Figure 4 ( A ) Immunoblot analysis of NEDD4L , MEKK2 , and MEKK3 immunoprecipitated with NEDD4L - specific antibody from lysates of HeLa cells pre - treated with 20μM MG - 132 for 6 h and then treated with IL - 17 for the indicated time . Data information : All experiments were performed three times , and representative data are shown . ( B ) Immunoblot analysis of NEDD4L precipitated with GST - tagged Null or GST - tagged MEKK2 recombinant protein . Data information : All experiments were performed three times , and representative data are shown . ( C ) Immunoblot of HA - tagged MEKK2 co - immunoprecipitated with anti - Myc antibody from lysates of HEK293T cells co - transfected with plasmids expressing HA - tagged MEKK2 and Myc - tagged wild - type NEDD4L or mutant NEDD4L with C2 ( ∆ C2 ) , WW ( ∆ WW ) or HECT ( ∆ HECT ) domain deleted . Data information : All experiments were performed three times , and representative data are shown . ( D ) Immunoblot analysis of MEKK2 and NEDD4L in Nedd4l + / + , and Neddl4 - / - treated with DMSO , 20μM MG - 132 , or 0 . 2 μM bafilomycin A1 ( Baf A1 ) for 6 h . Densitometry of MEKK2 was relative to actin . Data information : All experiments were performed three times , and representative data are shown . ( E ) Immunoblot analysis of MEKK2 incubated with NEDD4L or TRAF6 recombinant protein and the full complement of ubiquitination reaction components in vitro . Data information : All experiments were performed three times , and representative data are shown . ( F ) Immunoblot analysis of K48 - , K63 - linked , and total ubiquitination of MEKK2 following precipitation of MEKK2 with MEKK2 - specific antibody from lysates of Nedd4l + / + and Nedd4l - / - MEFs treated with MG - 132 . Data information : All experiments were performed three times , and representative data are shown . ( G ) Immunoblot analysis of total ubiquitination of Flag - tagged MEKK2 following precipitation of MEKK2 with Flag - tagged specific antibody from lysates of HEK293T cells co - transfected with plasmids expressing Flag - tagged MEKK2 and Myc - tagged wild - type NEDD4L or mutant NEDD4L with C2 ( ∆ C2 ) , WW ( ∆ WW ) or HECT ( ∆ HECT ) domain deleted . Data information : All experiments were performed three times , and representative data are shown . ( H ) Immunoblot analysis of total ubiquitination of Flag - tagged MEKK2 following immunoprecipitating MEKK2 with anti - Flag antibody from lysates of HEK293T cells co - transfected with plasmids expressing Flag - tagged MEKK2 , Myc - tagged NEDD4L , and HA - tagged wild - type or K6O , K11O , K27O , K29O , K33O , K48O , K63O mutant Ub . ( I ) Immunoblot analysis of total ubiquitination of Flag - tagged MEKK2 following immunoprecipitating MEKK2 with anti - Flag antibody from lysates of HEK293T cells co - transfected with plasmids expressing Flag - tagged MEKK2 , Myc - tagged NEDD4L , and HA - tagged wild - type or K6R , K11R , K27R , K29R , K33R , K48R , K63R mutant Ub . Data information : All experiments were performed three times , and representative data are shown . ( J ) Immunoblot analysis of total ubiquitination of Flag - tagged MEKK2 following immunoprecipitating MEKK2 with anti - Flag antibody from lysates of HEK293T cells co - transfected with plasmids expressing Flag - tagged MEKK2 , increased dosage of Myc - tagged NEDD4L , and HA - tagged wild - type , K27O , or K63O mutant Ub . Data information : All experiments were performed three times , and representative data are shown .

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

Figure 4(A)Immunoblot analysis of NEDD4L, MEKK2, and MEKK3 immunoprecipitated with NEDD4L-specific antibody from lysates of HeLa cells pre-treated with 20μM MG-132 for 6 h and then treated with IL-17 for the indicated time. Data information: All experiments were performed three times, and representative data are shown.(B)Immunoblot analysis of NEDD4L precipitated with GST-tagged Null or GST-tagged MEKK2 recombinant protein. Data information: All experiments were performed three times, and representative data are shown.(C)Immunoblot of HA-tagged MEKK2 co-immunoprecipitated with anti-Myc antibody from lysates of HEK293T cells co-transfected with plasmids expressing HA-tagged MEKK2 and Myc-tagged wild-type NEDD4L or mutant NEDD4L with C2 (∆C2), WW (∆WW) or HECT (∆HECT) domain deleted. Data information: All experiments were performed three times, and representative data are shown.(D)Immunoblot analysis of MEKK2 and NEDD4L in Nedd4l+/+, and Neddl4- /- treated with DMSO, 20μM MG-132, or 0.2 μM bafilomycin A1 (Baf A1) for 6 h. Densitometry of MEKK2 was relative to actin. Data information: All experiments were performed three times, and representative data are shown.(E)Immunoblot analysis of MEKK2 incubated with NEDD4L or TRAF6 recombinant protein and the full complement of ubiquitination reaction components in vitro. Data information: All experiments were performed three times, and representative data are shown.(F)Immunoblot analysis of K48-, K63-linked, and total ubiquitination of MEKK2 following precipitation of MEKK2 with MEKK2-specific antibody from lysates of Nedd4l+/+ and Nedd4l-/- MEFs treated with MG-132. Data information: All experiments were performed three times, and representative data are shown.(G)Immunoblot analysis of total ubiquitination of Flag-tagged MEKK2 following precipitation of MEKK2 with Flag-tagged specific antibody from lysates of HEK293T cells co-transfected with plasmids expressing Flag-tagged MEKK2 and Myc-tagged wild-type NEDD4L or mutant NEDD4L with C2 (∆C2), WW (∆WW) or HECT (∆HECT) domain deleted. Data information: All experiments were performed three times, and representative data are shown.(H)Immunoblot analysis of total ubiquitination of Flag-tagged MEKK2 following immunoprecipitating MEKK2 with anti-Flag antibody from lysates of HEK293T cells co-transfected with plasmids expressing Flag-tagged MEKK2, Myc-tagged NEDD4L, and HA-tagged wild-type or K6O, K11O, K27O, K29O, K33O, K48O, K63O mutant Ub. (I)Immunoblot analysis of total ubiquitination of Flag-tagged MEKK2 following immunoprecipitating MEKK2 with anti-Flag antibody from lysates of HEK293T cells co-transfected with plasmids expressing Flag-tagged MEKK2, Myc-tagged NEDD4L, and HA-tagged wild-type or K6R, K11R, K27R, K29R, K33R, K48R, K63R mutant Ub. Data information: All experiments were performed three times, and representative data are shown.(J)Immunoblot analysis of total ubiquitination of Flag-tagged MEKK2 following immunoprecipitating MEKK2 with anti-Flag antibody from lysates of HEK293T cells co-transfected with plasmids expressing Flag-tagged MEKK2, increased dosage of Myc-tagged NEDD4L, and HA-tagged wild-type, K27O, or K63O mutant Ub. Data information: All experiments were performed three times, and representative data are shown.


Figure 5 ( A , Immunoblot analysis of phosphorylated p38 ( p - p38 ) , phosphorylated NF - κB p65 ( P - P65 ) , MEKK2 and actin in HeLa cells ( A ) transfected with Mekk2 siRNA ( siMekk2 ) or control RNA ( siNC ) and stimulated with IL - 17 ( HeLa cells 50 ng / ml , for indicated time . Data information : Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . All experiments were performed two times , and representative data are shown . B ) Immunoblot analysis of phosphorylated p38 ( p - p38 ) , phosphorylated NF - κB p65 ( P - P65 ) , MEKK2 and actin in MEFs ( B ) transfected with Mekk2 siRNA ( siMekk2 ) or control RNA ( siNC ) and stimulated with IL - 17 MEFs 100ng / ml ) for indicated time . Data information : Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . All experiments were performed two times , and representative data are shown . ( C , Realtime PCR analysis of Il - 6 , Cxcl2 , and Ccl20 mRNA expression in HeLa cells ( C ) transfected with siRNA ( siMekk2 ) or control RNA ( siNC ) and stimulated with IL - 17 ( HeLa cells 50 ng / ml , for indicated time ( n = 3 technical replicates ) . Data information : Data are shown as the mean ± s . e . m . of technical replicates . All experiments were performed two times , and representative data are shown . D ) Realtime PCR analysis of Il - 6 , Cxcl2 , and Ccl20 mRNA expression in MEFs ( D ) transfected with siRNA ( siMekk2 ) or control RNA ( siNC ) and stimulated with IL - 17 MEFs 100ng / ml ) for indicated time ( n = 3 technical replicates ) . Data information : Data are shown as the mean ± s . e . m . of technical replicates . All experiments were performed two times , and representative data are shown . ( E ) Immunoblot analysis of phosphorylated p38 , phosphorylated NF - κB p65 ( P - P65 ) , NEDD4L , MEKK2 and actin in HeLa cells transfected with control RNA ( siNC ) , Nedd4l siRNA ( siNedd4l ) , Mekk2 siRNA ( siMekk2 ) or Nedd4l siRNA plus Mekk2 siRNA ( siNedd4l + siMekk2 ) , and stimulated with IL - 17 ( 50 ng / ml ) for indicated time . Data information : Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . All experiments were performed two times , and representative data are shown . ( F ) Immunoblot analysis of phosphorylated p38 , phosphorylated NF - κB p65 ( p - p65 ) , NEDD4L , MEKK2 and actin in Nedd4l + / + and Nedd4l - / - MEFs transfected with Mekk2 siRNA ( siMekk2 ) or control RNA ( siNc ) , and stimulated with IL - 17 ( 100 ng / ml ) for indicated time . Data information : Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . All experiments were performed two times , and representative data are shown . ( G , Realtime PCR analysis of Il - 6 , Cxcl2 , and Ccl20 mRNA expression in HeLa cells ( G ) treated as described in ( E ) ( n = 3 technical replicates ) . Data information : Data are shown as the mean ± s . e . m . of technical replicates . All experiments were performed two times , and representative data are shown . H ) Realtime PCR analysis of Il - 6 , Cxcl2 , and Ccl20 mRNA expression in MEFs ( H ) treated as described in ( F ) ( n = 3 technical replicates ) . Data information : Data are shown as the mean ± s . e . m . of technical replicates . All experiments were performed two times , and representative data are shown .

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 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 O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 phosphorylated p38 (p-p38), phosphorylated NF-κB p65 (P-P65), MEKK2 and actin in HeLa cells (A) transfected with Mekk2 siRNA (siMekk2) or control RNA (siNC) and stimulated with IL-17 (HeLa cells 50 ng/ml, for indicated time. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. All experiments were performed two times, and representative data are shown.B) Immunoblot analysis of phosphorylated p38 (p-p38), phosphorylated NF-κB p65 (P-P65), MEKK2 and actin in MEFs (B) transfected with Mekk2 siRNA (siMekk2) or control RNA (siNC) and stimulated with IL-17 MEFs 100ng/ml) for indicated time. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. All experiments were performed two times, and representative data are shown.(C, Realtime PCR analysis of Il-6, Cxcl2, and Ccl20 mRNA expression in HeLa cells (C) transfected with siRNA (siMekk2) or control RNA (siNC) and stimulated with IL-17 (HeLa cells 50 ng/ml, for indicated time (n = 3 technical replicates). Data information: Data are shown as the mean ± s.e.m. of technical replicates. All experiments were performed two times, and representative data are shown.D) Realtime PCR analysis of Il-6, Cxcl2, and Ccl20 mRNA expression in MEFs (D) transfected with siRNA (siMekk2) or control RNA (siNC) and stimulated with IL-17 MEFs 100ng/ml) for indicated time (n = 3 technical replicates). Data information: Data are shown as the mean ± s.e.m. of technical replicates. All experiments were performed two times, and representative data are shown.(E) Immunoblot analysis of phosphorylated p38, phosphorylated NF-κB p65 (P-P65), NEDD4L, MEKK2 and actin in HeLa cells transfected with control RNA (siNC), Nedd4l siRNA (siNedd4l), Mekk2 siRNA(siMekk2) or Nedd4l siRNA plus Mekk2 siRNA (siNedd4l+ siMekk2), and stimulated with IL-17(50 ng/ml) for indicated time. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. All experiments were performed two times, and representative data are shown.(F) Immunoblot analysis of phosphorylated p38, phosphorylated NF-κB p65 (p-p65), NEDD4L, MEKK2 and actin in Nedd4l+/+ and Nedd4l-/-MEFs transfected with Mekk2 siRNA (siMekk2) or control RNA (siNc), and stimulated with IL-17 (100 ng/ml) for indicated time. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. All experiments were performed two times, and representative data are shown.(G, Realtime PCR analysis of Il-6, Cxcl2, and Ccl20 mRNA expression in HeLa cells (G) treated as described in (E) (n = 3 technical replicates). Data information: Data are shown as the mean ± s.e.m. of technical replicates. All experiments were performed two times, and representative data are shown.H) Realtime PCR analysis of Il-6, Cxcl2, and Ccl20 mRNA expression in MEFs (H) treated as described in (F) (n = 3 technical replicates). Data information: Data are shown as the mean ± s.e.m. of technical replicates. All experiments were performed two times, and representative data are shown.


Figure 6 ( A ) Immunoblot analysis of Ser520 phosphorylation of MEKK2 immunoprecipitated from HeLa cells stimulated with IL - 17 ( 50 ng / ml ) for indicated time , and then incubated with or without calf intestinal alkaline phosphatasetime ( CIAP ) for 30 mins . Data information : All experiments were performed two times , and representative data are shown . ( B ) Realtime PCR analysis of Il - 6 , Cxcl2 , and Ccl20 mRNA expression in HeLa cells transfected with HA - tagged MEKK2 S520A or control vector , and stimulated with IL - 17 ( 50 ng / ml ) for indicated time ( n = 3 technical replicates ) . Data information : Data are shown as the mean ± s . e . m . of technical replicates . All experiments were performed two times , and representative data are shown . ( C ) Immunoblot analysis of phosphorylated p38 , phosphorylated NF - κB p65 ( P - P65 ) , HA , and actin in HeLa cells treated as in ( B ) . Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . Data information : All experiments were performed two times , and representative data are shown . ( D ) Immunoblot analysis of MEKK2 and TAK1 co - immunoprecipitated with anti - TAK1 antibody from lysates of HeLa cells treated with 50ng / ml IL - 17 for indicated time . Data information : All experiments were performed two times , and representative data are shown . ( E ) Immunoblot analysis of Ser520 phosphorylation of MEKK2 immunoprecipitated from HeLa cells transfected with Tak1 - specific ( siTak1 ) or control RNA ( siNC ) , and then stimulated with IL - 17 ( 50 ng / ml ) for indicated time . Data information : All experiments were performed two times , and representative data are shown . ( F ) Immunoblot analysis of Myc - tagged NEDD4L co - immunoprecipitated with anti - HA antibody from lysates of HEK293T cells co - transfected with plasmids expressing Myc - tagged NEDD4L and HA - tagged wild - type MEKK2 or mutant MEKK2 ( S520A ) . Data information : All experiments were performed two times , and representative data are shown . ( G ) Immunoblot analysis of total ubiquitination of HA - tagged MEKK2 and HA - tagged MEKK2 ( S520A ) following precipitation of MEKK2 with anti - HA antibody from lysates of HEK293T cells co - transfected with plasmids expressing HA - tagged MEKK2 or HA - tagged MEKK2 ( S520A ) together with Myc - tagged NEDD4L and Flag - tagged Ub . Data information : All experiments were performed two times , and representative data are shown . ( H ) Immunoblot analysis of HA - tagged MEKK2 , HA - tagged MEKK2 ( S520A ) , Myc - tagged NEDD4L , and actin in HEK293T cells transfected with the indicated plasmids with anti - HA , anti - Myc , or anti - actin antibody . Data information : All experiments were performed two times , and representative data are shown .

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 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 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 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 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 6(A)Immunoblot analysis of Ser520 phosphorylation of MEKK2 immunoprecipitated from HeLa cells stimulated with IL-17 (50 ng/ml) for indicated time, and then incubated with or without calf intestinal alkaline phosphatasetime (CIAP) for 30 mins. Data information: All experiments were performed two times, and representative data are shown.(B) Realtime PCR analysis of Il-6, Cxcl2, and Ccl20 mRNA expression in HeLa cells transfected with HA-tagged MEKK2 S520A or control vector, and stimulated with IL-17 (50 ng/ml) for indicated time (n = 3 technical replicates). Data information: Data are shown as the mean ± s.e.m. of technical replicates. All experiments were performed two times, and representative data are shown.(C) Immunoblot analysis of phosphorylated p38, phosphorylated NF-κB p65 (P-P65), HA, and actin in HeLa cells treated as in (B). Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. Data information: All experiments were performed two times, and representative data are shown.(D)Immunoblot analysis of MEKK2 and TAK1 co-immunoprecipitated with anti-TAK1 antibody from lysates of HeLa cells treated with 50ng/ml IL-17 for indicated time. Data information: All experiments were performed two times, and representative data are shown.(E)Immunoblot analysis of Ser520 phosphorylation of MEKK2 immunoprecipitated from HeLa cells transfected with Tak1-specific (siTak1) or control RNA (siNC), and then stimulated with IL-17 (50 ng/ml) for indicated time. Data information: All experiments were performed two times, and representative data are shown.(F)Immunoblot analysis of Myc-tagged NEDD4L co-immunoprecipitated with anti-HA antibody from lysates of HEK293T cells co-transfected with plasmids expressing Myc-tagged NEDD4L and HA-tagged wild-type MEKK2 or mutant MEKK2 (S520A). Data information: All experiments were performed two times, and representative data are shown.(G)Immunoblot analysis of total ubiquitination of HA-tagged MEKK2 and HA-tagged MEKK2(S520A) following precipitation of MEKK2 with anti-HA antibody from lysates of HEK293T cells co-transfected with plasmids expressing HA-tagged MEKK2 or HA-tagged MEKK2(S520A) together with Myc-tagged NEDD4L and Flag-tagged Ub. Data information: All experiments were performed two times, and representative data are shown.(H)Immunoblot analysis of HA-tagged MEKK2, HA-tagged MEKK2 (S520A), Myc-tagged NEDD4L, and actin in HEK293T cells transfected with the indicated plasmids with anti-HA, anti-Myc, or anti-actin antibody. Data information: All experiments were performed two times, and representative data are shown.


Figure 1B , phase contrast images of the morphological changes in MDR cells , residual MDR cells and regrown cells for Du145TXR or MCF - 7ADR . Red circles mark magnified areas ( bottom ) . Scale bars , 50 μm ( low magnification images ) . C - D , cell viability of residual MDR cells ( RMCs ) and MDR cells ( MCs ) of ( C ) Du145TXR or ( D ) MCF - 7ADR cells using the same combination treatment for 24 hours on the indicated days . Student ' s t test was used to analyze statistical differences . Mean with ± SD . Data information : Results are representative of three independent experiments . F - G , drug tolerant rate of ( F ) Du145 / Du145TXR ( 20 nM taxol / 20 nM taxol plus 50 μM verapamil ) or ( G ) MCF - 7 / MCF - 7ADR ( 200 nM adriamycin / 200 nM adriamycin plus 50 μM verapamil ) cells treated with drugs on the indicated days . Drug tolerant rate defined by comparing cell viability followed treatment , RMCs VS . MCs or CCs VS . PeCs , > 1 representing drug tolerant . Induction Time indicates the time of entering DTP state ; Sustaining Time indicates the time of maintaining DTP state . Blue colors represent control groups ; Red colors represent MDR cancer cells . Mean with ± SD .

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

Figure 1B, phase contrast images of the morphological changes in MDR cells, residual MDR cells and regrown cells for Du145TXR or MCF-7ADR. Red circles mark magnified areas (bottom). Scale bars, 50 μm (low magnification images).C-D, cell viability of residual MDR cells (RMCs) and MDR cells (MCs) of (C) Du145TXR or (D) MCF-7ADR cells using the same combination treatment for 24 hours on the indicated days. Student's t test was used to analyze statistical differences. Mean with ± SD. Data information: Results are representative of three independent experiments.F-G, drug tolerant rate of (F) Du145/Du145TXR (20 nM taxol/20 nM taxol plus 50 μM verapamil) or (G) MCF-7/MCF-7ADR (200 nM adriamycin/200 nM adriamycin plus 50 μM verapamil) cells treated with drugs on the indicated days. Drug tolerant rate defined by comparing cell viability followed treatment, RMCs VS. MCs or CCs VS. PeCs, > 1 representing drug tolerant. Induction Time indicates the time of entering DTP state; Sustaining Time indicates the time of maintaining DTP state. Blue colors represent control groups; Red colors represent MDR cancer cells. Mean with ± SD.


Figure 2D , heat map based on scoring rank indicated consistently expressed genes identified by RNA - seq of MCs VS . MPCs in Du145TXR and MCF - 7ADR . F , immunoblotting of NPC1L1 in MCs , MPCs and RCs of Du145TXR or MCF - 7ADR . G , immunoblotting of NPC1L1 in Du145TXR or MCF - 7ADR cells transfected with siNPC1L1 or siScramble . H - I , colony formation assay and quantification of ( H ) Du145TXR or ( I ) MCF - 7ADR cells transfected with siNPC1L1 or siScramble followed by treatment with indicated agents . One - way ANOVA was used to analyze statistical differences . Data information : Results are representative of three independent experiments .

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 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

Figure 2D, heat map based on scoring rank indicated consistently expressed genes identified by RNA-seq of MCs VS. MPCs in Du145TXR and MCF-7ADR.F, immunoblotting of NPC1L1 in MCs, MPCs and RCs of Du145TXR or MCF-7ADR. G, immunoblotting of NPC1L1 in Du145TXR or MCF-7ADR cells transfected with siNPC1L1 or siScramble.H-I, colony formation assay and quantification of (H) Du145TXR or (I) MCF-7ADR cells transfected with siNPC1L1 or siScramble followed by treatment with indicated agents. One-way ANOVA was used to analyze statistical differences. Data information: Results are representative of three independent experiments.


Figure 3A - C , flow cytometric analysis of ROS levels in Du145TXR or MCF - 7ADR cells treated with indicated agents for 12 and 36 hours . ( A ) Representative images and quantification of ROS in ( B ) Du145TXR or ( C ) MCF - 7ADR cells are shown . One - way ANOVA was used to analyze statistical differences . Mean with ± SD Data information : Results are representative of three independent experiments . D - E , cell viability of ( D ) Du145TXR or ( E ) MCF - 7ADR treated with indicated agents for 72 hours . One - way ANOVA was used to analyze statistical differences . Mean with ± SD . Data information : Results are representative of three independent experiments . J - K , cell viability of ( J ) Du145TXR or ( K ) MCF - 7ADR cells cultured in normal or serum free medium followed by treatment with indicated agents for 72 hours . One - way ANOVA was used to analyze statistical differences . Mean with ± SD . Data information : Results are representative of three independent experiments .

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Figure 3A-C, flow cytometric analysis of ROS levels in Du145TXR or MCF-7ADR cells treated with indicated agents for 12 and 36 hours. (A) Representative images and quantification of ROS in (B) Du145TXR or (C) MCF-7ADR cells are shown. One-way ANOVA was used to analyze statistical differences. Mean with ± SD Data information: Results are representative of three independent experiments.D-E, cell viability of (D) Du145TXR or (E) MCF-7ADR treated with indicated agents for 72 hours. One-way ANOVA was used to analyze statistical differences. Mean with ± SD. Data information: Results are representative of three independent experiments.J-K, cell viability of (J) Du145TXR or (K) MCF-7ADR cells cultured in normal or serum free medium followed by treatment with indicated agents for 72 hours. One-way ANOVA was used to analyze statistical differences. Mean with ± SD . Data information: Results are representative of three independent experiments.


Figure 4C - D , endocytosis analysis of ( C ) Du145TXR or ( D ) MCF - 7ADR cells treated with indicated agents for 8 hours , followed by staining with 250 μg / mL FITC - DEX for 4 hours . Scale bars , 10 μm . Data information : Results are representative of three independent experiments . E - F , phase contrast images of the morphological changes in ( E ) Du145TXR and ( F ) MCF - 7ADR cells cultured in normal or serum free medium followed by treatment with indicated agents for 12 hours . Scale bars , 10 or 25 μm . The red arrows indicate vacuoles . Data information : Results are representative of three independent experiments . I - J , colony formation assay and quantification of ( I ) Du145TXR or ( J ) MCF - 7ADR cells treated with indicated agents . One - way ANOVA was used to analyze statistical differences . Data information : Results are representative of three independent experiments .

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 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

Figure 4C-D, endocytosis analysis of (C) Du145TXR or (D) MCF-7ADR cells treated with indicated agents for 8 hours, followed by staining with 250 μg/mL FITC-DEX for 4 hours. Scale bars, 10 μm. Data information: Results are representative of three independent experiments.E-F, phase contrast images of the morphological changes in (E) Du145TXR and (F) MCF-7ADR cells cultured in normal or serum free medium followed by treatment with indicated agents for 12 hours. Scale bars, 10 or 25 μm. The red arrows indicate vacuoles. Data information: Results are representative of three independent experiments.I-J, colony formation assay and quantification of (I) Du145TXR or (J) MCF-7ADR cells treated with indicated agents. One-way ANOVA was used to analyze statistical differences. Data information: Results are representative of three independent experiments.


Figure 5C , immunoblot analysis of NPC1L1 and NRF2 in Du145TXR or MCF - 7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents for 24 hours . Data information : Results are representative of three independent experiments . F - G , relative luciferase activities of wild - type ( WT ) or mutant ( MUT ) NPC1L1 promoter ( - 205 ~ - 215 , # 1 ) reporter constructs in ( F ) Du145TXR or ( G ) MCF - 7ADR cells treated with indicated agents . Student ' s t test was used to analyze statistical differences . Mean with ± SD . Data information : Results are representative of three independent experiments . J - K , colony formation assay and quantification of ( J ) Du145TXR or ( K ) MCF - 7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents . One - way ANOVA was used to analyze statistical differences . Data information : Results are representative of three independent experiments .

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

Figure 5C, immunoblot analysis of NPC1L1 and NRF2 in Du145TXR or MCF-7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents for 24 hours. Data information: Results are representative of three independent experiments.F-G, relative luciferase activities of wild-type (WT) or mutant (MUT) NPC1L1 promoter (-205~-215, #1) reporter constructs in (F) Du145TXR or (G) MCF-7ADR cells treated with indicated agents. Student's t test was used to analyze statistical differences. Mean with ± SD. Data information: Results are representative of three independent experiments.J-K, colony formation assay and quantification of (J) Du145TXR or (K) MCF-7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents. One-way ANOVA was used to analyze statistical differences. Data information: Results are representative of three independent experiments.


Figure 6immunohistochemical staining of Ki67 in indicated groups . ( D ) Representative images are shown . Scale bar , 50 μm . , immunohistochemical staining of cleaved - caspase 3 ( CC3 ) in indicated groups . ( F ) Representative images are shown . Scale bar , 50 μm . ( I ) Corresponding images and isolated tumors are shown . The red dotted line represents cured tumors .

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Figure 6immunohistochemical staining of Ki67 in indicated groups. (D) Representative images are shown. Scale bar, 50 μm. , immunohistochemical staining of cleaved-caspase 3 (CC3) in indicated groups. (F) Representative images are shown. Scale bar, 50 μm.(I) Corresponding images and isolated tumors are shown. The red dotted line represents cured tumors.


Figure 1A Lysates of HeLa cells stably expressing GFP alone or GFP - FXR1 were subjected to immunoprecipitation using GFP - Trap beads ( GFP - IP ) , analysed by Western blot and quantified ( shown a mean value , * P < 0 . 05 , * * P < 0 . 01 ; N = 3 ) . B Lysates of HeLa cells stably expressing GFP alone or 3xGFP - Nup85 were immunoprecipitated using GFP - Trap beads ( GFP - IP ) , analysed by Western blot and quantified ( SE , short exposure , LE , long exposure ) ( shown a mean value , * P < 0 . 05 ; N = 3 ) . C Immunoprecipitation from HEK293T cell lysates using FXR1 antibody or IgG analysed by Western blot . The arrow points to the heavy chain of IgG ( IgG HC ) ( shown a mean value , * P < 0 . 05 ; N = 3 ) . D HeLa cells were treated with indicated siRNAs , synchronized by double thymidine block , and released for 12 hours and analysed by immunofluorescence microscopy for the lamin B receptor ( LBR ) to label the NE , and FXR1 . E HeLa cells stably expressing GFP - FXR1 were analysed by immunofluorescence microscopy for GFP and mAb414 , which labels FG - Nups . The magnified framed regions are shown in the corresponding numbered panels . The arrowheads indicate NE and cytoplasmic localization of GFP - FXR1 . F HeLa cells stably expressing GFP - Nup107 were synchronized by double thymidine block and released for 12 hours , permeabilized with Triton / SDS or digitonin for antibodies to access the nuclear and cytoplasmic or cytoplamsic side of the nucleus , respectively , and analysed by immunofluorescence microscopy .

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Figure 1A Lysates of HeLa cells stably expressing GFP alone or GFP-FXR1 were subjected to immunoprecipitation using GFP-Trap beads (GFP-IP), analysed by Western blot and quantified (shown a mean value, *P < 0.05, **P < 0.01; N = 3).B Lysates of HeLa cells stably expressing GFP alone or 3xGFP-Nup85 were immunoprecipitated using GFP-Trap beads (GFP-IP), analysed by Western blot and quantified (SE, short exposure, LE, long exposure) (shown a mean value, *P < 0.05; N = 3).C Immunoprecipitation from HEK293T cell lysates using FXR1 antibody or IgG analysed by Western blot. The arrow points to the heavy chain of IgG (IgG HC) (shown a mean value, *P < 0.05; N = 3).D HeLa cells were treated with indicated siRNAs, synchronized by double thymidine block, and released for 12 hours and analysed by immunofluorescence microscopy for the lamin B receptor (LBR) to label the NE, and FXR1.E HeLa cells stably expressing GFP-FXR1 were analysed by immunofluorescence microscopy for GFP and mAb414, which labels FG-Nups. The magnified framed regions are shown in the corresponding numbered panels. The arrowheads indicate NE and cytoplasmic localization of GFP-FXR1.F HeLa cells stably expressing GFP-Nup107 were synchronized by double thymidine block and released for 12 hours, permeabilized with Triton/SDS or digitonin for antibodies to access the nuclear and cytoplasmic or cytoplamsic side of the nucleus, respectively, and analysed by immunofluorescence microscopy.


Figure 2A HeLa cells were treated with the indicated siRNAs , synchronized by double thymidine block and release for 12 hours and analysed by immunofluorescence microscopy . The magnified framed regions are shown in the corresponding numbered panels . B HeLa cells stably expressing GFP , GFP - FXR1 wild type ( WT ) and GFP - FXR1 mutated in the sequence recognized by FXR1 siRNA - 1 ( GFP - FXR1 - MUT - siRNA1 ) were treated with the indicated siRNAs , synchronized by double thymidine block , released for 24 hours and then analysed by immunofluorescence microscopy . The percentage of cells with cytoplasmic nucleoporin granules was quantified , 1000 cells were analysed for each graph ( mean ± SD , * * P < 0 . 01 ; * * * P < 0 . 001 , N = 3 ) . HeLa cells were treated with the indicated siRNAs , synchronized by double thymidine block , released for 12 hours and analysed by immunofluorescence microscopy . Nups present in different NPC subcomplexes are depicted in the color code corresponding to the NPC scheme shown on the right . Additional or complementary representative images and channels of cells depicted in ( C ) HeLa cells were treated with the indicated siRNAs , synchronized by double thymidine block , released for 12 hours and analysed by immunofluorescence microscopy . Nuclear intensity of FG - Nups labelled by mAb414 ( D ) , RanBP2 ( E ) and GFP - Nup107 ( F ) was quantified . 1800 cells were analysed for each graph ( mean ± SD , * P < 0 . 05 ; * * P < 0 . 01 ; N = 3 ) . G , H Asynchronously proliferating U2OS cells were treated with the indicated siRNAs and analysed by immunofluorescence microscopy . The percentage of cells with cytoplasmic nucleoporin granules ( G ) was quantified and nuclear intensity of FG - Nups labelled by mAb414 ( H ) was quantified . 1600 cells were analysed in ( G ) and 2100 cells were analysed in ( H ) ( mean ± SD , * P < 0 . 05 ; * * * P < 0 . 001 ; N = 3 ) .

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Figure 2A HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block and release for 12 hours and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels.B HeLa cells stably expressing GFP, GFP-FXR1 wild type (WT) and GFP-FXR1 mutated in the sequence recognized by FXR1 siRNA-1 (GFP-FXR1-MUT-siRNA1) were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 hours and then analysed by immunofluorescence microscopy. The percentage of cells with cytoplasmic nucleoporin granules was quantified, 1000 cells were analysed for each graph (mean ±SD, **P < 0.01; ***P < 0.001, N = 3).HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Nups present in different NPC subcomplexes are depicted in the color code corresponding to the NPC scheme shown on the right. Additional or complementary representative images and channels of cells depicted in (C)HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Nuclear intensity of FG-Nups labelled by mAb414 (D), RanBP2 (E) and GFP-Nup107 (F) was quantified. 1800 cells were analysed for each graph (mean ±SD, *P < 0.05; **P < 0.01; N = 3).G, H Asynchronously proliferating U2OS cells were treated with the indicated siRNAs and analysed by immunofluorescence microscopy. The percentage of cells with cytoplasmic nucleoporin granules (G) was quantified and nuclear intensity of FG-Nups labelled by mAb414 (H) was quantified. 1600 cells were analysed in (G) and 2100 cells were analysed in (H) (mean ±SD, *P < 0.05; *** P<0.001; N = 3).


Figure 3A HeLa cells were treated with the indicated siRNAs , synchronized by double thymidine block , released for 24 hours and analysed by immunofluorescence microscopy . The magnified framed regions are shown in the corresponding numbered panels . Arrowheads point to nuclear blebs observed in FXR1 - deficient cells . B , HeLa cells stably expressing GFP , GFP - FXR1 wild type ( WT ) and GFP - FXR1 mutated in the sequence recognized by FXR1 siRNA - 1 ( GFP - FXR1 - MUT - siRNA1 ) were treated with the indicated siRNAs , synchronized by double thymidine block , released for 24 hours and analysed by Western blot ( B ) C HeLa cells stably expressing GFP , GFP - FXR1 wild type ( WT ) and GFP - FXR1 mutated in the sequence recognized by FXR1 siRNA - 1 ( GFP - FXR1 - MUT - siRNA1 ) were treated with the indicated siRNAs , synchronized by double thymidine block , released for 24 hours and analysed by immunofluorescence microscopy ( C ) . The percentage of cells with irregular nuclei was quantified , 1000 cells were analysed ( mean ± SD , * * P < 0 . 01 , * * * P < 0 . 001 ; N = 3 ) . HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs , synchronized by double thymidine block , released for 12 hours and analysed by immunofluorescence microscopy . Time from prophase till anaphase ( D ) , from prophase till metaphase ( E ) , from metaphase till anaphase ( F ) and from anaphase till chromatin decondensation ( G ) was quantified . HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs , synchronized by double thymidine block , released for 12 hours and analysed by immunofluorescence microscopy . The selected frames of the movies are depicted and time is shown in minutes ( H ) . Arrowheads point to nuclear blebs appearing during nuclear expansion of FXR1 - deficient cells . HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs , synchronized by double thymidine block , released for 12 hours and analysed by immunofluorescence microscopy . Percentage of daughter cells with irregular nuclei was quantified in ( I ) HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs , synchronized by double thymidine block , released for 12 hours and analysed by immunofluorescence microscopy . time from anaphase till nuclear blebs was quantified in ( J ) .

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Figure 3A HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 hours and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels. Arrowheads point to nuclear blebs observed in FXR1-deficient cells.B, HeLa cells stably expressing GFP, GFP-FXR1 wild type (WT) and GFP-FXR1 mutated in the sequence recognized by FXR1 siRNA-1 (GFP-FXR1-MUT-siRNA1) were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 hours and analysed by Western blot (B)C HeLa cells stably expressing GFP, GFP-FXR1 wild type (WT) and GFP-FXR1 mutated in the sequence recognized by FXR1 siRNA-1 (GFP-FXR1-MUT-siRNA1) were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 hours and analysed by immunofluorescence microscopy (C). The percentage of cells with irregular nuclei was quantified, 1000 cells were analysed (mean ±SD, **P < 0.01, ***P < 0.001; N = 3).HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Time from prophase till anaphase (D), from prophase till metaphase (E), from metaphase till anaphase (F) and from anaphase till chromatin decondensation (G) was quantified.HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. The selected frames of the movies are depicted and time is shown in minutes (H). Arrowheads point to nuclear blebs appearing during nuclear expansion of FXR1-deficient cells.HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Percentage of daughter cells with irregular nuclei was quantified in (I)HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. time from anaphase till nuclear blebs was quantified in (J).


Figure 4A - D HeLa cells stably expressing GFP - Nup107 were treated with indicated siRNAs , synchronized by double thymidine block , released and analysed by live video spinning disk confocal microscopy ( A ) . The selected frames of the movies are depicted and time is shown in minutes . The onset of anaphase is indicated . The magnified framed regions with time indicated in minutes are shown in ( B ) . White arrowheads point to the cytoplasmic GFP - NUP107 granules appearing during nuclear expansion of control and FXR1 - deficient cells , yellow arrowheads point to the fussion events of GFP - NUP107 granules with NE in control cells . The percentage of cells with cytoplasmic GFP - Nup107 granules was quantified in ( C ) . Time from anaphase till GFP - Nup107 cytoplasmic granule formation was quantified in ( D ) . 57 cells were analysed ( mean ± SD , * P < 0 . 05 ; ns = non - significant ; N = 3 ) .

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Figure 4A-D HeLa cells stably expressing GFP-Nup107 were treated with indicated siRNAs, synchronized by double thymidine block, released and analysed by live video spinning disk confocal microscopy (A). The selected frames of the movies are depicted and time is shown in minutes. The onset of anaphase is indicated. The magnified framed regions with time indicated in minutes are shown in (B). White arrowheads point to the cytoplasmic GFP-NUP107 granules appearing during nuclear expansion of control and FXR1-deficient cells, yellow arrowheads point to the fussion events of GFP-NUP107 granules with NE in control cells. The percentage of cells with cytoplasmic GFP-Nup107 granules was quantified in (C). Time from anaphase till GFP-Nup107 cytoplasmic granule formation was quantified in (D). 57 cells were analysed (mean ±SD, *P < 0.05; ns = non-significant; N = 3).


Figure 5A HeLa cells stably expressing GFP - Nup133 were synchronized by double thymidine block , released for 12 hours , treated or not with NaAsO2 to induce stress granule formation and with 1 , 6 Hexanediol , and analysed by immunofluorescence microscopy . The magnified framed regions are shown in the corresponding numbered panels . B , C HeLa cells stably expressing GFP - Nup133 were treated with the indicated siRNAs , synchronized by double thymidine block , released for 12 hours , treated with or without 1 , 6 Hexanediol and analysed by immunofluorescence microscopy . The magnified framed regions are shown in the corresponding numbered panels . The percentage of cells with cytoplasmic GFP - Nup133 granules was quantified in ( C ) , 3100 cells were analysed ( mean ± SD , * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ; N = 3 ) .

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Figure 5A HeLa cells stably expressing GFP-Nup133 were synchronized by double thymidine block, released for 12 hours, treated or not with NaAsO2 to induce stress granule formation and with 1,6 Hexanediol, and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels.B, C HeLa cells stably expressing GFP-Nup133 were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours, treated with or without 1,6 Hexanediol and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels. The percentage of cells with cytoplasmic GFP-Nup133 granules was quantified in (C), 3100 cells were analysed (mean ±SD, **P < 0.01; ***P < 0.001; ****P < 0.0001; N = 3).


Figure 6A HeLa cells stably expressing GFP alone or GFP - FXR1 were immunoprecipitated using GFP - Trap beads ( GFP - IP ) , analysed by Western blot and quantified ( mean , * P < 0 . 05 ; N = 3 ) . B , C HeLa cells were treated with the indicated siRNAs , synchronized by double thymidine block , released for 12 hours and analysed by immunofluorescence microscopy . Arrowheads indicate blebbed regions of nuclei . The percentage of interphasic cells with Nup granules was quantified ( C ) , 900 cells were analysed ( mean ± SD , * * * P < 0 . 001 ; N = 3 ) . D Lysates of HeLa cells stably expressing GFP alone or GFP - FXR1 were subjected to immunoprecipitation using GFP - Trap beads ( GFP - IP ) , analysed by Western blot and quantified ( mean , * P < 0 . 05 ; * * P < 0 . 01 ; N = 3 ) . E , F HeLa cells were treated with the indicated siRNAs , synchronized by double thymidine block and released for 12 hours and analysed by immunofluorescence microscopy for FXR1 and mAb414 . The magnified framed regions are shown in the corresponding numbered panels in ( E ) . The percentage of cells with cytoplasmic Nup granules was quantified in ( F ) , 2700 cells were analysed ( mean ± SD , * * * P < 0 . 001 ; N = 3 ) .

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

Figure 6A HeLa cells stably expressing GFP alone or GFP-FXR1 were immunoprecipitated using GFP-Trap beads (GFP-IP), analysed by Western blot and quantified (mean, *P < 0.05; N = 3).B, C HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Arrowheads indicate blebbed regions of nuclei. The percentage of interphasic cells with Nup granules was quantified (C), 900 cells were analysed (mean ±SD, ***P < 0.001; N = 3).D Lysates of HeLa cells stably expressing GFP alone or GFP-FXR1 were subjected to immunoprecipitation using GFP-Trap beads (GFP-IP), analysed by Western blot and quantified (mean, *P < 0.05; **P < 0.01; N = 3).E, F HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block and released for 12 hours and analysed by immunofluorescence microscopy for FXR1 and mAb414. The magnified framed regions are shown in the corresponding numbered panels in (E). The percentage of cells with cytoplasmic Nup granules was quantified in (F), 2700 cells were analysed (mean ±SD, ***P < 0.001; N = 3).


Figure 7A , B HeLa cells stably expressing GFP - Nup133 were treated with the indicated siRNAs , synchronized by double thymidine block , released for 12 hours , treated with nocodazole to induce granule formation and washed - out as indicated and analysed by immunofluorescence microscopy . The percentage of cells with cytoplasmic GFP - Nup133 granules was quantified in ( B ) , 5200 cells were analysed ( mean ± SD , * * P < 0 . 01 ; * * * P < 0 . 001 ; N = 3 ) . C - E HeLa cells stably expressing GFP - Nup107 were treated with the indicated siRNAs , synchronized by double thymidine block , released for 12 hours , treated with nocodazole to induce granule formation and washed - out as in ( A ) and analysed by live video spinning disk confocal microscopy . The selected frames of the movies are depicted and time is shown in minutes . The magnified framed regions are depicted in the lower rows . White arrowheads point to individual GFP - Nup107 - positive granules . Yellow arrowheads point to the granules undergoing fusion events . The percentage of cells with fusion / fission events of GFP - Nup107 granules was quantified in ( D ) , 815 cells were analysed ( mean ± SD , * * P < 0 . 01 ; N = 3 ) . The number of fusion / fission events per cell was quantified in ( E ) , 815 cells were analysed ( mean ± SD ; N = 3 ) .

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Figure 7A, B HeLa cells stably expressing GFP-Nup133 were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours, treated with nocodazole to induce granule formation and washed-out as indicated and analysed by immunofluorescence microscopy. The percentage of cells with cytoplasmic GFP-Nup133 granules was quantified in (B), 5200 cells were analysed (mean ±SD, **P < 0.01; ***P < 0.001; N = 3).C-E HeLa cells stably expressing GFP-Nup107 were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours, treated with nocodazole to induce granule formation and washed-out as in (A) and analysed by live video spinning disk confocal microscopy. The selected frames of the movies are depicted and time is shown in minutes. The magnified framed regions are depicted in the lower rows. White arrowheads point to individual GFP-Nup107-positive granules. Yellow arrowheads point to the granules undergoing fusion events. The percentage of cells with fusion/fission events of GFP-Nup107 granules was quantified in (D), 815 cells were analysed (mean ±SD, **P < 0.01; N = 3). The number of fusion/fission events per cell was quantified in (E), 815 cells were analysed (mean ±SD; N = 3).


Figure 8Human FXS - patient derived fibroblasts ( FXS - 1 , FXS - 2 ) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by Western blot ( A ) Human FXS - patient derived fibroblasts ( FXS - 1 , FXS - 2 ) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy Examples of Nup localization defects are shown in ( B ) Human FXS - patient derived fibroblasts ( FXS - 1 , FXS - 2 ) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy The percentage of cells with cytoplasmic Nup granules was quantified in ( C ) , 283 cells were analysed ( mean ± SD , * * * P < 0 . 001 ; N = 3 ) . Human FXS - patient derived fibroblasts ( FXS - 1 , FXS - 2 ) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy examples of nuclear architecture defects are shown in ( D ) . Human induced pluripotent stem cells ( iPSCs ) derived from a FXS patient ( FXS - iPSC ) and the isogenic rescue cells ( C1 _ 2 - iPSC ) were analysed by immunofluorescence microscopyHuman induced pluripotent stem cells ( iPSCs ) derived from a FXS patient ( FXS - iPSC ) and the isogenic rescue cells ( C1 _ 2 - iPSC ) were analysed by immunofluorescence microscopy The percentage of cells with cytoplasmic Nup133 granules was quantified in ( F ) , 5500 cells were analysed ( mean ± SD , * * P < 0 . 01 ; N = 3 ) . Human induced pluripotent stem cells ( iPSCs ) derived from a FXS patient ( FXS - iPSC ) and the isogenic rescue cells ( C1 _ 2 - iPSC ) were analysed by immunofluorescence microscopy Examples of co - localization events of re - expressed FMRP and Nup133 are shown in ( G ) . H , I Mouse Embryonic Fibroblasts ( MEFs ) derived from the Fmr1 knock - out ( KO ) mice and wild type controls were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy ( H ) . The percentage of cells with cytoplasmic nucleoporin granules was quantified in ( I ) , 2400 cells were analysed ( mean ± SD , * P < 0 . 05 ; N = 3 ) .

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Figure 8Human FXS-patient derived fibroblasts (FXS-1, FXS-2) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by Western blot (A)Human FXS-patient derived fibroblasts (FXS-1, FXS-2) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy Examples of Nup localization defects are shown in (B)Human FXS-patient derived fibroblasts (FXS-1, FXS-2) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy The percentage of cells with cytoplasmic Nup granules was quantified in (C), 283 cells were analysed (mean ±SD, ***P < 0.001; N = 3).Human FXS-patient derived fibroblasts (FXS-1, FXS-2) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy examples of nuclear architecture defects are shown in (D).Human induced pluripotent stem cells (iPSCs) derived from a FXS patient (FXS-iPSC) and the isogenic rescue cells (C1_2-iPSC) were analysed by immunofluorescence microscopyHuman induced pluripotent stem cells (iPSCs) derived from a FXS patient (FXS-iPSC) and the isogenic rescue cells (C1_2-iPSC) were analysed by immunofluorescence microscopy The percentage of cells with cytoplasmic Nup133 granules was quantified in (F), 5500 cells were analysed (mean ±SD, **P < 0.01; N = 3).Human induced pluripotent stem cells (iPSCs) derived from a FXS patient (FXS-iPSC) and the isogenic rescue cells (C1_2-iPSC) were analysed by immunofluorescence microscopy Examples of co-localization events of re-expressed FMRP and Nup133 are shown in (G).H, I Mouse Embryonic Fibroblasts (MEFs) derived from the Fmr1 knock-out (KO) mice and wild type controls were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy (H). The percentage of cells with cytoplasmic nucleoporin granules was quantified in (I), 2400 cells were analysed (mean ±SD, *P < 0.05; N = 3).


Figure 9A HeLa cells were transfected with the import / export reporter plasmid XRGG - GFP , treated with the indicated siRNAs and synchronized in early G1 phase by Monastrol release . Dexamethasone was added for 3 hours to induce XRGG - GFP nuclear import . Following wash - out the nuclear export of XRGG - GFP was analysed by live video spinning disk confocal microscopy . The selected frames of the movies are depicted in Figure EV5C . The percentage of cytoplasmic XRGG - GFP over time was quantified in Figure EV5D and quantifications of individual cells from the 20 and 30 minutes time points are depicted in ( A ) , 199 cells were analysed ( mean ± SD , * P < 0 . 05 ; * * P < 0 . 01 ; N = 3 ) . B HeLa cells stably expressing GFP - Nup133 were treated with the indicated siRNAs , synchronized by double thymidine block , released for 12 hours and analysed by immunofluorescence microscopy . The magnified framed regions are shown in the corresponding numbered panels . C , D Asynchronously proliferating HeLa cells were treated with indicated siRNAs and analysed by immunofluorescence microscopy ( C ) . The percentage of p - Rb positive cells was quantified in ( D ) , 2800 cells were analysed ( mean ± SD , * * P < 0 . 01 ; N = 3 ) . Asynchronously proliferating HeLa cells were treated with indicated siRNAs , incubated with EdU during 30min and analysed by immunofluorescence microscopy . The percentage of p - Rb and / or EdU positive cells was quantified in ( E ) , 2100 cells were analysed ( mean ± SD , * P < 0 . 05 ; * * P < 0 . 01 ; N = 3 ) F Asynchronously proliferating HeLa cells were treated with indicated siRNAs , incubated with EdU during 30min and analysed by immunofluorescence microscopy . the percentage of p - Rb and / or cyclin B positive cells was quantified in (

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Figure 9A HeLa cells were transfected with the import/export reporter plasmid XRGG-GFP, treated with the indicated siRNAs and synchronized in early G1 phase by Monastrol release. Dexamethasone was added for 3 hours to induce XRGG-GFP nuclear import. Following wash-out the nuclear export of XRGG-GFP was analysed by live video spinning disk confocal microscopy. The selected frames of the movies are depicted in Figure EV5C. The percentage of cytoplasmic XRGG-GFP over time was quantified in Figure EV5D and quantifications of individual cells from the 20 and 30 minutes time points are depicted in (A), 199 cells were analysed (mean ±SD, *P < 0.05; **P < 0.01; N = 3).B HeLa cells stably expressing GFP-Nup133 were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels.C, D Asynchronously proliferating HeLa cells were treated with indicated siRNAs and analysed by immunofluorescence microscopy (C). The percentage of p-Rb positive cells was quantified in (D), 2800 cells were analysed (mean ±SD, **P < 0.01; N = 3).Asynchronously proliferating HeLa cells were treated with indicated siRNAs, incubated with EdU during 30min and analysed by immunofluorescence microscopy. The percentage of p-Rb and/or EdU positive cells was quantified in (E), 2100 cells were analysed (mean ±SD, *P < 0.05; **P < 0.01; N = 3)F Asynchronously proliferating HeLa cells were treated with indicated siRNAs, incubated with EdU during 30min and analysed by immunofluorescence microscopy. the percentage of p-Rb and/or cyclin B positive cells was quantified in (


Figure 1Activated GFAP + astrocytes in white matter MS lesions with inflammatory infiltrates and demyelination show an upregulation of OTUB1 ( arrows ) . Photomicrographs are obtained from case 9 ( A ) and case 5 ( B ) listed in Table EV1 . Data are representative for all 10 MS patients analyzed . Double immunofluorescence with rabbit anti - OTUB1 ( Cy3 ) and mouse anti - GFAP ( FITC ) ; original magnification × 400 ; scale bars correspond to 50 µm .

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Figure 1Activated GFAP+ astrocytes in white matter MS lesions with inflammatory infiltrates and demyelination show an upregulation of OTUB1 (arrows). Photomicrographs are obtained from case 9 (A) and case 5 (B) listed in Table EV1. Data are representative for all 10 MS patients analyzed. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-GFAP (FITC); original magnification ×400; scale bars correspond to 50 µm.


Figure 2WB analysis of OTUB1 expression in cultured primary astrocytes from OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice . Direct ex vivo WB analysis of OTUB1 expression in astrocytes isolated from adult OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice . Normal CNS architecture in OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice . Myelination is normal in the brain and spinal cord of an OTUB1fl / fl and a GFAP - Cre OTUB1fl / fl mouse . CV - LFB staining ; original magnification × 50 ; scale bars correspond to 100 µm . All photographs are representative of three mice per group . Absolute numbers of CD45 + cells in the spinal cord of OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice ( n = 5 for both groups , mean + SEM ) . Absolute numbers of different subpopulations of leukocytes infiltrating the spinal cord of OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice were analyzed by flow cytometry ( n = 5 for both groups , mean + SEM ) . In an OTUB1fl / fl and a GFAP - Cre OTUB1fl / fl mouse , OTUB1 is strongly expressed by NeuN + neurons in the brain and spinal cord . Double immunofluorescence with rabbit anti - OTUB1 ( Cy3 ) and mouse anti - NeuN ( FITC ) . Single NogoA + oligodendrocytes in the brain and spinal cord ( arrows ) of an OTUB1fl / fl and a GFAP - Cre OTUB1fl / fl mouse express OTUB1 . Double immunofluorescence with rabbit anti - OTUB1 ( Cy3 ) and mouse anti - NogoA ( Alexa Fluor 488 ) . GFAP + astrocytes are of normal morphology and distribution in the brain and spinal cord of a non - immunized OTUB1fl / fl mouse and a GFAP - Cre OTUB1fl / fl mouse . Note that GFAP + astrocytes of an OTUB1fl / fl mouse do not express OTUB1 . GFAP + astrocytes of a GFAP - Cre OTUB1fl / fl mouse are also OTUB1 - negative and represent a negative control for OTUB1 staining . Double immunofluorescence with rabbit anti - OTUB1 ( Cy3 ) and mouse anti - GFAP ( FITC ) .

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Figure 2WB analysis of OTUB1 expression in cultured primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice.Direct ex vivo WB analysis of OTUB1 expression in astrocytes isolated from adult OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice.Normal CNS architecture in OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice. Myelination is normal in the brain and spinal cord of an OTUB1fl/fl and a GFAP-Cre OTUB1fl/fl mouse. CV-LFB staining; original magnification ×50; scale bars correspond to 100 µm. All photographs are representative of three mice per group.Absolute numbers of CD45+ cells in the spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice (n = 5 for both groups, mean + SEM). Absolute numbers of different subpopulations of leukocytes infiltrating the spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were analyzed by flow cytometry (n = 5 for both groups, mean + SEM).In an OTUB1fl/fl and a GFAP-Cre OTUB1fl/fl mouse, OTUB1 is strongly expressed by NeuN+ neurons in the brain and spinal cord. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-NeuN (FITC). Single NogoA+ oligodendrocytes in the brain and spinal cord (arrows) of an OTUB1fl/fl and a GFAP-Cre OTUB1fl/fl mouse express OTUB1. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-NogoA (Alexa Fluor 488). GFAP+ astrocytes are of normal morphology and distribution in the brain and spinal cord of a non-immunized OTUB1fl/fl mouse and a GFAP-Cre OTUB1fl/fl mouse. Note that GFAP+ astrocytes of an OTUB1fl/fl mouse do not express OTUB1. GFAP+ astrocytes of a GFAP-Cre OTUB1fl/fl mouse are also OTUB1-negative and represent a negative control for OTUB1 staining. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-GFAP (FITC).


Figure 3Activated GFAP + astrocytes in the spinal cord upregulate OTUB1 in EAE at maximal disease activity ( day 15 p . i . ) in an OTUB1fl / fl mouse ( arrowheads ) , but not in a GFAP - Cre OTUB1fl / fl mouse . Note the OTUB1 - expressing neuron in an OTUB1fl / fl mouse which is surrounded by GFAP - expressing processes of an activated astrocyte ( asterisk ) . At day 22 p . i . , astrocytes have downregulated OTUB1 expression while neurons express OTUB1 in an OTUB1fl / fl mouse ( asterisk ) . Astrocytes in a GFAP - Cre OTUB1fl / fl mouse are OTUB1 - negative while neurons express OTUB1 ( asterisk ) . At day 22 p . i . , demyelination in a GFAP - Cre OTUB1fl / fl mouse is much more severe and extended as compared to an OTUB1fl / fl mouse . Inflammation persists in the GFAP - Cre OTUB1fl / fl mouse , whereas it has resolved in an OTUB1fl / fl mouse . All photographs are representative of three mice per group ; original magnification × 400 ; scale bars correspond to 50 µm . ( A , C ) Double immunofluorescence with rabbit anti - OTUB1 ( Cy3 ) and mouse anti - GFAP ( FITC ) . ( B , D ) CV - LFB staining . EAE was induced in GFAP - Cre OTUB1fl / fl mice ( n = 29 ) and OTUB1fl / fl control littermates ( n = 29 ) by MOG35 - 55 peptide immunization with pertussis toxin . Graph represents data pooled from 4 experiments with 7 - 8 mice per group and shows the mean clinical scores + SEM . Statistical analysis performed using Mann - Whitney U test ; * p < 0 . 05 . EAE was induced in GFAP - Cre OTUB1fl / fl mice ( n = 12 ) and OTUB1fl / fl control littermates ( n = 12 ) by MOG35 - 55 peptide immunization without pertussis toxin . Graph represents the mean clinical scores + SEM . Statistical analysis performed using Mann - Whitney U test ; * p < 0 . 05 .

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Figure 3Activated GFAP+ astrocytes in the spinal cord upregulate OTUB1 in EAE at maximal disease activity (day 15 p.i.) in an OTUB1fl/fl mouse (arrowheads), but not in a GFAP-Cre OTUB1fl/fl mouse. Note the OTUB1-expressing neuron in an OTUB1fl/fl mouse which is surrounded by GFAP-expressing processes of an activated astrocyte (asterisk). At day 22 p.i., astrocytes have downregulated OTUB1 expression while neurons express OTUB1 in an OTUB1fl/fl mouse (asterisk). Astrocytes in a GFAP-Cre OTUB1fl/fl mouse are OTUB1-negative while neurons express OTUB1 (asterisk). At day 22 p.i., demyelination in a GFAP-Cre OTUB1fl/fl mouse is much more severe and extended as compared to an OTUB1fl/fl mouse. Inflammation persists in the GFAP-Cre OTUB1fl/fl mouse, whereas it has resolved in an OTUB1fl/fl mouse. All photographs are representative of three mice per group; original magnification ×400; scale bars correspond to 50 µm. (A, C) Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-GFAP (FITC). (B, D) CV-LFB staining.EAE was induced in GFAP-Cre OTUB1fl/fl mice (n = 29) and OTUB1fl/fl control littermates (n = 29) by MOG35-55 peptide immunization with pertussis toxin. Graph represents data pooled from 4 experiments with 7-8 mice per group and shows the mean clinical scores + SEM. Statistical analysis performed using Mann-Whitney U test; * p < 0.05. EAE was induced in GFAP-Cre OTUB1fl/fl mice (n = 12) and OTUB1fl/fl control littermates (n = 12) by MOG35-55 peptide immunization without pertussis toxin. Graph represents the mean clinical scores + SEM. Statistical analysis performed using Mann-Whitney U test; * p < 0.05.


Figure 4Percentages of CD4 + and CD8 + T cells infiltrating the spinal cord of OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice were analyzed by flow cytometry at day 15 ( n = 7 for OTUB1fl / fl group ; n = 9 for GFAP - Cre OTUB1fl / fl group ) and 22 ( n = 4 for OTUB1fl / fl group ; n = 5 for GFAP - Cre OTUB1fl / fl group ) p . i . Representative dot plots are shown . Absolute number of infiltrating leukocytes in the spinal cord of OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice at day 15 ( n = 7 for OTUB1fl / fl group ; n = 9 for GFAP - Cre OTUB1fl / fl group ) and 22 ( n = 4 for OTUB1fl / fl group ; n = 5 for GFAP - Cre OTUB1fl / fl group ) p . i . was calculated based on flow cytometry results . Data show mean + SEM . Infiltrating CD4 + T cells were further analyzed by flow cytometry for their ability to produce GM - CSF , IFN - γ , and IL - 17 at day 15 and 22 p . i . , respectively . Representative dot plots are shown . Absolute number of infiltrating GM - CSF - , IFN - γ - , and IL - 17 - producing CD4 + T cells at day 15 ( n = 7 for OTUB1fl / fl group ; n = 9 for GFAP - Cre OTUB1fl / fl group ) and 22 ( n = 4 for OTUB1fl / fl group ; n = 5 for GFAP - Cre OTUB1fl / fl group ) p . i . was calculated based on flow cytometry results ( mean + SEM ) . Relative expression of GM - CSF , IFN - γ , IL - 17 , TNF , IL - 6 , CXCL1 , CXCL10 , CXCL11 , CCL2 and NOS2 mRNA in the spinal cord of OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice at day 15 and 22 p . i . was determined by quantitative real - time PCR . Data are presented as the relative increase over unimmunized control mice ( n = 3 for all groups ) . Data show mean + SEM . Astrocytes were isolated from unimmunized and diseased OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice at day 15 p . i . , respectively . Relative expression of CXCL10 , CXCL11 , CCL2 , and NOS2 mRNA was determined by quantitative real - time PCR . Data are presented as relative increase of genes at day 15 p . i . over gene expression of astrocytes from unimmunized control mice ( n = 3 for all groups ) . Data show mean + SEM . Primary astrocytes isolated from OTUB1fl / fl ( n = 3 ) and GFAP - Cre OTUB1fl / fl ( n = 3 ) mice were left untreated or stimulated with IFN - γ ( 10 ng / ml ) , IL - 17 ( 50 ng / ml ) , and TNF ( 10 ng / ml ) , respectively , for 16 h . mRNA levels of CXCL10 , CXCL11 , CCL2 , and NOS2 were detected by quantitative real - time PCR .

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Figure 4Percentages of CD4+ and CD8+ T cells infiltrating the spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were analyzed by flow cytometry at day 15 (n = 7 for OTUB1fl/fl group; n = 9 for GFAP-Cre OTUB1fl/fl group) and 22 (n = 4 for OTUB1fl/fl group; n = 5 for GFAP-Cre OTUB1fl/fl group) p.i. Representative dot plots are shown.Absolute number of infiltrating leukocytes in the spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice at day 15 (n = 7 for OTUB1fl/fl group; n = 9 for GFAP-Cre OTUB1fl/fl group) and 22 (n = 4 for OTUB1fl/fl group; n = 5 for GFAP-Cre OTUB1fl/fl group) p.i. was calculated based on flow cytometry results. Data show mean + SEM.Infiltrating CD4+ T cells were further analyzed by flow cytometry for their ability to produce GM-CSF, IFN-γ, and IL-17 at day 15 and 22 p.i., respectively. Representative dot plots are shown. Absolute number of infiltrating GM-CSF-, IFN-γ-, and IL-17-producing CD4+ T cells at day 15 (n = 7 for OTUB1fl/fl group; n = 9 for GFAP-Cre OTUB1fl/fl group) and 22 (n = 4 for OTUB1fl/fl group; n = 5 for GFAP-Cre OTUB1fl/fl group) p.i. was calculated based on flow cytometry results (mean + SEM).Relative expression of GM-CSF, IFN-γ, IL-17, TNF, IL-6, CXCL1, CXCL10, CXCL11, CCL2 and NOS2 mRNA in the spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice at day 15 and 22 p.i. was determined by quantitative real-time PCR. Data are presented as the relative increase over unimmunized control mice (n = 3 for all groups). Data show mean + SEM.Astrocytes were isolated from unimmunized and diseased OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice at day 15 p.i., respectively. Relative expression of CXCL10, CXCL11, CCL2, and NOS2 mRNA was determined by quantitative real-time PCR. Data are presented as relative increase of genes at day 15 p.i. over gene expression of astrocytes from unimmunized control mice (n = 3 for all groups). Data show mean + SEM.Primary astrocytes isolated from OTUB1fl/fl (n = 3) and GFAP-Cre OTUB1fl/fl (n = 3) mice were left untreated or stimulated with IFN-γ (10 ng/ml), IL-17 (50 ng/ml), and TNF (10 ng/ml), respectively, for 16 h. mRNA levels of CXCL10, CXCL11, CCL2, and NOS2 were detected by quantitative real-time PCR.


Figure 5Primary astrocytes from OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice were treated with IFN - γ ( 10 ng / ml ) for indicated times and analyzed by western blot with indicated antibodies . Primary astrocytes from OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice were treated with IFN - γ ( 10 ng / ml ) for indicated times . Cytoplasmic and nuclear extracts were separated and analyzed by WB with indicated antibodies . Astrocytes were selectively isolated by MACS from OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice at days 0 and 15 p . i . , respectively . Relative expression of STAT1 mRNA was determined by quantitative real - time PCR . Data show the relative increase of STAT1 mRNA over that of unimmunized control mice ( n = 3 for all groups ) ( mean + SEM ) . Primary astrocytes from OTUB1fl / fl ( n = 3 ) and GFAP - Cre OTUB1fl / fl ( n = 3 ) mice were stimulated with IFN - γ ( 10 ng / ml ) for indicated times . mRNA was isolated and analyzed by quantitative real - time PCR for SOCS1 . Data show the relative increase over untreated controls ( mean + SEM ) . Primary astrocytes from wild type mice were stimulated with IFN - γ ( 10 ng / ml ) for indicated times . Proteins were immunoprecipitated from whole cell lysates with anti - OTUB1 antibody and analyzed by WB with antibodies against JAK1 , JAK2 , SOCS1 , and OTUB1 . Primary astrocytes from OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice were left untreated or treated with IFN - γ ( 10 ng / ml ) for 30 min . Proteins were immunoprecipitated with anti - SOCS1 antibody and analyzed by WB for SOCS1 , OTUB1 , and K48 Ubiquitin . Primary astrocytes from GFAP - Cre OTUB1fl / fl mice were cotransfected with SOCS1 - MYC , K48 ubiquitin - HA , and OTUB1 - GFP / OTUB1 - ∆ N - GFP / OTUB1 - C91S - GFP plasmids . Twenty - four hours after transfection , cells were left untreated or stimulated with IFN - γ ( 10 ng / ml ) for 30 min before lysation . Proteins were immunoprecipitated with anti - MYC antibody and analyzed by WB for MYC , GFP , and HA .

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Figure 5Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were treated with IFN-γ (10 ng/ml) for indicated times and analyzed by western blot with indicated antibodies.Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were treated with IFN-γ (10 ng/ml) for indicated times. Cytoplasmic and nuclear extracts were separated and analyzed by WB with indicated antibodies.Astrocytes were selectively isolated by MACS from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice at days 0 and 15 p.i., respectively. Relative expression of STAT1 mRNA was determined by quantitative real-time PCR. Data show the relative increase of STAT1 mRNA over that of unimmunized control mice (n = 3 for all groups) (mean + SEM).Primary astrocytes from OTUB1fl/fl (n = 3) and GFAP-Cre OTUB1fl/fl (n = 3) mice were stimulated with IFN-γ (10 ng/ml) for indicated times. mRNA was isolated and analyzed by quantitative real-time PCR for SOCS1. Data show the relative increase over untreated controls (mean + SEM).Primary astrocytes from wild type mice were stimulated with IFN-γ (10 ng/ml) for indicated times. Proteins were immunoprecipitated from whole cell lysates with anti-OTUB1 antibody and analyzed by WB with antibodies against JAK1, JAK2, SOCS1, and OTUB1.Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were left untreated or treated with IFN-γ (10 ng/ml) for 30 min. Proteins were immunoprecipitated with anti-SOCS1 antibody and analyzed by WB for SOCS1, OTUB1, and K48 Ubiquitin.Primary astrocytes from GFAP-Cre OTUB1fl/fl mice were cotransfected with SOCS1-MYC, K48 ubiquitin-HA, and OTUB1-GFP/OTUB1-∆N-GFP/OTUB1-C91S-GFP plasmids. Twenty-four hours after transfection, cells were left untreated or stimulated with IFN-γ (10 ng/ml) for 30 min before lysation. Proteins were immunoprecipitated with anti-MYC antibody and analyzed by WB for MYC, GFP, and HA.


Figure 6Primary astrocytes from OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice were transfected with SOCS1 - MYC plasmids . Twenty - four hours after transfection , cells were treated with cycloheximide ( CHX , 100 μg / ml ) for indicated times . SOCS1 protein levels were analyzed by WB ( upper panel ) . The lower panel shows the relative protein levels of SOCS1 normalized to Tubulin ( n = 3 for both groups ) . Primary astrocytes from GFAP - Cre OTUB1fl / fl mice were transfected with SOCS1 - MYC plasmids or cotransfected with SOCS1 - MYC + OTUB1 - GFP plasmids . Twenty - four hours after transfection , cells were treated with CHX ( 100 μg / ml ) for indicated times . SOCS1 protein levels were analyzed by WB ( upper panel ) . The lower panel shows the relative protein levels of SOCS1 normalized to Tubulin ( n = 3 for both groups ) ( mean + SEM ) . Primary astrocytes from GFAP - Cre OTUB1fl / fl mice were cotransfected with SOCS1 - MYC and OTUB1 - GFP / OTUB1 - ∆ N - GFP / OTUB1 - C91S - GFP plasmids . Twenty - four hours after transfection , cells were treated with CHX ( 100 μg / ml ) for indicated times . SOCS1 protein levels were analyzed by WB ( left panel ) . The right panel shows the relative protein levels of SOCS1 normalized to Tubulin ( n = 4 for both groups ) . Data represent mean + SEM . Primary astrocytes from OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice were stimulated with IFN - γ ( 10 ng / ml ) for four hours . Thereafter , cells were treated with CHX ( 100 μg / ml ) for indicated times . Whole cell lysates were analyzed by WB with indicated antibodies . Primary astrocytes from OTUB1fl / fl mice were transfected with SOCS1 - MYC plasmids . Five hours later , astrocytes were subsequently transfected with different siRNAs or left untreated . Twenty - four hours after siRNA transfection , protein was isolated and analyzed by WB for SOCS1 expression . Primary astrocytes from OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice were transfected with nonsense siRNA and SOCS1 siRNA - 2 . Twenty - four hours after transfection , cells were stimulated with IFN - γ ( 10 μg / ml ) for 16 hours before mRNA isolation . Quantitative real - time PCR was performed to detect mRNA levels of CXCL10 , CCL2 , and NOS2 ( n = 4 for all groups ) . Data show mean + SEM .

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Figure 6Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were transfected with SOCS1-MYC plasmids. Twenty-four hours after transfection, cells were treated with cycloheximide (CHX, 100 μg/ml) for indicated times. SOCS1 protein levels were analyzed by WB (upper panel). The lower panel shows the relative protein levels of SOCS1 normalized to Tubulin (n = 3 for both groups).Primary astrocytes from GFAP-Cre OTUB1fl/fl mice were transfected with SOCS1-MYC plasmids or cotransfected with SOCS1-MYC + OTUB1-GFP plasmids. Twenty-four hours after transfection, cells were treated with CHX (100 μg/ml) for indicated times. SOCS1 protein levels were analyzed by WB (upper panel). The lower panel shows the relative protein levels of SOCS1 normalized to Tubulin (n = 3 for both groups) (mean + SEM).Primary astrocytes from GFAP-Cre OTUB1fl/fl mice were cotransfected with SOCS1-MYC and OTUB1-GFP/OTUB1-∆N-GFP/OTUB1-C91S-GFP plasmids. Twenty-four hours after transfection, cells were treated with CHX (100 μg/ml) for indicated times. SOCS1 protein levels were analyzed by WB (left panel). The right panel shows the relative protein levels of SOCS1 normalized to Tubulin (n = 4 for both groups). Data represent mean + SEM.Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were stimulated with IFN-γ (10 ng/ml) for four hours. Thereafter, cells were treated with CHX (100 μg/ml) for indicated times. Whole cell lysates were analyzed by WB with indicated antibodies.Primary astrocytes from OTUB1fl/fl mice were transfected with SOCS1-MYC plasmids. Five hours later, astrocytes were subsequently transfected with different siRNAs or left untreated. Twenty-four hours after siRNA transfection, protein was isolated and analyzed by WB for SOCS1 expression.Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were transfected with nonsense siRNA and SOCS1 siRNA-2. Twenty-four hours after transfection, cells were stimulated with IFN-γ (10 μg/ml) for 16 hours before mRNA isolation. Quantitative real-time PCR was performed to detect mRNA levels of CXCL10, CCL2, and NOS2 (n = 4 for all groups). Data show mean + SEM.


Figure 7Primary astrocytes from OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice were treated with IFN - β ( 10 ng / ml ) for indicated times . Whole cell lysates were analyzed by WB with indicated antibodies . Primary astrocytes from OTUB1fl / fl ( n = 3 ) and GFAP - Cre OTUB1fl / fl ( n = 3 ) mice were treated with IFN - β ( 10 ng / ml ) for 16 hours . Quantitative real - time PCR was performed to detect OTUB1 ( B ) , CXCL10 ( C ) , CXCL11 ( D ) , CCL2 ( E ) , and NOS2 ( F ) mRNA levels . Data show mean + SEM .

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Figure 7Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were treated with IFN-β (10 ng/ml) for indicated times. Whole cell lysates were analyzed by WB with indicated antibodies.Primary astrocytes from OTUB1fl/fl (n = 3) and GFAP-Cre OTUB1fl/fl (n = 3) mice were treated with IFN-β (10 ng/ml) for 16 hours. Quantitative real-time PCR was performed to detect OTUB1 (B), CXCL10 (C), CXCL11 (D), CCL2 (E), and NOS2 (F) mRNA levels. Data show mean + SEM.


Figure 1Sucrose gradient UV profile of 60S / 40S / IAPV - IRES reaction mixture after an overnight run . The peaks corresponding to 80S and 40S were used for RNA extraction and UREA - PAGE shown in the inset . Representative cryo - EM image where roughly half of the particles correspond to 40S ( blue ) and the other half to 80S ( orange ) .

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Figure 1Sucrose gradient UV profile of 60S/40S/IAPV-IRES reaction mixture after an overnight run. The peaks corresponding to 80S and 40S were used for RNA extraction and UREA-PAGE shown in the inset.Representative cryo-EM image where roughly half of the particles correspond to 40S (blue) and the other half to 80S (orange).


Figure 1Size exclusion chromatography profiles of recombinant proteins used in this study . AU : arbitrary units ; WT : wild type ; FL : full - length .

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Figure 1Size exclusion chromatography profiles of recombinant proteins used in this study. AU: arbitrary units; WT: wild type; FL: full-length.


Figure 2Analysis of the interaction between CtUba4 and GST - CtUrm1 ( ratio 2 : 1 or 0 . 5 : 1 when marked with an asterisk ) in the presence of nucleotide derivates by GST pull - down resolved by SDS - PAGE and visualized with Coomassie stain . Functional analyses of ScUba4 mutant yeast using APM - gel retardation ( top ) and viability in response to rapamycin ( bottom ) . PAGE was supplemented with APM to retard the migration of thiolated tRNAs and allow their visualization by northern blot using an anti - tRNA < sub > UU < em > C < / em > < / sub > < sup > Gl < em > u < / em > < / sup > ; probe . APM : ( [ N - Acryloyl - amino ] phenyl ) mercuric chloride ; YPD : yeast extract peptone dextrose . All residue numbering follows the CtUba4 sequence , but the respective ScUba4 numbering is added in subscript .

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Figure 2Analysis of the interaction between CtUba4 and GST-CtUrm1 (ratio 2:1 or 0.5:1 when marked with an asterisk) in the presence of nucleotide derivates by GST pull-down resolved by SDS-PAGE and visualized with Coomassie stain.Functional analyses of ScUba4 mutant yeast using APM-gel retardation (top) and viability in response to rapamycin (bottom). PAGE was supplemented with APM to retard the migration of thiolated tRNAs and allow their visualization by northern blot using an anti-tRNAUUCGlu; probe. APM: ([N-Acryloyl-amino]phenyl)mercuric chloride; YPD: yeast extract peptone dextrose. All residue numbering follows the CtUba4 sequence, but the respective ScUba4 numbering is added in subscript.