figf5 ( b ) Serum and amino - acid starvation of HeLa cells for 5 h reduces LC3 - II levels ( above ) , but increases autolysosome numbers detected using tfLC3 ( below ) . With tfLC3 , GFP - and RFP - positive puncta represent autophagosomes before lysosomal fusion , whereas RFP - positive / GFP - negative puncta represent autolysosomes - GFP is more rapidly quenched by low lysosomal pH ( see Methods ) . Increased autolysosomes indicate enhanced starvation - induced flux of LC3 to lysosomes . ( c ) ARL8B knockdown increases autophagosomal synthesis . siRNA - transfected HeLa cells were incubated for 48 h , then left untreated or incubated with bafilomycin A1 . LC3 - II levels versus actin were quantified ( bottom graphs ) . Asterisk : nonspecific band . ( d ) ARL8B overexpression inhibits autophagosome synthesis and degradation . HeLa cells overexpressing ARL8B or empty vector were analysed as in ( c ) . ( e ) HeLa cells were co - transfected with either ARL8B overexpression construct or siRNA ( non - targeting siRNA and empty peGFP vector were transfection controls ) together with mCherry - LC3 for 48 h . After fixation , cells were stained for endogenous LAMP1 and DNA ( DAPI ) . Representative maximum - intensity projections of serial confocal optical sections are shown . Co - localization panels show overlapping mCherry - LC3 and LAMP1 signals . ( f - h ) Quantification of autophagosome - lysosome fusion in HeLa cells . Percentages of autolysosomes ( positive for both mCherry - LC3 and LAMP1 ) to autophagosomes ( positive for mCherry - LC3 and negative for LAMP1 ) were quantified . In ( h ) , we analysed cells treated for 2 h with nocodazole before fixation , which dispersed the perinuclear lysosomal cluster ( see Fig . 2a ) . ( i ) Autophagosomal and autolysosomal numbers in tfLC3 - expressing cells after ARL8B overexpression and knockdown ( Supplementary Fig . S5o shows representative cells . ) . In ( f - i ) we analysed 20 cells per group in three independent experiments . Values are means ± s . e . m of three independent experiments carried out in triplicate . * P0 . 05 , * * P0 . 01 , * * * P0 . 005 Student ' s t - test ; other comparisons not significant ( n . s . ) . Uncropped images of blots are shown in Supplementary Fig . S7 .

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

figf5(b) Serum and amino-acid starvation of HeLa cells for 5 h reduces LC3-II levels (above), but increases autolysosome numbers detected using tfLC3 (below). With tfLC3, GFP- and RFP-positive puncta represent autophagosomes before lysosomal fusion, whereas RFP-positive/GFP-negative puncta represent autolysosomes-GFP is more rapidly quenched by low lysosomal pH (see Methods). Increased autolysosomes indicate enhanced starvation-induced flux of LC3 to lysosomes.(c) ARL8B knockdown increases autophagosomal synthesis. siRNA-transfected HeLa cells were incubated for 48 h, then left untreated or incubated with bafilomycin A1. LC3-II levels versus actin were quantified (bottom graphs). Asterisk: nonspecific band.(d) ARL8B overexpression inhibits autophagosome synthesis and degradation. HeLa cells overexpressing ARL8B or empty vector were analysed as in (c).(e) HeLa cells were co-transfected with either ARL8B overexpression construct or siRNA (non-targeting siRNA and empty peGFP vector were transfection controls) together with mCherry-LC3 for 48 h. After fixation, cells were stained for endogenous LAMP1 and DNA (DAPI). Representative maximum-intensity projections of serial confocal optical sections are shown. Co-localization panels show overlapping mCherry-LC3 and LAMP1 signals. (f-h) Quantification of autophagosome-lysosome fusion in HeLa cells. Percentages of autolysosomes (positive for both mCherry-LC3 and LAMP1) to autophagosomes (positive for mCherry-LC3 and negative for LAMP1) were quantified. In (h), we analysed cells treated for 2 h with nocodazole before fixation, which dispersed the perinuclear lysosomal cluster (see Fig. 2a). (i) Autophagosomal and autolysosomal numbers in tfLC3-expressing cells after ARL8B overexpression and knockdown (Supplementary Fig. S5o shows representative cells.). In (f-i) we analysed 20 cells per group in three independent experiments. Values are means ± s.e.m of three independent experiments carried out in triplicate. *P0.05, **P0.01, ***P0.005 Student's t-test; other comparisons not significant (n.s.). Uncropped images of blots are shown in Supplementary Fig. S7.


Figure 1 ( A ) ELISA of supernatant IL - 1β for unprimed THP - 1 cells ( UT ) or LPS - primed ( 500 ng / ml , 3 hrs ) THP - 1 cells then infected with ZIKV ( MOI = 1 ) for 36 hrs . Uninfected cells serves as mock control . ( B ) Immunoblot analysis of supernatant ( Sup ) and cell extracts ( Lys ) of THP - 1 cells , left untreated or primed by LPS ( 500 ng / ml , 3 hrs ) , after ZIKV infection ( MOI = 1 ) for 36 hrs . ( C ) ELISA of IL - 1β in the supernatants of wild type ( WT ) , NLRP3 knockout ( KO ) # 1 or AIM2 KO # 1 THP - 1 cells left untreated ( UT ) , or pretreated with LPS ( 500 ng / ml , 3 hrs ) followed by ATP ( 5 mM , 6 hrs ) stimulation , or stimulated with poly ( dA : dT ) ( 1mg / ml , 6 hrs ) , or infected with ZIKV ( MOI = 1 , 36 hrs ) . ( D ) ELISA of IL - 1β in the supernatants of two clones of NLRP3 KO THP - 1 cells with or without ZIKV infection ( MOI = 1 ) at the indicated time points . ( E ) ELISA of IL - 1β in the supernatants of PBMCs from two donors transfected with control ( ctrl ) siRNA or NLRP3 siRNA followed by ZIKV infection ( MOI = 1 ) for 36 hrs . ( F ) ELISA of IL - 1β in the supernatant of WT , NLRP3 KO or AIM2 KO THP - 1 cells with ZIKV ( MR766 or GZ01 ) ( MOI = 1 ) or DENV - 2 ( MOI = 1 ) infection for 36 hrs . ( G ) ELISA of IL - 1β in the supernatants of THP - 1 cells pre - infected with ZIKV for 24 hrs or mock control . The cells were then treated with LPS ( 500 ng / ml , 3 hrs ) followed by ATP ( 5 mM ) treatment for 6 hrs . ( H ) Immunoblot analysis of supernatant ( Sup ) and cell extracts ( Lys ) of THP - 1 cells pre - infected with ZIKV ( MOI = 1 ) or mock control for 24 hrs . The cells were then treated with LPS ( 500 ng / ml , 3 hrs ) followed by ATP ( 5 mM ) stimulation for 6 hrs . ( I ) Immunoblot analysis of supernatants and cell extracts of 293T cells transfected with plasmids expressing NLRP3 , ASC , pro - caspase - 1 and pro - IL - 1β together with NS1 . ( J ) Immunoblot analysis of protein extracts of Flag - tagged NS1 - inducible THP - 1 cells treated with increasing doses of doxycycline ( Dox ) for 24 hrs . ( K ) ELISA of IL - 1β in the supernatants of Flag - NS1 - inducible THP - 1 cells left unprimed then treated with ATP ( 5 mM , 6 hrs ) or poly ( I : C ) ( 2 mg / ml , 6 hrs ) , or pre - treated with LPS ( 500 ng / ml , 3 hrs ) followed by ATP ( 5 mM , 6 hrs ) or poly ( I : C ) ( 2 mg / ml , 6 hrs ) stimulation . ( L ) Immunoblot analysis of supernatants ( Sup ) and cell extracts ( Lys ) of Flag - NS1 - inducible THP - 1 cells pre - treated with LPS ( 500 ng / ml , 3 hrs ) followed by ATP ( 5 mM , 6 hrs ) or poly ( I : C ) ( 2 mg / ml , 6 hrs ) stimulation .

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

Figure 1(A) ELISA of supernatant IL-1β for unprimed THP-1 cells (UT) or LPS-primed (500 ng/ml, 3 hrs) THP-1 cells then infected with ZIKV (MOI=1) for 36 hrs. Uninfected cells serves as mock control.(B) Immunoblot analysis of supernatant (Sup) and cell extracts (Lys) of THP-1 cells, left untreated or primed by LPS (500 ng/ml, 3 hrs), after ZIKV infection (MOI=1) for 36 hrs.(C) ELISA of IL-1β in the supernatants of wild type (WT), NLRP3 knockout (KO) #1 or AIM2 KO#1 THP-1 cells left untreated (UT), or pretreated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM, 6 hrs) stimulation, or stimulated with poly (dA:dT) (1mg/ml, 6 hrs), or infected with ZIKV (MOI=1, 36 hrs).(D) ELISA of IL-1β in the supernatants of two clones of NLRP3 KO THP-1 cells with or without ZIKV infection (MOI=1) at the indicated time points.(E) ELISA of IL-1β in the supernatants of PBMCs from two donors transfected with control (ctrl) siRNA or NLRP3 siRNA followed by ZIKV infection (MOI=1) for 36 hrs.(F) ELISA of IL-1β in the supernatant of WT, NLRP3 KO or AIM2 KO THP-1 cells with ZIKV (MR766 or GZ01) (MOI=1) or DENV-2 (MOI=1) infection for 36 hrs.(G) ELISA of IL-1β in the supernatants of THP-1 cells pre-infected with ZIKV for 24 hrs or mock control. The cells were then treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM) treatment for 6 hrs.(H) Immunoblot analysis of supernatant (Sup) and cell extracts (Lys) of THP-1 cells pre-infected with ZIKV (MOI=1) or mock control for 24 hrs. The cells were then treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM) stimulation for 6 hrs.(I) Immunoblot analysis of supernatants and cell extracts of 293T cells transfected with plasmids expressing NLRP3, ASC, pro-caspase-1 and pro-IL-1β together with NS1.(J) Immunoblot analysis of protein extracts of Flag-tagged NS1-inducible THP-1 cells treated with increasing doses of doxycycline (Dox) for 24 hrs.(K) ELISA of IL-1β in the supernatants of Flag-NS1-inducible THP-1 cells left unprimed then treated with ATP (5 mM, 6 hrs) or poly (I:C) (2 mg/ml, 6 hrs), or pre-treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM, 6 hrs) or poly (I:C) (2 mg/ml, 6 hrs) stimulation.(L) Immunoblot analysis of supernatants (Sup) and cell extracts (Lys) of Flag-NS1-inducible THP-1 cells pre-treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM, 6 hrs) or poly (I:C) (2 mg/ml, 6 hrs) stimulation.


Figure 2 ( A ) Relative qRT - PCR analysis of ZIKV RNA in THP - 1 cells left untreated or treated with LPS ( 500 ng / ml , 3 hrs ) and ATP ( 5 mM , 6 hrs ) , followed by ZIKV infection ( MOI = 1 ) for the indicated time points . ( B ) Relative qRT - PCR analysis of ZIKV RNA in wild type ( WT ) or NLRP3 knockout ( KO ) THP - 1 cells infected with ZIKV ( MOI = 1 ) for the indicated time points . ( C ) Immunoblot analysis of extracts of WT or Nlrp3 - / - BMDMs by the indicated antibodies . ( D ) Relative qRT - PCR analysis of ZIKV RNA in WT or Nlrp3 - / - BMDMs infected with ZIKV ( MOI = 1 ) for the indicated time points . ( E ) Plaque titration of ZIKV in supernatants of WT or Nlrp3 - / - BMDMs infected with ZIKV ( MOI = 1 ) for the indicated time points . ( F ) Immunoblot analysis of extracts of WT or caspase - 1 KO THP - 1 cells by the indicated antibodies . ( G ) Relative qRT - PCR analysis of ZIKV RNA in WT or caspase - 1 KO THP - 1 cells infected with ZIKV ( MOI = 1 ) for the indicated time points . ( H ) Relative qRT - PCR analysis of ZIKV RNA in NS - 1 - inducible THP - 1 cells treated with or without doxycycline ( Dox ) , then left untreated or treated with Ac - YVAD - cmk ( 20 μΜ ) for 3 hrs followed by ZIKV infection ( MOI = 1 ) for the indicated time points . ( I ) Relative qRT - PCR analysis of NLRP3 knockdown efficiency in PBMCs from two donors . ( J ) Relative qRT - PCR analysis of ZIKV RNA in PBMCs transfected with control siRNA or NLRP3 siRNA then infected with ZIKV ( MOI = 1 ) for 36 hrs . ( K ) qRT - PCR analysis of ZIKV RNA in PBMCs treated with DMSO or Ac - YVAD - cmk ( 20μM ) for 3hrs followed by ZIKV infection ( MOI = 1 ) for 36 hrs .

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Figure 2(A) Relative qRT-PCR analysis of ZIKV RNA in THP-1 cells left untreated or treated with LPS (500 ng/ml, 3 hrs) and ATP (5 mM, 6 hrs), followed by ZIKV infection (MOI=1) for the indicated time points.(B) Relative qRT-PCR analysis of ZIKV RNA in wild type (WT) or NLRP3 knockout (KO) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.(C) Immunoblot analysis of extracts of WT or Nlrp3-/- BMDMs by the indicated antibodies.(D) Relative qRT-PCR analysis of ZIKV RNA in WT or Nlrp3-/- BMDMs infected with ZIKV (MOI=1) for the indicated time points.(E) Plaque titration of ZIKV in supernatants of WT or Nlrp3-/- BMDMs infected with ZIKV (MOI=1) for the indicated time points.(F) Immunoblot analysis of extracts of WT or caspase-1 KO THP-1 cells by the indicated antibodies.(G) Relative qRT-PCR analysis of ZIKV RNA in WT or caspase-1 KO THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.(H) Relative qRT-PCR analysis of ZIKV RNA in NS-1-inducible THP-1 cells treated with or without doxycycline (Dox), then left untreated or treated with Ac-YVAD-cmk (20 μΜ) for 3 hrs followed by ZIKV infection (MOI=1) for the indicated time points.(I) Relative qRT-PCR analysis of NLRP3 knockdown efficiency in PBMCs from two donors.(J) Relative qRT-PCR analysis of ZIKV RNA in PBMCs transfected with control siRNA or NLRP3 siRNA then infected with ZIKV (MOI=1) for 36 hrs.(K) qRT-PCR analysis of ZIKV RNA in PBMCs treated with DMSO or Ac-YVAD-cmk (20μM) for 3hrs followed by ZIKV infection (MOI=1) for 36 hrs.


Figure 3 ( A ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with HA - NS1 together with Flag - NLRP3 , Flag - ASC , Flag - caspase - 1 and Flag - IL - 1β . WCL , whole cell lysates . ( B ) Co - immunoprecipitation and immunoblot analysis of extracts of NS1 - inducible THP - 1 cells , which expressed Flag - tagged NS1 , treated with or without doxycycline ( Dox ) ( 100 ng / ml ) . ( C ) Immunoblot analysis of extracts of THP - 1 cells treated with LPS ( 500 ng / ml , 3 hrs ) and ATP ( 5 mM , 6 hrs ) followed by increasing doses of Dox treatment . ( D ) Immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 together with increasing amount of HA - NS1 . Below , caspase - 1 mRNA RT - PCR ; RPL13A mRNA RT - PCR serves as a loading control . ( E ) Immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 together with increasing amount of HA - DENV - 2 - NS1 . ( F ) Immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 together with empty vector or HA - NS1 followed by cycloheximide ( CHX ) treatment ( 100 μg / ml ) for the indicated time points . ( G ) Immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 together with empty vector or HA - NS1 then treated with MG132 ( 10 mM ) , Lactacystin ( 5 μM ) , Carfilzomib ( 100 mM ) or CQ ( 50 mM ) for 6 hrs . ( H ) Co - immunoprecipitation and immunoblot analysis of extracts of Flag - NS1 - inducible THP - 1 cells treated with or without Dox ( 100 ng / ml ) . ( I ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with plasmids expressing Flag - caspase - 1 and indicated HA - tagged WT ubiquitin or mutants ( K6 - , K11 - , K27 - , K29 - , K33 - , K48 - and K63 - Only ) , together with empty vector or Myc - NS1 , followed by MG132 ( 10 mM ) treatment for 6 hrs .

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Figure 3(A) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with HA-NS1 together with Flag-NLRP3, Flag-ASC, Flag-caspase-1 and Flag-IL-1β. WCL, whole cell lysates.(B) Co-immunoprecipitation and immunoblot analysis of extracts of NS1-inducible THP-1 cells, which expressed Flag-tagged NS1, treated with or without doxycycline (Dox) (100 ng/ml).(C) Immunoblot analysis of extracts of THP-1 cells treated with LPS (500 ng/ml, 3 hrs) and ATP (5 mM, 6 hrs) followed by increasing doses of Dox treatment.(D) Immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 together with increasing amount of HA-NS1. Below, caspase-1 mRNA RT-PCR; RPL13A mRNA RT-PCR serves as a loading control.(E) Immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 together with increasing amount of HA-DENV-2-NS1.(F) Immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 together with empty vector or HA-NS1 followed by cycloheximide (CHX) treatment (100 μg/ml) for the indicated time points.(G) Immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 together with empty vector or HA-NS1 then treated with MG132 (10 mM), Lactacystin (5 μM), Carfilzomib (100 mM) or CQ (50 mM) for 6 hrs.(H) Co-immunoprecipitation and immunoblot analysis of extracts of Flag-NS1-inducible THP-1 cells treated with or without Dox (100 ng/ml).(I) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with plasmids expressing Flag-caspase-1 and indicated HA-tagged WT ubiquitin or mutants (K6-, K11-, K27-, K29-, K33-, K48- and K63-Only), together with empty vector or Myc-NS1, followed by MG132 (10 mM) treatment for 6 hrs.


Figure 4 ( A ) Immunoblot analysis of extracts of wild type ( WT ) and USP8 knockout ( KO ) 293T cells transfected with Flag - caspase - 1 together with empty vector or Myc - NS1 . ( B ) Immunoblot analysis of extracts of transfected with Flag - caspase - 1 together with empty vector or Myc - USP8 then treated with or without MG132 ( 10 mM ) for 6 hrs . ( C ) Co - immunoprecipitation and immunoblot analysis of extracts of Flag - NS1 - inducible THP - 1 cells treated with or without Dox ( 100 ng / ml ) then transfected with or without poly ( I : C ) ( 2 mg / ml , 6 hrs ) . ( D ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 and Myc - USP8 together with HA - NS1 followed by MG132 treatment ( 10 mM ) for 6 hrs . ( E ) Co - immunoprecipitation and immunoblot analysis of extracts of THP - 1 cells infected with ZIKV ( MOI = 1 ) for 24 hrs . ( F ) Co - immunoprecipitation and immunoblot analysis of extracts of WT and USP8 KO THP - 1 cells infected with ZIKV ( MOI = 1 ) for 24 hrs . ( G ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with plasmids expressing Flag - caspase - 1 and indicated HA - tagged WT ubiquitin ( Ub ) or mutants ( K6 - , K11 - , K27 - , K29 - , K33 - , K48 - and K63 - Only ) , together with empty vector or Myc - USP8 , followed by MG132 ( 10 mM ) treatment for 6 hrs . ( H ) Immunoblot analysis of extracts of WT or USP8 KO 293T cells transfected with Flag - caspase - 1 , HA - K11 - Ub together with empty vector or Myc - NS1 , followed by two - step immunoprecipitation with anti - Flag beads and immunoblot analysis with anti - HA . ( I ) Co - immunoprecipitation and immunoblot analysis of extracts of USP8 KO 293T cells transfected with Flag - caspase - 1 and HA - K11 - Ub together with WT or inactive mutant C786A of USP8 .

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Figure 4(A) Immunoblot analysis of extracts of wild type (WT) and USP8 knockout (KO) 293T cells transfected with Flag-caspase-1 together with empty vector or Myc-NS1.(B) Immunoblot analysis of extracts of transfected with Flag-caspase-1 together with empty vector or Myc-USP8 then treated with or without MG132 (10 mM) for 6 hrs.(C) Co-immunoprecipitation and immunoblot analysis of extracts of Flag-NS1-inducible THP-1 cells treated with or without Dox (100 ng/ml) then transfected with or without poly (I:C) (2 mg/ml, 6 hrs).(D) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 and Myc-USP8 together with HA-NS1 followed by MG132 treatment (10 mM) for 6 hrs.(E) Co-immunoprecipitation and immunoblot analysis of extracts of THP-1 cells infected with ZIKV (MOI=1) for 24 hrs.(F) Co-immunoprecipitation and immunoblot analysis of extracts of WT and USP8 KO THP-1 cells infected with ZIKV (MOI=1) for 24 hrs.(G) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with plasmids expressing Flag-caspase-1 and indicated HA-tagged WT ubiquitin (Ub) or mutants (K6-, K11-, K27-, K29-, K33-, K48- and K63-Only), together with empty vector or Myc-USP8, followed by MG132 (10 mM) treatment for 6 hrs.(H) Immunoblot analysis of extracts of WT or USP8 KO 293T cells transfected with Flag-caspase-1, HA-K11-Ub together with empty vector or Myc-NS1, followed by two-step immunoprecipitation with anti-Flag beads and immunoblot analysis with anti-HA.(I) Co-immunoprecipitation and immunoblot analysis of extracts of USP8 KO 293T cells transfected with Flag-caspase-1 and HA-K11-Ub together with WT or inactive mutant C786A of USP8.


Figure 5 ( A ) Immunoblot analysis of extracts of 293T cells transfected with empty vector or HA - NS1 together with Flag - caspae - 1 wild type ( WT ) and mutants ( K37R , K44R or K134R ) . Lower panel shows the intensity analysis of the bands from the three independent experiments . ( B ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with HA - K11 - ubiquitin ( Ub ) together with Flag - caspase - 1 ( WT , K37R , K44R , K134R ) . ( C ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 ( WT or K134R ) and HA - K11 - Ub , together with empty vector or Myc - NS1 . Lower panel shows the intensity analysis of the bands from the three independent experiments . ( D ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 ( WT or K134R ) and HA - K11 - Ub , together with empty vector or Myc - USP8 . Lower panel shows the intensity analysis of the bands from the three independent experiments .

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

Figure 5(A) Immunoblot analysis of extracts of 293T cells transfected with empty vector or HA-NS1 together with Flag-caspae-1 wild type (WT) and mutants (K37R, K44R or K134R). Lower panel shows the intensity analysis of the bands from the three independent experiments.(B) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with HA-K11-ubiquitin (Ub) together with Flag-caspase-1 (WT, K37R, K44R, K134R).(C) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 (WT or K134R) and HA-K11-Ub, together with empty vector or Myc-NS1. Lower panel shows the intensity analysis of the bands from the three independent experiments.(D) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 (WT or K134R) and HA-K11-Ub, together with empty vector or Myc-USP8. Lower panel shows the intensity analysis of the bands from the three independent experiments.


Figure 6 ( A ) Relative qRT - PCR analysis of IFNB , IFIT2 and IFIT1 mRNA levels in wild type ( WT ) or caspase - 1 knockout ( KO ) THP - 1 cells infected with ZIKV ( MOI = 1 ) for the indicated time points . ( B ) Relative qRT - PCR analysis of IFNB mRNA levels in THP - 1 cells left untreated or treated with LPS ( 500 ng / ml , 3 hrs ) and ATP ( 5 mM , 6 hrs ) , followed by ZIKV infection ( MOI = 1 ) for the indicated time points . ( C ) Relative qRT - PCR analysis of IFNB mRNA levels in wild type ( WT ) or NLRP3 knockout ( KO ) THP - 1 cells infected with ZIKV ( MOI = 1 ) for the indicated time points . ( D ) Relative qRT - PCR analysis of IFNB , IFIT2 and IFIT1 mRNA levels in Flag - NS1 - inducible THP - 1 cells treated with or without Dox then left untreated or treated with YVAD ( 20 μM ) for 3 hrs , followed by ZIKV infection ( MOI = 1 ) for the indicated time points . ( E ) Relative qRT - PCR analysis of IFNB mRNA in PBMCs transfected with control siRNA or NLRP3 siRNA then infected with ZIKV ( MOI = 1 ) for 36 hrs . ( F ) Relative qRT - PCR analysis of IFNB mRNA in PBMCs treated with DMSO or Ac - YVAD - cmk ( 20μM ) for 3hrs followed by ZIKV infection ( MOI = 1 ) for 36 hrs . ( G ) ELISA of IFN - β in the supernatants of PBMCs treated with DMSO or Ac - YVAD - cmk ( 20μM ) for 3hrs followed by ZIKV infection ( MOI = 1 ) for 36 hrs . ( H ) ELISA of IL - 1β production in brain of WT or Nlrp3 - / - 1 - day - old neonatal mice ( n = 3 per group ) intracerebrally injected with 1 × 105 plaque - forming units ( PFU ) of ZIKV for 5 days . ( I ) Relative qRT - PCR analysis of IFNB mRNA levels in brain tissues of WT or Nlrp3 - / - neonatal mice with or without ZIKV infection ( J ) ELISA of IFN - β production in brains of WT or Nlrp3 - / - neonatal mice ( K ) Relative qRT - PCR analysis of ZIKV RNA levels in brain tissues of WT or Nlrp3 - / - neonatal mice with or without ZIKV infection ( L ) Plaque titration of ZIKV in brain tissues of WT or Nlrp3 - / - neonatal mice ( M ) Body weight gain of WT and Nlrp3 - / - neonatal mice ( n = 4 per group ) inoculated with ZIKV ( 1 × 105 PFUs ) .

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Figure 6(A) Relative qRT-PCR analysis of IFNB, IFIT2 and IFIT1 mRNA levels in wild type (WT) or caspase-1 knockout (KO) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.(B) Relative qRT-PCR analysis of IFNB mRNA levels in THP-1 cells left untreated or treated with LPS (500 ng/ml, 3 hrs) and ATP (5 mM, 6 hrs), followed by ZIKV infection (MOI=1) for the indicated time points.(C) Relative qRT-PCR analysis of IFNB mRNA levels in wild type (WT) or NLRP3 knockout (KO) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.(D) Relative qRT-PCR analysis of IFNB , IFIT2 and IFIT1 mRNA levels in Flag-NS1-inducible THP-1 cells treated with or without Dox then left untreated or treated with YVAD (20 μM) for 3 hrs, followed by ZIKV infection (MOI=1) for the indicated time points.(E) Relative qRT-PCR analysis of IFNB mRNA in PBMCs transfected with control siRNA or NLRP3 siRNA then infected with ZIKV (MOI=1) for 36 hrs.(F) Relative qRT-PCR analysis of IFNB mRNA in PBMCs treated with DMSO or Ac-YVAD-cmk (20μM) for 3hrs followed by ZIKV infection (MOI=1) for 36 hrs.(G) ELISA of IFN-β in the supernatants of PBMCs treated with DMSO or Ac-YVAD-cmk (20μM) for 3hrs followed by ZIKV infection (MOI=1) for 36 hrs.(H) ELISA of IL-1β production in brain of WT or Nlrp3-/- 1-day-old neonatal mice (n=3 per group) intracerebrally injected with 1×105 plaque-forming units (PFU) of ZIKV for 5 days.(I) Relative qRT-PCR analysis of IFNB mRNA levels in brain tissues of WT or Nlrp3-/- neonatal mice with or without ZIKV infection(J) ELISA of IFN-β production in brains of WT or Nlrp3-/- neonatal mice(K) Relative qRT-PCR analysis of ZIKV RNA levels in brain tissues of WT or Nlrp3-/- neonatal mice with or without ZIKV infection(L) Plaque titration of ZIKV in brain tissues of WT or Nlrp3-/- neonatal mice(M) Body weight gain of WT and Nlrp3-/- neonatal mice (n=4 per group) inoculated with ZIKV (1×105 PFUs).


Figure 7 ( A ) Relative qRT - PCR analysis of ZIKV RNA levels in wild type ( WT ) or cGAS knockout ( KO ) THP - 1 cells after ZIKV infection ( MOI = 1 ) for indicated time points . ( B ) Relative qRT - PCR analysis of IFNΒ , IFIT2 and IFIT1 mRNA levels in WT or cGAS KO THP - 1 cells after ZIKV infection ( MOI = 1 ) for indicated time points . ( C ) Relative qRT - PCR analysis of ZIKV RNA and IFNB mRNA levels in PBMCs transduced with Cas9 and small guide RNA targeting GFP , cGAS or NLRP3 and infected with ZIKV ( MOI = 1 ) for 36 hrs . ( D ) Relative qRT - PCR analysis of ZIKV RNA and IFNΒ mRNA levels in WT or cGAS KO THP - 1 cells treated with or without YVAD ( 20 μM ) , followed by ZIKV infection ( MOI = 1 ) for indicated times points . ( E ) Immunoblot analysis of extracts of Flag - cGAS - overexpressed THP - 1 cells treated with or without YVAD ( 20 μM ) then infected with ZIKV ( MOI = 5 ) for 24 hrs or infected with HSV - 1 ( MOI = 1 ) for 24 hrs . Immunoblot analysis of WT , NLRP3 KO ( F ) THP - 1 cells infected with ZIKV ( MOI = 1 ) for the indicated time points . Immunoblot analysis of WT , caspase - 1 KO ( G ) THP - 1 cells infected with ZIKV ( MOI = 1 ) for the indicated time points . ( H ) Immunoblot analysis of extracts of WT or Nlrp3 - / - BMDMs infected with ZIKV ( MOI = 1 ) for the indicated time points . ( I ) Immunoblot analysis of PBMCs from Donor # 1 transfected with control siRNA or NLRP3 siRNA followed by ZIKV infection ( MOI = 1 ) for the indicated time points . ( J ) Immunoblot analysis of extracts of 293T cells transfected with Myc - cGAS ( tagged on C - terminal ) , GFP - ASC , Flag - caspase - 1 , together with empty vector or HA - NS1 . ( K ) Relative qRT - PCR analysis of ZIKV RNA and IFNB mRNA levels in WT , cGAS KO THP - 1 cells as well as cGAS KO THP - 1 cells reconstituted with WT or D140A / D157A mutant of cGAS , followed by ZIKV infection ( MOI = 1 ) for 24 hrs .

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Figure 7(A) Relative qRT-PCR analysis of ZIKV RNA levels in wild type (WT) or cGAS knockout (KO) THP-1 cells after ZIKV infection (MOI=1) for indicated time points.(B) Relative qRT-PCR analysis of IFNΒ, IFIT2 and IFIT1 mRNA levels in WT or cGAS KO THP-1 cells after ZIKV infection (MOI=1) for indicated time points.(C) Relative qRT-PCR analysis of ZIKV RNA and IFNB mRNA levels in PBMCs transduced with Cas9 and small guide RNA targeting GFP, cGAS or NLRP3 and infected with ZIKV (MOI=1) for 36 hrs.(D) Relative qRT-PCR analysis of ZIKV RNA and IFNΒ mRNA levels in WT or cGAS KO THP-1 cells treated with or without YVAD (20 μM), followed by ZIKV infection (MOI=1) for indicated times points.(E) Immunoblot analysis of extracts of Flag-cGAS-overexpressed THP-1 cells treated with or without YVAD (20 μM) then infected with ZIKV (MOI=5) for 24 hrs or infected with HSV-1 (MOI=1) for 24 hrs.Immunoblot analysis of WT, NLRP3 KO (F) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.Immunoblot analysis of WT, caspase-1 KO (G) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.(H) Immunoblot analysis of extracts of WT or Nlrp3-/- BMDMs infected with ZIKV (MOI=1) for the indicated time points.(I) Immunoblot analysis of PBMCs from Donor#1 transfected with control siRNA or NLRP3 siRNA followed by ZIKV infection (MOI=1) for the indicated time points.(J) Immunoblot analysis of extracts of 293T cells transfected with Myc-cGAS (tagged on C-terminal), GFP-ASC, Flag-caspase-1, together with empty vector or HA-NS1.(K) Relative qRT-PCR analysis of ZIKV RNA and IFNB mRNA levels in WT, cGAS KO THP-1 cells as well as cGAS KO THP-1 cells reconstituted with WT or D140A/D157A mutant of cGAS, followed by ZIKV infection (MOI=1) for 24 hrs.


Figure 1Western blot analysis showing increased RNF8 protein level in HeLa cells after siRNA - mediated p97 depletion under physiological conditions and after IR ( 10 Gy ) . Graph represents the quantifications of ( A ) ( * * * P < 0 . 001 ; unpaired t - test , n = 3 , mean + SEM ) . Western blot analysis showing increased RNF8 protein level in HeLa cells after p97 chemical inhibition ( CB5083 , 10µM for 6h ) under physiological conditions and after IR ( 10 Gy ) . Graph represents the quantifications of ( C ) ( * P < 0 . 05 , * * * * P < 0 . 0001 ; unpaired t - test , n = 3 , mean + SEM ) . Western blot analysis showing increased RNF8 protein level in HEK293 cells after doxycycline - induced mild expression of the p97EQ variant under physiological conditions and after IR ( 10 Gy ) . Graph represents the quantifications of ( E ) ( * * P < 0 . 01 , * * * * P < 0 . 0001 ; unpaired t - test , n = 4 , mean + SEM ) . Western blot analysis of CHX chase kinetics showing reduced RNF8 degradation rate in HEK293 cells after siRNA - mediated p97 depletion . Graph represents the quantifications of ( H ) ( * * * P < 0 . 001 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 3 , mean + SEM ) and Western blot for efficacy of siRNA depletion of p97 ( right ) . Western blot analysis of Flag - RNF8 denaturing - IP in HEK293 cells showing K48 - linked hyper - ubiquitination of RNF8 after siRNA - mediated p97 depletion . Western blot analysis of Strep - p97 Co - IP in HEK293 cells showing increased RNF8 interaction with p97EQ variant as compared to p97WT under physiological conditions and after IR ( 10 Gy ) . Graph represents the quantifications of ( K ) ( * P < 0 . 05 ; unpaired t - test , n = 2 , mean + SEM ) .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 1Western blot analysis showing increased RNF8 protein level in HeLa cells after siRNA-mediated p97 depletion under physiological conditions and after IR (10 Gy).   Graph represents the quantifications of (A) (***P<0.001; unpaired t-test, n=3, mean +SEM).   Western blot analysis showing increased RNF8 protein level in HeLa cells after p97 chemical inhibition (CB5083, 10µM for 6h) under physiological conditions and after IR (10 Gy).   Graph represents the quantifications of (C) (*P<0.05, ****P<0.0001; unpaired t-test, n=3, mean +SEM).   Western blot analysis showing increased RNF8 protein level in HEK293 cells after doxycycline-induced mild expression of the p97EQ variant under physiological conditions and after IR (10 Gy).   Graph represents the quantifications of (E) (**P<0.01, ****P<0.0001; unpaired t-test, n=4, mean +SEM).   Western blot analysis of CHX chase kinetics showing reduced RNF8 degradation rate in HEK293 cells after siRNA-mediated p97 depletion.   Graph represents the quantifications of (H) (***P<0.001, ****P<0.0001; two-way ANOVA, n=3, mean +SEM) and Western blot for efficacy of siRNA depletion of p97 (right).   Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing K48-linked hyper-ubiquitination of RNF8 after siRNA-mediated p97 depletion.Western blot analysis of Strep-p97 Co-IP in HEK293 cells showing increased RNF8 interaction with p97EQ variant as compared to p97WT under physiological conditions and after IR (10 Gy).   Graph represents the quantifications of (K) (*P<0.05; unpaired t-test, n=2, mean +SEM).


Figure 2Western blot analysis of Flag - RNF8 denaturing - IP in HEK293 cells showing the ubiquitination pattern of RNF8 - WT and RNF8 - RING * variant , under siRNA - mediated luciferase ( siLuc ) or p97 - depleted conditions ( sip97 ) . Western blot analysis of CHX chase kinetics in HeLa cells showing the degradation kinetics of RNF8 and inhibition of RNF8 degradation by simultaneous proteasome inhibition ( MG132 , 10µM ) . Graph represents the quantifications of ( B ) ( ns ; not significant , P > 0 . 05 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 2 , mean + SEM ) . Western blot analysis of Flag - RNF8 denaturing - IP in HEK293 cells showing hyper - ubiquitination of RNF8 after proteasome inhibition ( MG132 , 10µM for 6h ) . Western blot analysis of CHX chase kinetics in U2OS cells , comparing the degradation rate of Flag - RNF8 - WT and Flag - RNF8 - RING * . Endogenous RNF8 was depleted by shRNF8 targeting only endogenous RNF8 . Graph represents the quantifications of ( E ) ( ns P > 0 . 05 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 3 , mean + SEM ) .

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

Figure 2Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing the ubiquitination pattern of RNF8-WT and RNF8-RING* variant, under siRNA-mediated luciferase (siLuc) or p97-depleted conditions (sip97).Western blot analysis of CHX chase kinetics in HeLa cells showing the degradation kinetics of RNF8 and inhibition of RNF8 degradation by simultaneous proteasome inhibition (MG132, 10µM).   Graph represents the quantifications of (B) (ns; not significant, P>0.05, ****P<0.0001; two-way ANOVA, n=2, mean +SEM).   Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing hyper-ubiquitination of RNF8 after proteasome inhibition (MG132, 10µM for 6h).Western blot analysis of CHX chase kinetics in U2OS cells, comparing the degradation rate of Flag-RNF8-WT and Flag-RNF8-RING*. Endogenous RNF8 was depleted by shRNF8 targeting only endogenous RNF8.   Graph represents the quantifications of (E) (ns P>0.05, ****P<0.0001; two-way ANOVA, n=3, mean +SEM).


Figure 3Western blot analysis of Strep - p97 Co - IP in nuclear fraction of HEK293 cells showing interaction of p97 with ATX3 under physiological conditions and after IR ( 10 Gy ) . Western blot analysis of Flag - ATX3 Co - IP in HEK293 cells showing interaction of ATX3 with p97 , RNF8 and PSα6 under physiological conditions and after IR ( 10 Gy ) . Western blot analysis of Flag - ATX3 WT or Flag - ATX3 VBM * Co - IP in HEK293 cells showing interaction of ATX3 with p97 , RNF8 and p97 core co - factors Npl4 - Ufd1 . ( * represents unspecific bands ) . Western blot analysis of Strep - p97EQ Co - IP in HEK293 cells , in presence or absence ( CRISPR knockout ; ∆ ATX3 ) of ATX3 , showing increased interaction of RNF8 with p97 in absence of ATX3 , under physiological conditions and after IR ( 10 Gy ) . Graph represents quantifications of ( F ) ( * P < 0 . 05 , * * P < 0 . 01 ; unpaired t - test , n = 3 , mean + SEM ) .

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

Figure 3Western blot analysis of Strep-p97 Co-IP in nuclear fraction of HEK293 cells showing interaction of p97 with ATX3 under physiological conditions and after IR (10 Gy).Western blot analysis of Flag-ATX3 Co-IP in HEK293 cells showing interaction of ATX3 with p97, RNF8 and PSα6 under physiological conditions and after IR (10 Gy).Western blot analysis of Flag-ATX3 WT or Flag-ATX3 VBM* Co-IP in HEK293 cells showing interaction of ATX3 with p97, RNF8 and p97 core co-factors Npl4-Ufd1. (* represents unspecific bands).Western blot analysis of Strep-p97EQ Co-IP in HEK293 cells, in presence or absence (CRISPR knockout; ∆ATX3) of ATX3, showing increased interaction of RNF8 with p97 in absence of ATX3, under physiological conditions and after IR (10 Gy).   Graph represents quantifications of (F) (*P<0.05, **P<0.01; unpaired t-test, n=3, mean +SEM).


Figure 4Western blot analysis of CHX chase kinetics in HeLa cells showing accelerated endogenous RNF8 degradation in the soluble fraction ( Cytosol and Nucleosol ) of ∆ ATX3 cell extract . Arrow represents the main RNF8 band and asterisks represents unspecific bands . Graphs represent the quantifications of ( A ) . RNF8 level at starting point ( 0h ) was shown without equalisation ( left ) . In order to nullify the difference in RNF8 level at starting point ( 0h ) , we equalised RNF8 level to 100 % and then compared the degradation rate ( right ) . ( * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 2 , mean + SEM ) . Western blot analysis of CHX chase showing the kinetics of endogenous RNF8 degradation in the soluble fraction ( Cytosol and Nucleosol ) of ATX3 knockdown HeLa cells . The degradation was completely blocked after simultaneous inhibition of proteasome ( MG132 , 10µM ) . Western blot analysis of Flag - RNF8 denaturing - IP in HEK293 cells showing RNF8 hyper - ubiquitination in siRNA - mediated p97 or ATX3 - depleted conditions . Western blot analysis of Flag - RNF8 denaturing - IP in HeLa cells showing RNF8 hyper - ubiquitination in ∆ ATX3 condition . RNF8 hyper - ubiquitination was suppressed by reintroduction of GFP - ATX3 - WT but not with GFP - ATX3 - C14A or GFP - ATX3 - UIM * .

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

Figure 4Western blot analysis of CHX chase kinetics in HeLa cells showing accelerated endogenous RNF8 degradation in the soluble fraction (Cytosol and Nucleosol) of ∆ATX3 cell extract. Arrow represents the main RNF8 band and asterisks represents unspecific bands.   Graphs represent the quantifications of (A). RNF8 level at starting point (0h) was shown without equalisation (left). In order to nullify the difference in RNF8 level at starting point (0h), we equalised RNF8 level to 100% and then compared the degradation rate (right). (**P<0.01, ***P<0.001, ****P<0.0001; two-way ANOVA, n=2, mean +SEM).   Western blot analysis of CHX chase showing the kinetics of endogenous RNF8 degradation in the soluble fraction (Cytosol and Nucleosol) of ATX3 knockdown HeLa cells. The degradation was completely blocked after simultaneous inhibition of proteasome (MG132, 10µM).Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing RNF8 hyper-ubiquitination in siRNA-mediated p97 or ATX3-depleted conditions.Western blot analysis of Flag-RNF8 denaturing-IP in HeLa cells showing RNF8 hyper-ubiquitination in ∆ATX3 condition. RNF8 hyper-ubiquitination was suppressed by reintroduction of GFP-ATX3-WT but not with GFP-ATX3-C14A or GFP-ATX3-UIM*.


Figure 5Western blot analysis of CHX chase showing accelerated degradation of RNF8 in soluble fraction ( cytoplasm + nucleoplasm ) of HeLa cells , expressing DOX - inducible GFP - ATX3 - C14A variant as compared to GFP - ATX3 - WT . Endogenous ATX3 was depleted with siRNA targeting 3 ' UTR region of ATXN3 . Graph represents the quantifications of ( A ) . In order to nullify the difference in RNF8 level at starting point ( 0h ) , we equalised RNF8 level to 100 % and then compared the degradation rate . RNF8 level at starting point ( 0h ) was also shown without equalisation . ( * P < 0 . 05 , * * P < 0 . 01 ; two - way ANOVA , n = 2 , mean + SEM ) . Western blot analysis of CHX chase showing accelerated degradation of RNF8 in soluble fraction ( cytoplasm + nucleoplasm ) of HeLa cells , expressing DOX - inducible ATX3 - UIM * variant as compared to ATX3 - WT . Endogenous ATX3 was depleted with siRNA ATX3 _ 3 ' UTR . Graph represents the quantifications of ( C ) . In order to nullify the difference in RNF8 level at starting point ( 0h ) , we equalised RNF8 level to 100 % and then compared the degradation rate . RNF8 level at starting point ( 0h ) was also shown without equalisation . ( ns P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 2 , mean + SEM ) . Western blot analysis showing endogenous RNF8 protein level in soluble fraction ( Cytosol and Nucleosol ) of HeLa cells under indicated conditions . RNF8 level was significantly reduced after siRNA mediated ATX3 depletion and then was significantly rescued by DOX - inducible expression of GFP - ATX3 - WT but not with GFP - ATX3 - VBM . Graphs represent the quantifications of ( E ) . ( ns P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 ; unpaired t - test , n = 3 , mean + SEM ) . Representative IF images showing γH2AX - positive micronuclei ( white arrows ) under physiological conditions with indicated siRNA mediated depletion . ( Scale bar represents 10 µm ) Graph represents quantification of ( G ) , showing percentage of cells with γH2AX - positive micronuclei in U2OS cells . ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; unpaired t - test , n = 3 , mean + SEM , at least 200 randomly selected nuclei were counted per condition per experiment ) .

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

Figure 5Western blot analysis of CHX chase showing accelerated degradation of RNF8 in soluble fraction (cytoplasm+nucleoplasm) of HeLa cells, expressing DOX-inducible GFP-ATX3-C14A variant as compared to GFP-ATX3-WT. Endogenous ATX3 was depleted with siRNA targeting 3'UTR region of ATXN3.   Graph represents the quantifications of (A). In order to nullify the difference in RNF8 level at starting point (0h), we equalised RNF8 level to 100% and then compared the degradation rate. RNF8 level at starting point (0h) was also shown without equalisation. (*P<0.05, **P<0.01; two-way ANOVA, n=2, mean +SEM).   Western blot analysis of CHX chase showing accelerated degradation of RNF8 in soluble fraction (cytoplasm + nucleoplasm) of HeLa cells, expressing DOX-inducible ATX3-UIM* variant as compared to ATX3-WT. Endogenous ATX3 was depleted with siRNA ATX3_3'UTR.   Graph represents the quantifications of (C). In order to nullify the difference in RNF8 level at starting point (0h), we equalised RNF8 level to 100% and then compared the degradation rate. RNF8 level at starting point (0h) was also shown without equalisation. (ns P>0.05, *P<0.05, **P<0.01, ****P<0.0001; two-way ANOVA, n=2, mean +SEM).   Western blot analysis showing endogenous RNF8 protein level in soluble fraction (Cytosol and Nucleosol) of HeLa cells under indicated conditions. RNF8 level was significantly reduced after siRNA mediated ATX3 depletion and then was significantly rescued by DOX-inducible expression of GFP-ATX3-WT but not with GFP-ATX3-VBM.   Graphs represent the quantifications of (E). (ns P>0.05, * P<0.05, **P<0.01; unpaired t-test, n=3, mean +SEM).   Representative IF images showing γH2AX-positive micronuclei (white arrows) under physiological conditions with indicated siRNA mediated depletion. (Scale bar represents 10 µm)   Graph represents quantification of (G), showing percentage of cells with γH2AX-positive micronuclei in U2OS cells. (* P<0.05, **P<0.01, ***P<0.001; unpaired t-test, n=3, mean +SEM, at least 200 randomly selected nuclei were counted per condition per experiment).


Figure 6Quantifications of neutral comet assay showing the level of DSBs under indicated siRNA - depleted conditions , before and after IR ( 4 Gy ) . ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 3 , mean + SEM ) . Colony forming assay ( CFA ) showing the IR sensitivity of HeLa cells under siRNA - mediated p97 or ATX3 - depleted conditions . ( n = 3 , mean ± SD ) . CFA showing the IR sensitivity of HeLa - WT cells compared to HeLa ∆ ATX3 cells . ( n = 3 , mean ± SD ) . CFA showing the rescue of cell survival in IR treated , ATX3 - depleted ( siRNA ) , HeLa cells , after DOX - inducible expression of ATX3 - WT but not with ATX3 - C14A or ATX3 - UIM * variant . ( ns P > 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 3 , mean ± SD ) . CFA showing the rescue of cell survival in IR treated , ATX3 - depleted ( siRNA ) , HeLa cells , after DOX - inducible expression of ATX3 - WT but not with ATX3 - VBM variant . ( ns P > 0 . 05 , * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 3 , mean ± SD ) . Representative IF micrographs in U2OS cells showing IRIF formation of GFP - RNF8 - WT , 53BP1 and γH2AX ( first panel ) and GFP - RNF8 - ∆ RING , 53BP1 and γH2AX ( second panel ) under indicated siRNA - depleted or p97 - inhibited ( CB5083 ) conditions . ( Scale bar ; 10µm ) . Quantification of ( F ) for GFP - RNF8 - WT . Graph represents the percentage of cells with increased GFP - RNF8 - WT foci ( white bars ) or percentage of cells with > 5 53BP1 foci ( grey bars ) . ( * * * P < 0 . 001 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 2 , mean + SEM , at least 100 nuclei per condition and experiment ) . Quantification of ( F ) for GFP - RNF8 - ∆ RING . Graph represents the percentage of cells with increased GFP - RNF8 - ∆ RING foci ( white bars ) or percentage of cells with > 5 53BP1 foci ( grey bars ) . ( ns P > 0 . 05 , * P < 0 . 01 , * * P < 0 . 01 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 2 , mean + SEM , at least 100 nuclei per condition and experiment ) . Representative IF images and quantification showing endogenous K63 - Ub and γ - H2AX at UV - A micro - laser - induced DNA damage tracks in U2OS cells under indicated siRNA - depleted conditions . ( Scale bar ; 10µm ) . Quantification of ( I ) . Graph represents the average intensity of the K63 - Ub ( ns P > 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; unpaired t - test , n = 2 , mean + SEM , more than 50 nuclei were analysed ) .

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

Figure 6Quantifications of neutral comet assay showing the level of DSBs under indicated siRNA-depleted conditions, before and after IR (4 Gy). (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; two-way ANOVA, n=3, mean +SEM).Colony forming assay (CFA) showing the IR sensitivity of HeLa cells under siRNA-mediated p97 or ATX3-depleted conditions. (n=3, mean ±SD).   CFA showing the IR sensitivity of HeLa-WT cells compared to HeLa∆ATX3 cells. (n=3, mean ±SD).   CFA showing the rescue of cell survival in IR treated, ATX3-depleted (siRNA), HeLa cells, after DOX-inducible expression of ATX3-WT but not with ATX3-C14A or ATX3-UIM* variant. (ns P>0.05, **P<0.01, ****P<0.0001; two-way ANOVA, n=3, mean ±SD).   CFA showing the rescue of cell survival in IR treated, ATX3-depleted (siRNA), HeLa cells, after DOX-inducible expression of ATX3-WT but not with ATX3-VBM variant. (ns P>0.05, *P<0.05, ***P<0.001, ****P<0.0001; two-way ANOVA, n=3, mean ±SD).   Representative IF micrographs in U2OS cells showing IRIF formation of GFP-RNF8-WT, 53BP1 and γH2AX (first panel) and GFP-RNF8-∆RING, 53BP1 and γH2AX (second panel) under indicated siRNA-depleted or p97-inhibited (CB5083) conditions. (Scale bar; 10µm).   Quantification of (F) for GFP-RNF8-WT. Graph represents the percentage of cells with increased GFP-RNF8-WT foci (white bars) or percentage of cells with >5 53BP1 foci (grey bars). (***P<0.001, ****P<0.0001; two-way ANOVA, n=2, mean +SEM, at least 100 nuclei per condition and experiment).   Quantification of (F) for GFP-RNF8-∆RING. Graph represents the percentage of cells with increased GFP-RNF8-∆RING foci (white bars) or percentage of cells with >5 53BP1 foci (grey bars). (ns P>0.05, *P<0.01, **P<0.01, ****P<0.0001; two-way ANOVA, n=2, mean +SEM, at least 100 nuclei per condition and experiment).   Representative IF images and quantification showing endogenous K63-Ub and γ-H2AX at UV-A micro-laser-induced DNA damage tracks in U2OS cells under indicated siRNA-depleted conditions. (Scale bar; 10µm).   Quantification of (I). Graph represents the average intensity of the K63-Ub (ns P>0.05, **P<0.01, ***P<0.001; unpaired t-test, n=2, mean +SEM, more than 50 nuclei were analysed).


Figure 7Representative IF images showing the average intensity of endogenous RNF168 at UV - A micro - laser - induced DNA damage tracks in U2OS cells . ( Scale bar ; 10µm ) . Graph represents the quantification of ( A ) . ( * * * P < 0 . 001 ; unpaired t - test , n = 3 , mean + SEM , more than 50 nuclei were analysed ) . Representative IF images showing the average intensity of endogenous 53BP1 at UV - A micro - laser - induced DNA damage tracks in U2OS cells . ( Scale bar ; 10µm ) . Graph represents the quantification of ( C ) . ( ns P > 0 . 05 , * * * P < 0 . 001 ; unpaired t - test , n = 2 , mean + SEM , at least 80 nuclei per condition and experiment ) . Western blot analysis of Flag - histone H1 denaturing - IP , showing ubiquitination pattern of histone H1 after 30 min of IR ( 10 Gy ) treatment under indicated siRNA - depleted conditions . Representative IF images showing the average intensity of endogenous BRCA1 at UV - A micro - laser - induced DNA damage tracks in U2OS cells . ( Scale bar ; 10µm ) . Graph represents the quantification of ( F ) . ( ns P > 0 . 05 , * * * P < 0 . 001 ; unpaired t - test , n = 2 , mean + SEM , at least 100 nuclei per condition and experiment ) .

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

Figure 7Representative IF images showing the average intensity of endogenous RNF168 at UV-A micro-laser-induced DNA damage tracks in U2OS cells. (Scale bar; 10µm).   Graph represents the quantification of (A). (***P<0.001; unpaired t-test, n=3, mean +SEM, more than 50 nuclei were analysed).   Representative IF images showing the average intensity of endogenous 53BP1 at UV-A micro-laser-induced DNA damage tracks in U2OS cells. (Scale bar; 10µm).   Graph represents the quantification of (C). (ns P>0.05, ***P<0.001; unpaired t-test, n=2, mean +SEM, at least 80 nuclei per condition and experiment).   Western blot analysis of Flag-histone H1 denaturing-IP, showing ubiquitination pattern of histone H1 after 30 min of IR (10 Gy) treatment under indicated siRNA-depleted conditions.Representative IF images showing the average intensity of endogenous BRCA1 at UV-A micro-laser-induced DNA damage tracks in U2OS cells. (Scale bar; 10µm).   Graph represents the quantification of (F). (ns P>0.05, ***P<0.001; unpaired t-test, n=2, mean +SEM, at least 100 nuclei per condition and experiment).


Figure 8GFP based reporter assay showing the efficiency of NHEJ repair pathway under indicated siRNA - depleted conditions in HEK293 cells . Graph represents the quantifications from three independent experiments ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; one - way ANOVA , n = 3 , mean + SEM ) . GFP based reporter assay showing the efficiency of HR repair pathway under indicated siRNA - depleted conditions in HEK293 cells . Graph represents the quantifications from three independent experiments ( ns P > 0 . 05 , * * P < 0 . 01 ; one - way ANOVA , n = 3 , mean + SEM ) . CFA showing the IR sensitivity of 53BP1 - proficient and deficient MCF7 cells under indicated siRNA - depleted conditions . Data was collected from two individual experiments with triplicates . ( ns P > 0 . 05 ; two - way ANOVA , n = 2 , mean ± SEM ) . CFA showing the IR sensitivity of BRCA2 - proficient and deficient DLD1 cells under indicated siRNA - depleted conditions . Data was collected from two individual experiments with triplicates . ( * * P < 0 . 01 , * * * P < 0 . 001 ; two - way ANOVA , n = 2 , mean ± SEM ) . Representative IF - images showing Rad51 foci in S - phase ( Edu positive ) U2OS nucleus after 5h of IR ( 2Gy ) exposure . ( Scale bar ; 10µm ) . Graph represents the quantification of ( E ) showing average number of Rad51 foci per S - phase ( Edu positive ) nucleus . ( ns P > 0 . 05 , * * * * P < 0 . 0001 ; unpaired t - test , n = 2 , mean + SEM , at least 50 nuclei per condition and experiment ) . CFA showing the rescue of cell survival in IR treated , ATX3 - depleted ( siRNA ) , HeLa cells , after co - depletion ( siRNA ) of RNF8 . ( ns P > 0 . 05 , * P < 0 . 05 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 3 , mean ± SEM ) .

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

Figure 8GFP based reporter assay showing the efficiency of NHEJ repair pathway under indicated siRNA-depleted conditions in HEK293 cells. Graph represents the quantifications from three independent experiments (*P<0.05, **P<0.01, ***P<0.001; one-way ANOVA, n=3, mean +SEM).GFP based reporter assay showing the efficiency of HR repair pathway under indicated siRNA-depleted conditions in HEK293 cells. Graph represents the quantifications from three independent experiments (ns P>0.05, **P<0.01; one-way ANOVA, n=3, mean +SEM).CFA showing the IR sensitivity of 53BP1-proficient and deficient MCF7 cells under indicated siRNA-depleted conditions. Data was collected from two individual experiments with triplicates. (ns P>0.05; two-way ANOVA, n=2, mean ±SEM).CFA showing the IR sensitivity of BRCA2-proficient and deficient DLD1 cells under indicated siRNA-depleted conditions. Data was collected from two individual experiments with triplicates. (**P<0.01, ***P<0.001; two-way ANOVA, n=2, mean ±SEM).Representative IF-images showing Rad51 foci in S-phase (Edu positive) U2OS nucleus after 5h of IR (2Gy) exposure. (Scale bar; 10µm).   Graph represents the quantification of (E) showing average number of Rad51 foci per S-phase (Edu positive) nucleus. (ns P>0.05, ****P<0.0001; unpaired t-test, n=2, mean +SEM, at least 50 nuclei per condition and experiment).   CFA showing the rescue of cell survival in IR treated, ATX3-depleted (siRNA), HeLa cells, after co-depletion (siRNA) of RNF8. (ns P>0.05, *P<0.05, ****P<0.0001; two-way ANOVA, n=3, mean ±SEM).


Figure 1 . ( A and B ) Immunoblot of 9 - mo - old quadricepsmuscle lysates from control ( cont ) or VCP - WT , - RH9 , or - RH12 mutant transgenic lines with p62 ( A ) or LC3 ( B ) antibodies . Note the increase in p62 and LC3II isoforms in mutant animals . Densitometric quantification is from more than six 9 - mo - old animals / group . LC3 and p62 levels are normalized to loading control . Error bars represent the standard error from six independent experiments . * * , P < 0 . 001 . AU , arbitrary unit . ( C ) p62 immunostaining of tibialis anterior ( TA ) or quadriceps ( Quad ) muscle from 9 - mo - old control , VCP - WT , or one of two VCP - RH transgenic lines ( RH9 or RH12 ) . Note accumulations of p62 in the middle of myofibers or along subsarcolemmal regions . ( D ) Histochemistry and immunohistochemistry of quadriceps muscle from VCP - RH - expressing transgenic mice . Hematoxylin and eosin ( H & amp ; amp ; E ) staining of a 15 - mo - old animal with an RV , p62 immunostaining of an RV from a 12 - mo - old animal , LC3 immunostaining of an RV , and subsarcolemmal accumulations of LC3 from a 12 - mo - old animal . The bracket highlights one LC3 - positive myofiber . A single muscle fiber is outlined in white . ( C and D ) Arrows denote vacuoles or accumulations of p62 or LC3 . Bars , 30 µm .

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

Figure 1.(A and B) Immunoblot of 9-mo-old quadricepsmuscle lysates from control (cont) or VCP-WT, -RH9, or -RH12 mutant transgenic lines with p62 (A) or LC3 (B) antibodies. Note the increase in p62 and LC3II isoforms in mutant animals. Densitometric quantification is from more than six 9-mo-old animals/group. LC3 and p62 levels are normalized to loading control. Error bars represent the standard error from six independent experiments. **, P < 0.001. AU, arbitrary unit.(C) p62 immunostaining of tibialis anterior (TA) or quadriceps (Quad) muscle from 9-mo-old control, VCP-WT, or one of two VCP-RH transgenic lines (RH9 or RH12). Note accumulations of p62 in the middle of myofibers or along subsarcolemmal regions.(D) Histochemistry and immunohistochemistry of quadriceps muscle from VCP-RH-expressing transgenic mice. Hematoxylin and eosin (H&amp;E) staining of a 15-mo-old animal with an RV, p62 immunostaining of an RV from a 12-mo-old animal, LC3 immunostaining of an RV, and subsarcolemmal accumulations of LC3 from a 12-mo-old animal. The bracket highlights one LC3-positive myofiber. A single muscle fiber is outlined in white. (C and D) Arrows denote vacuoles or accumulations of p62 or LC3. Bars, 30 µm.


Figure 2 . ( A ) Lysates from scrambled siRNA or VCP - targeted siRNA - treated U20S cells immunoblotted with antibodies to VCP , LC3 , p62 , or α - tubulin . Densitometric analysis is graphically represented from four independent experiments . LC3 and p62 levels are normalized to loading control . ( B ) Fluorescent microscopy images of siRNA scrambled control ( Scr ) or VCP - targeted siRNA KD cells expressing GFP - LC3 . Basal numbers of GFP - LC3 puncta / cell were counted . ( C ) Lysates from control or myc - tagged VCP - WT , ATPase - inactive VCP - EQ , or one of two IBMPFD mutants , VCP - RH or - AE , expressed for 16 h from stably transfected tetracycline - inducible U20S cells and immunoblotted with antibodies to myc , LC3 , p62 , or α - tubulin . Densitometric analysis is graphically represented from four independent experiments . LC3 and p62 levels are normalized to loading control . Note the increase in LC3II and p62 in VCP siRNA KD and mutant - expressing cells . ( D ) Fluorescent microscopy images of tetracycline - inducible U20S cells expressing GFP - LC3 treated with and without Baf for 4 h ( DMSO control [ left ] or Baf + [ right ] ) . Cells are control U20S cells and VCP - WT , - RH , - AE , and - EQ . ( E ) The number of GFP - LC3 puncta / cell was counted for control U20S and VCP - WT , - EQ , - RH , and - AE with and without Baf for 4 h ( DMSO or Baf ) . Note the increase in basal GFP - LC3 puncta in mutant - expressing cells . ( A - C and E ) Error bars represent the standard error from four independent experiments . * , P < 0 . 01 ; and * * , P < 0 . 001 . Bars , 25 µm .

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

Figure 2.(A) Lysates from scrambled siRNA or VCP-targeted siRNA-treated U20S cells immunoblotted with antibodies to VCP, LC3, p62, or α-tubulin. Densitometric analysis is graphically represented from four independent experiments. LC3 and p62 levels are normalized to loading control.(B) Fluorescent microscopy images of siRNA scrambled control (Scr) or VCP-targeted siRNA KD cells expressing GFP-LC3. Basal numbers of GFP-LC3 puncta/cell were counted.(C) Lysates from control or myc-tagged VCP-WT, ATPase-inactive VCP-EQ, or one of two IBMPFD mutants, VCP-RH or -AE, expressed for 16 h from stably transfected tetracycline-inducible U20S cells and immunoblotted with antibodies to myc, LC3, p62, or α-tubulin. Densitometric analysis is graphically represented from four independent experiments. LC3 and p62 levels are normalized to loading control. Note the increase in LC3II and p62 in VCP siRNA KD and mutant-expressing cells.(D) Fluorescent microscopy images of tetracycline-inducible U20S cells expressing GFP-LC3 treated with and without Baf for 4 h (DMSO control [left] or Baf+ [right]). Cells are control U20S cells and VCP-WT, -RH, -AE, and -EQ. (E) The number of GFP-LC3 puncta/cell was counted for control U20S and VCP-WT, -EQ, -RH, and -AE with and without Baf for 4 h (DMSO or Baf). Note the increase in basal GFP-LC3 puncta in mutant-expressing cells. (A-C and E) Error bars represent the standard error from four independent experiments. *, P < 0.01; and **, P < 0.001. Bars, 25 µm.


Figure 3 . ( A ) Epifluorescent images for GFP and mRFP in U20S or VCP - WT - , VCP - RH - , VCP - AE - , or VCP - EQ - expressing cells transfected with mRFP - GFP - LC3 ( tfLC3 ) and treated with rapamycin for 2 h to induce autolysosome formation . ( B ) siRNA control ( Scr ) or VCP - KD - or Baf - treated control U20S cells transfected with tfLC3 and treated with rapamycin for 2 h to induce autolysosome formation . ( A and B ) Open arrows denote autophagosomes ( both GFP and mRFP fluorescence ) , whereas closed arrows highlight autolysosomes ( mRFP only fluorescence ) . ( C ) The graph represents Pearson ' s coefficient of GFP and mRFP colocalization from 10 independent fields of cells in two different experiments . Error bars represent the standard error from 20 fields in two independent experiments . * , P < 0 . 001 . ( D ) Lysates from U20S or tetracycline - inducible VCP - WT , - RH , or - AE cells treated with vehicle or Baf for 4 h and immunoblotted for LC3 and α - tubulin . Note that Baf treatment does not increase the LC3II levels in IBMPFD mutant ( RH and AE ) - expressing cells . Bars , 15 µm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3.(A) Epifluorescent images for GFP and mRFP in U20S or VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells transfected with mRFP-GFP-LC3 (tfLC3) and treated with rapamycin for 2 h to induce autolysosome formation. (B) siRNA control (Scr) or VCP-KD- or Baf-treated control U20S cells transfected with tfLC3 and treated with rapamycin for 2 h to induce autolysosome formation. (A and B) Open arrows denote autophagosomes (both GFP and mRFP fluorescence), whereas closed arrows highlight autolysosomes (mRFP only fluorescence). (C) The graph represents Pearson's coefficient of GFP and mRFP colocalization from 10 independent fields of cells in two different experiments. Error bars represent the standard error from 20 fields in two independent experiments. *, P < 0.001.(D) Lysates from U20S or tetracycline-inducible VCP-WT, -RH, or -AE cells treated with vehicle or Baf for 4 h and immunoblotted for LC3 and α-tubulin. Note that Baf treatment does not increase the LC3II levels in IBMPFD mutant (RH and AE)-expressing cells. Bars, 15 µm.


Figure 4 . ( A ) Lysates from siRNA - treated U20S cells ( scramble control [ Scr ] or VCP siRNA KD ) after autophagic induction via nutrient deprivation for 0 , 2 , or 4 h and immunoblotting for LC3 and α - tubulin . LC3II degrades over time in control but not in KD cells . One of two experiments is shown . ( B ) Lysates from U20S or tetracycline - inducible VCP - WT , - EQ , - RH , or - AE cells after autophagic induction via nutrient deprivation for 0 , 2 , 4 , or 6 h and immunoblotting for LC3 and α - tubulin . LC3II degrades in control and VCP - WT but degrades less efficiently in mutant ( EQ , RH , or AE ) - expressing cells . ( C ) Graphical representation of densitometric evaluation of LC3II and α - tubulin at each time point from three independent experiments .

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

Figure 4.(A) Lysates from siRNA-treated U20S cells (scramble control [Scr] or VCP siRNA KD) after autophagic induction via nutrient deprivation for 0, 2, or 4 h and immunoblotting for LC3 and α-tubulin. LC3II degrades over time in control but not in KD cells. One of two experiments is shown.(B) Lysates from U20S or tetracycline-inducible VCP-WT, -EQ, -RH, or -AE cells after autophagic induction via nutrient deprivation for 0, 2, 4, or 6 h and immunoblotting for LC3 and α-tubulin. LC3II degrades in control and VCP-WT but degrades less efficiently in mutant (EQ, RH, or AE)-expressing cells. (C) Graphical representation of densitometric evaluation of LC3II and α-tubulin at each time point from three independent experiments.


Figure 5 . ( A ) Epifluorescent images for GFP - LC3 ( LC3 ) and LTR of VCP - WT - , VCP - RH - , VCP - AE - , or VCP - EQ - expressing cells transfected with GFP - LC3 and treated with rapamycin for 2 h to induce autolysosome formation . Open arrows highlight autolysosomes ( GFP and LTR colocalized ) , and closed arrows show autophagosomes ( GFP only ) . ( B ) Pearson ' s coefficient of GFP and LTR colocalization from 10 independent fields of cells in two different experiments . ( C ) Epifluorescent images for GFP - LC3 and endogenous Lamp1 immunohistochemistry of U20S , VCP - WT - , VCP - RH - , VCP - AE - , or VCP - EQ - expressing cells or U20S cells cotreated with Baf transfected with GFP - LC3 and treated with rapamycin for 2 h to induce autolysosome formation . Arrows highlight autolysosomes ( GFP and Lamp1 colocalized ) . ( A and C ) The boxed regions in the merge field are enlarged in the adjacent panels . ( D ) Pearson ' s coefficient of GFP and Lamp1 colocalization from 10 independent fields of cells in two different experiments . ( B and D ) Error bars represent the standard error from 20 fields in two independent experiments . * , P < 0 . 001 . ( E ) Electron microscopy of tetracycline - inducible control or VCP - WT - , VCP - RH - , or VCP - AE - expressing U20S cells induced for 16 h and then treated with rapamycin for 2 h . Note the accumulation of autophagic structures in the mutant - expressing cell lines . Arrows denote autophagic structures .

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

Figure 5.(A) Epifluorescent images for GFP-LC3 (LC3) and LTR of VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells transfected with GFP-LC3 and treated with rapamycin for 2 h to induce autolysosome formation. Open arrows highlight autolysosomes (GFP and LTR colocalized), and closed arrows show autophagosomes (GFP only). (B) Pearson's coefficient of GFP and LTR colocalization from 10 independent fields of cells in two different experiments.(C) Epifluorescent images for GFP-LC3 and endogenous Lamp1 immunohistochemistry of U20S, VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells or U20S cells cotreated with Baf transfected with GFP-LC3 and treated with rapamycin for 2 h to induce autolysosome formation. Arrows highlight autolysosomes (GFP and Lamp1 colocalized). (A and C) The boxed regions in the merge field are enlarged in the adjacent panels. (D) Pearson's coefficient of GFP and Lamp1 colocalization from 10 independent fields of cells in two different experiments. (B and D) Error bars represent the standard error from 20 fields in two independent experiments. *, P < 0.001.(E) Electron microscopy of tetracycline-inducible control or VCP-WT-, VCP-RH-, or VCP-AE-expressing U20S cells induced for 16 h and then treated with rapamycin for 2 h. Note the accumulation of autophagic structures in the mutant-expressing cell lines. Arrows denote autophagic structures.


Figure 6 . ( A ) Immunofluorescence images of quadriceps muscle using p62 and Lamp1 or - 2 antibodies . Quadriceps section from a 9 - mo - oldVCP - WT mouse , age - matched VCP - RH transgenic , 12 - mo - oldVCP - RH transgenic , and IBMPFD patientmuscle biopsy are shown . Open arrows highlight p62 and Lamp1 / 2 colocalization , and closed arrows show regions of only p62immunofluorescence . Single myofibers containing RVs are outlined in the merge panels . ( B and C ) Transmission electron microscopy of tibialis anterior muscle from one of two independent lines ( B shows RH12 , and C shows RH9 lines ) of 12 - mo - old VCP - RH - expressing transgenic mice . Note the large vacuolated structures that are perinuclear or subsarcolemmal . Bars : ( A ) 30 µm ; ( B and C ) 500 nm .

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 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

Figure 6.(A) Immunofluorescence images of quadriceps muscle using p62 and Lamp1 or -2 antibodies. Quadriceps section from a 9-mo-oldVCP-WT mouse, age-matched VCP-RH transgenic, 12-mo-oldVCP-RH transgenic, and IBMPFD patientmuscle biopsy are shown. Open arrows highlight p62 and Lamp1/2 colocalization, and closed arrows show regions of only p62immunofluorescence. Single myofibers containing RVs are outlined in the merge panels.(B and C) Transmission electron microscopy of tibialis anterior muscle from one of two independent lines (B shows RH12, and C shows RH9 lines) of 12-mo-old VCP-RH-expressing transgenic mice. Note the large vacuolated structures that are perinuclear or subsarcolemmal. Bars: (A) 30 µm; (B and C) 500 nm.


Figure 7 . ( A ) Representative images using in vivo bioluminescence of control or IBMPFD mutant RH - expressing mice ( RH12 ) 2 d after electroporation ( baseline ) or after 24 h of nutrient deprivation ( starvation ) of polyQ80 ( Q80 ) - or polyQ19 - luciferase ( Q19 ) in the right and left tibialis anterior , respectively . The ratio of polyQ80 - luciferase / polyQ19 - luciferase is indicated below each image set . Values are in × 104 photons per second per square centimeter per steradian . ( B ) Box and whisker plot of the change ( Δ ) in the ratio of polyQ80 - luciferase / polyQ19 - luciferase activity in the left and right tibialis anterior muscle of control , VCP - WT , or one of two VCP - RH ( RH12 or RH9 ) transgenic mouse lines after 24 h of starvation . The graph is representative of three animals per group . The p - value for RH12 was 0 . 05 and 0 . 06 when compared with control animals or VCP - WT transgenic . The p - value for RH9 was 0 . 03 when compared with either control or VCP - WT transgenic . The p - value was 0 . 01 and 0 . 02 when combined RH12 and RH9 animals were compared with control or VCP - WT groups , respectively . There was no statistical difference between control or VCP - WT groups .

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

Figure 7.(A) Representative images using in vivo bioluminescence of control or IBMPFD mutant RH-expressing mice (RH12) 2 d after electroporation (baseline) or after 24 h of nutrient deprivation (starvation) of polyQ80 (Q80)- or polyQ19-luciferase (Q19) in the right and left tibialis anterior, respectively. The ratio of polyQ80-luciferase/polyQ19-luciferase is indicated below each image set. Values are in ×104 photons per second per square centimeter per steradian. (B) Box and whisker plot of the change (Δ) in the ratio of polyQ80-luciferase/polyQ19-luciferase activity in the left and right tibialis anterior muscle of control, VCP-WT, or one of two VCP-RH (RH12 or RH9) transgenic mouse lines after 24 h of starvation. The graph is representative of three animals per group. The p-value for RH12 was 0.05 and 0.06 when compared with control animals or VCP-WT transgenic. The p-value for RH9 was 0.03 when compared with either control or VCP-WT transgenic. The p-value was 0.01 and 0.02 when combined RH12 and RH9 animals were compared with control or VCP-WT groups, respectively. There was no statistical difference between control or VCP-WT groups.


Figure 8 . ( A ) Quantitation of mCherry - TDP - 43 distribution ( nuclear only or cytoplasmic ) in control U20S , VCP - WT - , VCP - RH - , VCP - AE - , or VCP - EQ - expressing cells , or control U20S cells treated with 10 µM Baf or 50 µM chloroquine ( Chlq ) for 4 h from 10 different fields from two independent experiments . Error bars represent the standard error from 20 fields in two independent experiments . * , P < 0 . 02 when compared with VCP - WT - expressing cells . ( B ) Epifluorescent images for mCherry - TDP - 43 ( red ) and DAPI ( blue ) in control U20S , VCP - WT - , VCP - RH - , or VCP - AE - expressing cells , or control U20S cells treated with Baf or chloroquine for 4 h . Arrows denote cytosolic TDP - 43 and perinuclear TDP - 43 inclusions . ( C , top ) Immunoblot for TDP - 43 , lamin A / C , and actin from nuclear or cytosolic lysate fractions of mCherry - TDP - 43 - transfected VCP - WT - , VCP - RH - , VCP - AE - , or VCP - EQ - expressing cells . Note the increase in cytosolic TDP - 43 from IBMPFD mutant - and EQ - expressing cells . ( bottom ) Immunoblot for TDP - 43 , lamin A / C , and actin from nuclear or cytosolic lysate fractions of untreated U20S cells transfected with mCherry - TDP - 43 or similarly transfected cells treated with 30 µg / ml ( cq1 ) or 120 µg / ml ( cq2 ) chloroquine diphosphate or 200 ng / ml Baf . Data are representative of three independent experiments . Bar , 15 µm .

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

Figure 8.(A) Quantitation of mCherry-TDP-43 distribution (nuclear only or cytoplasmic) in control U20S, VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells, or control U20S cells treated with 10 µM Baf or 50 µM chloroquine (Chlq) for 4 h from 10 different fields from two independent experiments. Error bars represent the standard error from 20 fields in two independent experiments. *, P < 0.02 when compared with VCP-WT-expressing cells. (B) Epifluorescent images for mCherry-TDP-43 (red) and DAPI (blue) in control U20S, VCP-WT-, VCP-RH-, or VCP-AE-expressing cells, or control U20S cells treated with Baf or chloroquine for 4 h. Arrows denote cytosolic TDP-43 and perinuclear TDP-43 inclusions.(C, top) Immunoblot for TDP-43, lamin A/C, and actin from nuclear or cytosolic lysate fractions of mCherry-TDP-43-transfected VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells. Note the increase in cytosolic TDP-43 from IBMPFD mutant- and EQ-expressing cells. (bottom) Immunoblot for TDP-43, lamin A/C, and actin from nuclear or cytosolic lysate fractions of untreated U20S cells transfected with mCherry-TDP-43 or similarly transfected cells treated with 30 µg/ml (cq1) or 120 µg/ml (cq2) chloroquine diphosphate or 200 ng/ml Baf. Data are representative of three independent experiments. Bar, 15 µm.


Figure 9 . ( A ) TDP - 43 immunostaining of quadriceps skeletal muscle from 15 - mo - old control ( Cont ) or VCP - WT , - RH9 , or - RH12 transgenic mice . A single myofiber is outlined in white . ( B ) TDP - 43 immunostaining of quadriceps skeletal muscle from 3 - mo - old mice treated with saline or 50 mg / kg / d intraperitoneal chloroquine ( Chlq ) for 4 wk . ( A and B ) Insets show one myonuclei from each field . Bars , 30 µm .

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Figure 9.(A) TDP-43 immunostaining of quadriceps skeletal muscle from 15-mo-old control (Cont) or VCP-WT, -RH9, or -RH12 transgenic mice. A single myofiber is outlined in white.(B) TDP-43 immunostaining of quadriceps skeletal muscle from 3-mo-old mice treated with saline or 50 mg/kg/d intraperitoneal chloroquine (Chlq) for 4 wk. (A and B) Insets show one myonuclei from each field. Bars, 30 µm.


Figure 1 ( A ) Muscular exophers contain organelles and large protein complexes . Arrows : white - exopher , blue - muscle cell , green - mitochondria , red - proteasome foci . MOM - mitochondrial outer membrane . ( B ) BMW actively releases significant amounts of exopher . The image shows the middle part of the worm ' s body with muscles marked with dashed lines . Arrows indicate representative exophers , and the asterisk indicates the position of the vulva . ( H ) Knockdown of two autophagy genes , atg - 7 and lgg - 1 significantly reduce the number of generated exophers . n = 91 - 103 ; N = 3 .

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Figure 1(A) Muscular exophers contain organelles and large protein complexes. Arrows: white - exopher, blue - muscle cell, green - mitochondria, red - proteasome foci. MOM - mitochondrial outer membrane. (B) BMW actively releases significant amounts of exopher. The image shows the middle part of the worm's body with muscles marked with dashed lines. Arrows indicate representative exophers, and the asterisk indicates the position of the vulva.(H) Knockdown of two autophagy genes, atg-7 and lgg-1 significantly reduce the number of generated exophers. n = 91-103; N = 3.


Figure 2 ( A ) The highest number of exophers is produced during the hermaphrodite reproductive period and in aging animals . Males do not produce exophers during the first days of adulthood and begin to generate a small number of exophers later in life . Starting n = 90 hermaphrodites and 150 males ; N = 3 . ( B ) Feminized hermaphrodites of thermosensitive a fem - 1 mutant strain do not produce exophers regardless of growth temperature . This phenotype can be partially rescued by mating fem - 1 mutants with males . n = 10 - 26 ; N = 2 . ( D ) Hermaphrodites sterilized via FUdR treatment produce no exophers or only a few per animal . n = 118 and 112 animals ; N = 3 . ( E ) Males and sterile hermaphrodites ( via FUdR treatment ) show the formation of spherical structures in the BWM that resemble mature exophers . MOM - mitochondrial outer membrane .

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Figure 2(A) The highest number of exophers is produced during the hermaphrodite reproductive period and in aging animals. Males do not produce exophers during the first days of adulthood and begin to generate a small number of exophers later in life. Starting n = 90 hermaphrodites and 150 males; N = 3.(B) Feminized hermaphrodites of thermosensitive a fem-1 mutant strain do not produce exophers regardless of growth temperature. This phenotype can be partially rescued by mating fem-1 mutants with males. n = 10-26; N = 2.(D) Hermaphrodites sterilized via FUdR treatment produce no exophers or only a few per animal. n = 118 and 112 animals; N = 3.(E) Males and sterile hermaphrodites (via FUdR treatment) show the formation of spherical structures in the BWM that resemble mature exophers. MOM - mitochondrial outer membrane.


Figure 3 ( B ) RNAi knockdown of genes regulating the egg laying rate and their presence in the uterus influences exopher production . n = 50 - 60 ; N = 2 . ( F ) Increased eggshell permeability caused by emb - 27 knockdown elevates exopher production . n = 103 and 89 ; N = 3 .

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Figure 3(B) RNAi knockdown of genes regulating the egg laying rate and their presence in the uterus influences exopher production. n = 50-60; N = 2.(F) Increased eggshell permeability caused by emb-27 knockdown elevates exopher production. n = 103 and 89; N = 3.


Figure 4 ( C , D ) Animals with overactive exopheresis have reduced exploratory behaviour presented as a reduction in straight line distance travelled ( C ) and by representative worms traces on the plate ( D ) . n = 11 - 34 ; N = 3 . ( E ) RNAi knockdown of the egg yolk precursor protein VIT - 1 increases the number of muscular exophers . n = 20 ; N = 2 . ( F ) Embryos from hermaphrodite mothers that produce a high number of exophers contain more egg yolk precursor protein VIT - 2 . Representative images of embryos with endogenous VIT - 2 : : GFP levels from mothers with different exopheresis activity are shown above the graph . n = 15 and 14 , N = 5 and 6 . Scale bar is 10 µm . ( G ) RNAi knockdown of RME - 2 yolk receptor abolish exopher production . n = 110 - 123 ; N = 3 . ( H ) Muscle - produced VIT - 2 is released from muscles via exophers . The image shows the midbody of worms expressing the proteasome subunit RPN - 5 tagged with RFP in BWM and VIT - 2 : : GFP endogenous expression . Arrows : white - exopher , blue - muscle cell . Dashed lines mark embryos present in the uterus . Scale bar is 10 µm .

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

Figure 4(C, D) Animals with overactive exopheresis have reduced exploratory behaviour presented as a reduction in straight line distance travelled (C) and by representative worms traces on the plate (D). n = 11-34; N = 3.(E) RNAi knockdown of the egg yolk precursor protein VIT-1 increases the number of muscular exophers. n = 20; N = 2.(F) Embryos from hermaphrodite mothers that produce a high number of exophers contain more egg yolk precursor protein VIT-2. Representative images of embryos with endogenous VIT-2::GFP levels from mothers with different exopheresis activity are shown above the graph. n = 15 and 14, N = 5 and 6. Scale bar is 10 µm.(G) RNAi knockdown of RME-2 yolk receptor abolish exopher production. n = 110-123; N = 3.(H) Muscle-produced VIT-2 is released from muscles via exophers. The image shows the midbody of worms expressing the proteasome subunit RPN-5 tagged with RFP in BWM and VIT-2::GFP endogenous expression. Arrows: white - exopher, blue - muscle cell. Dashed lines mark embryos present in the uterus. Scale bar is 10 µm.