected protein peaks , see Dataset EV3 . Proteins observed in multiple assembled peaks ( n = 659 ) are enriched in components of the proteasome and other known large complex assemblies .

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 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

Figure 3Peptide-level SEC chromatograms are grouped by UniprotKB identifier to detect co-elution signals indicative of protein elution ranges/peaks. Based on external size calibration of the apparent analyte molecular weight per SEC fraction, signals can be attributed to likely assembled or monomeric state (also see Appendix Figure S4). For TCPE, three distinct elution signals, numbered 1-3, are detected, two in the assembled and one in the monomer elution range.Proteins are observed eluting in 1-6 distinct peaks and with a wide range of apparent vs. monomeric molecular weight ratios (distributions, left panels). The molecular weight ratios of the three peaks detected for TCPE (displayed in A) are indicated. Many of the proteins eluting in a single peak (top panel and bar) appear assembled. Proteins eluting multiple times (27 % of the proteins) do so preferentially in the assembled range, suggesting frequent participation in multiple differently sized macromolecular assemblies (lower panels and pie chart). For a list of all detected protein peaks, see Dataset EV3.Proteins observed in multiple assembled peaks (n = 659) are enriched in components of the proteasome and other known large complex assemblies.


Figure 5For nearly half the CORUM complex hypotheses queried , two or more distinct subunit co - elution signals were detected ( See methods and Appendix information on CCprofiler ) . Among the four distinct co - elution signals detected from the eight canonical CSN subunits ' chromatograms ( here with CSN7A , not CSN7B ) are two distinct signals indicating distinct co - elution of two different complex variants . Distinct co - elution of holo - CSN ( observed at the expected fraction 35 ) and Mini - CSN CSN1 / 3 / 8 ( observed eluting offset only one fraction late , F45 , of the expected fraction , F44 ) . Expected fractions are estimated from the cumulative sum of one copy per component and external size calibration . Coloring adapted to highlight subversion components and their partitioning across holo - and sub - complex . CSN1 / 3 / 8 interact and form a submodule within the CSN holo - complex structure ( PDB accession 4D10 ) . The observations are consistent between the two whole workflow replicates ( see Appendix Figure S6A ) . Lower right panel , validation of distinct elution behavior of holo - CSN exclusive ( CSN7A ) and shared subunit ( CSN8 ) by immunoblotting . For full immunoblotting data ( CSN1 , CSN3 , CSN4 , CSN5 , CSN7A and CSN8 ) see Appendix Figure S7C ) . CSN1 / 3 / 8 display distinct fractionation patterns in co - fractionation experiments performed in other laboratories , specifically in orthogonal ion exchange fractionation of HEK293 lysates ( Wan et al . 2015 , upper panels ) and size exclusion chromatographic fractionation of U2OS lysates ( Kirkwood et al . 2013 , lower panel ) , in line with the CSN1 / 3 / 8 as distinct entity . Baculoviral co - expression of human CSN1 , CSN3 and CSN8 in Sf21VM insect cells , with CSN8 N - terminally Strep ( II ) - and CSN1 & CSN3 N - terminally His6 - tagged , followed by affinity purification and SDS - PAGE displays banding pattern in line with the formation of a stable trimer CSN1 / 3 / 8 .

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Figure 5For nearly half the CORUM complex hypotheses queried, two or more distinct subunit co-elution signals were detected (See methods and Appendix information on CCprofiler).Among the four distinct co-elution signals detected from the eight canonical CSN subunits' chromatograms (here with CSN7A, not CSN7B) are two distinct signals indicating distinct co-elution of two different complex variants.Distinct co-elution of holo-CSN (observed at the expected fraction 35) and Mini-CSN CSN1/3/8 (observed eluting offset only one fraction late, F45, of the expected fraction, F44). Expected fractions are estimated from the cumulative sum of one copy per component and external size calibration. Coloring adapted to highlight subversion components and their partitioning across holo- and sub-complex. CSN1/3/8 interact and form a submodule within the CSN holo-complex structure (PDB accession 4D10). The observations are consistent between the two whole workflow replicates (see Appendix Figure S6A). Lower right panel, validation of distinct elution behavior of holo-CSN exclusive (CSN7A) and shared subunit (CSN8) by immunoblotting. For full immunoblotting data (CSN1, CSN3, CSN4, CSN5, CSN7A and CSN8) see Appendix Figure S7C).CSN1/3/8 display distinct fractionation patterns in co-fractionation experiments performed in other laboratories, specifically in orthogonal ion exchange fractionation of HEK293 lysates (Wan et al. 2015, upper panels) and size exclusion chromatographic fractionation of U2OS lysates (Kirkwood et al. 2013, lower panel), in line with the CSN1/3/8 as distinct entity.Baculoviral co-expression of human CSN1, CSN3 and CSN8 in Sf21VM insect cells, with CSN8 N-terminally Strep(II)- and CSN1 & CSN3 N-terminally His6-tagged, followed by affinity purification and SDS-PAGE displays banding pattern in line with the formation of a stable trimer CSN1/3/8.


Figure 6Protein - level SEC chromatograms of the 22 canonical 26S proteasome subunits . Vertical black lines indicate the apexes of six distinct co - elution signals detected in complex - centric scoring ; two of which represent well - known co - occurring variants , the full 26S ( i ) and the 20S ( ii ) particle devoid the 19S lid and ATPase ( Indicated by structural models , PDB accession 5GJR ) and four of which , composed of predominantly 20S α and β subunits , appear at reduced size ( 222 - 107 kDa , fractions 39 , 40 , 42 and 46 ) . The observations are consistent between the two whole workflow replicates ( see Appendix Figure S6B ) . Subunit mass distribution across early and late assembly intermediate elution ranges suggests predominant components of the intermediary species accumulating in HEK293 cells .

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

Figure 6Protein-level SEC chromatograms of the 22 canonical 26S proteasome subunits. Vertical black lines indicate the apexes of six distinct co-elution signals detected in complex-centric scoring; two of which represent well-known co-occurring variants, the full 26S (i) and the 20S (ii) particle devoid the 19S lid and ATPase (Indicated by structural models, PDB accession 5GJR) and four of which, composed of predominantly 20S α and β subunits, appear at reduced size (222 - 107 kDa, fractions 39, 40, 42 and 46). The observations are consistent between the two whole workflow replicates (see Appendix Figure S6B).Subunit mass distribution across early and late assembly intermediate elution ranges suggests predominant components of the intermediary species accumulating in HEK293 cells.


Figure 7SECexplorer web - interface for querying custom protein sets for co - elution behavior in the SEC - SWATH - MS data , viewing chromatograms for interpretation and with algorithmic assistance . Zoom in to high MW peak shoulder of holo - COP9 Signalosome ( compare Figure 5 ) , where defined co - elution signals of CSN substrate components CUL4A and DDB2 suggest the partial resolution of substrate - bound and free pools of CSN holo - complex . Estimation of the fraction of holo - CSN in the likely substrate - bound pool versus the free pool , with eight measurements along the eight subunits and based on Gaussian deconvolution of two signals underlying the observed peak and shoulder ( also see Appendix Figure S7 ) .

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Figure 7SECexplorer web-interface for querying custom protein sets for co-elution behavior in the SEC-SWATH-MS data, viewing chromatograms for interpretation and with algorithmic assistance.Zoom in to high MW peak shoulder of holo-COP9 Signalosome (compare Figure 5), where defined co-elution signals of CSN substrate components CUL4A and DDB2 suggest the partial resolution of substrate-bound and free pools of CSN holo-complex.Estimation of the fraction of holo-CSN in the likely substrate-bound pool versus the free pool, with eight measurements along the eight subunits and based on Gaussian deconvolution of two signals underlying the observed peak and shoulder (also see Appendix Figure S7).


Figure 1A Statistical analysis of NFATc1 expression with respect to tumor grading in KPCmice tissues . B Immunohistochemistry detection of NFATc1 and p53 , illustrating G1 ‐ G2 and G3 in human PDAC tissues . Scale bars , 100 μm . C Statistical illustration of TMA analysis demonstrating NFATc1 strong nuclear positive tumors ( n = 161 patients ) with respect to functional status of p53 expression in human PDAC tissues . D Representative H & amp ; amp ; E ‐ stained sections as well as immunohistochemistry detection of HA ‐ NFATc1 and p53 in well ‐ and poorly differentiated pancreatic tissues of KNCmice . Scale bars , 100 μm . E Quantification of p53 expression with respect to the tumor grading by evaluating both the intensity of immunostaining and the number of cells with overexpression by means of an immunoreactivity score ( IRS ) in KNCmice ( every dot represents one mouse ) . F Incidence of livermetastases in KNCmice ( mean values ± SD ) .

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Figure 1A Statistical analysis of NFATc1 expression with respect to tumor grading in KPCmice tissues.B Immunohistochemistry detection of NFATc1 and p53, illustrating G1‐G2 and G3 in human PDAC tissues. Scale bars, 100 μm.C Statistical illustration of TMA analysis demonstrating NFATc1 strong nuclear positive tumors (n = 161 patients) with respect to functional status of p53 expression in human PDAC tissues.D Representative H&amp;E‐stained sections as well as immunohistochemistry detection of HA‐NFATc1 and p53 in well‐ and poorly differentiated pancreatic tissues of KNCmice. Scale bars, 100 μm.E Quantification of p53 expression with respect to the tumor grading by evaluating both the intensity of immunostaining and the number of cells with overexpression by means of an immunoreactivity score (IRS) in KNCmice (every dot represents one mouse).F Incidence of livermetastases in KNCmice (mean values ± SD).


Figure 2A Genome ‐ wide expression and GSEA analysis show p53 ‐ enriched signatures with " EMT " and " stem cell transcript " in KNCtumor cells . B Heat map showing p53 ‐ dependent regulation of " EMT " ‐ and " stemness " ‐ related genes in KNCtumor cells . Fold change relative to control cells is displayed in a green red color scheme for selected genes with FClog2 1 . 0 or FClog2 > − 1 . 0 . C Statistical analysis of wound healing assay performed in primary KNCtumor cells following p53 depletion . Time ‐ lapse analyzer software was used to quantify the wound closure rate . Means ± SD are shown from one out of three independent experiments . D Western blot analysis revealing protein expression levels upon p53 depletion in KNCtumor cells . E X ‐ fold mRNA expression of EMT ‐ related marker genes after p53 depletion in KNCtumor cells . Means ± SD from at least three independent experiments are shown . Asterisks display significance ( * P 0 . 05 ) . F Representative protein expression of EMT inducers in pancreatic lysates prepared from KNCmice with different tumor gradings .

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Figure 2A Genome‐wide expression and GSEA analysis show p53‐enriched signatures with "EMT" and "stem cell transcript" in KNCtumor cells.B Heat map showing p53‐dependent regulation of "EMT"‐ and "stemness"‐related genes in KNCtumor cells. Fold change relative to control cells is displayed in a green red color scheme for selected genes with FClog2 1.0 or FClog2 > −1.0.C Statistical analysis of wound healing assay performed in primary KNCtumor cells following p53 depletion. Time‐lapse analyzer software was used to quantify the wound closure rate. Means ± SD are shown from one out of three independent experiments.D Western blot analysis revealing protein expression levels upon p53 depletion in KNCtumor cells.E X‐fold mRNA expression of EMT‐related marker genes after p53 depletion in KNCtumor cells. Means ± SD from at least three independent experiments are shown. Asterisks display significance (*P 0.05).F Representative protein expression of EMT inducers in pancreatic lysates prepared from KNCmice with different tumor gradings.


Figure 3A Kaplan - Meier curves showing survival of KNC , KPNC , and KPΔNC mice under the control of p48 ‐ Cre pancreas ‐ specific promoters ( P 0 . 0001 for KPNC or KPΔNC cohorts versus KNC , log ‐ rank test , for pairwise combination ) . B Pathological features of KPNC and KP ∆ NC mice before tumor extraction . Pancreatic tissue is encircled . C , D HE and corresponding immunohistochemistry stainings of ( C ) KPNC and ( D ) KP ∆ NCmouse tumors . Scale bars , 100 μm . E Tumor grading of PDAC from KPNC mice ( n = 10 ) .

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Figure 3A Kaplan-Meier curves showing survival of KNC, KPNC, and KPΔNC mice under the control of p48‐Cre pancreas‐specific promoters (P 0.0001 for KPNC or KPΔNC cohorts versus KNC, log‐rank test, for pairwise combination).B Pathological features of KPNC and KP∆NC mice before tumor extraction. Pancreatic tissue is encircled.C, D HE and corresponding immunohistochemistry stainings of (C) KPNC and (D) KP∆NCmouse tumors. Scale bars, 100 μm.E Tumor grading of PDAC from KPNC mice (n = 10).


Figure 4A Quantitative RT ‐ PCR analysis of EMT ‐ related markers obtained from tumors derived from mice of the indicated genotypes ( n = 3 , means ± SD ) . B Relative mRNA expression of NFATc1 ‐ and EMT ‐ related marker genes on administration of CsA ( 1 μM ) in KPNC cells . Data represents means ± SD from at least three independent experiments . Asterisks show significance ( * P 0 . 05 ) . C Western blot analysis revealing protein expression levels in KPNC cells from the indicated proteins following transfection of siRNA control or siRNA toward NFATc1 , respectively .

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Figure 4A Quantitative RT‐PCR analysis of EMT‐related markers obtained from tumors derived from mice of the indicated genotypes (n = 3, means ± SD).B Relative mRNA expression of NFATc1‐ and EMT‐related marker genes on administration of CsA (1 μM) in KPNC cells. Data represents means ± SD from at least three independent experiments. Asterisks show significance (*P 0.05).C Western blot analysis revealing protein expression levels in KPNC cells from the indicated proteins following transfection of siRNA control or siRNA toward NFATc1, respectively.