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 1FACS gating strategy representative of 5 independent experiments adopted to sort CD11b + CD45int microglia distinctly from CD11b + CD45high resident macrophages and CD11b - CD45high lymphocytes . Analysis of relative transcript levels of CD11b + CD45int FACS - sorted microglia compared with whole brain tissue by qPCR . Gene expression levels of microglia ( Olfml3 , Fcrls , Tmem119 , Siglech , Gpr34 , P2ry12 ) , astrocyte ( Gfap , Gjb6 , Ntsr2 , Aldh1l1 ) , oligodendrocyte ( Mobp , Mog , Cldn1 ) , and neuron ( Tubb3 , Vglut1 , NeuN ) markers . Bars represent mean ( n = 4 ; pool of 1 female and 1 male per biological replicate ) of relative expression ( Gapdh as housekeeping gene ) ± SEM ( * p < 0 . 05 ; * * p < 0 . 01 by two - tailed Student ' s test ) . N . D . not detected . Representative quantification of CD206 expression in CD11b + CD45int microglia and CD11b + CD45high resident macrophages . Values denote the percentage of the mean ± SEM of 5 independent experiments . Representative images of 2 independent experiments showing microglia , resident macrophages and lymphocytes acquired with ImageStream imaging cytometer ( Amnis ) based on CD45 , CD11b and CD206 expression levels ( scale bar represents 7 µm ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O 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 1FACS gating strategy representative of 5 independent experiments adopted to sort CD11b+CD45int microglia distinctly from CD11b+CD45high resident macrophages and CD11b-CD45high lymphocytes.Analysis of relative transcript levels of CD11b+CD45int FACS-sorted microglia compared with whole brain tissue by qPCR. Gene expression levels of microglia (Olfml3, Fcrls, Tmem119, Siglech, Gpr34, P2ry12), astrocyte (Gfap, Gjb6, Ntsr2, Aldh1l1), oligodendrocyte (Mobp, Mog, Cldn1), and neuron (Tubb3, Vglut1, NeuN) markers. Bars represent mean (n=4; pool of 1 female and 1 male per biological replicate) of relative expression (Gapdh as housekeeping gene) ± SEM (*p < 0.05; **p < 0.01 by two-tailed Student's test). N.D. not detected.Representative quantification of CD206 expression in CD11b+CD45int microglia and CD11b+CD45high resident macrophages. Values denote the percentage of the mean ± SEM of 5 independent experiments.Representative images of 2 independent experiments showing microglia, resident macrophages and lymphocytes acquired with ImageStream imaging cytometer (Amnis) based on CD45, CD11b and CD206 expression levels (scale bar represents 7 µm).


Figure 2Three - four months old BL / 6 mice were treated with an acute dose of LPS ( 4 µg / g body ) or vehicle ( saline ) . Microglia ( pool of 2 mice per group per replicate ; 1 female and 1 male ) were FACS - sorted 24 hours later . Gene expression levels of microglia homeostatic ( Olfml3 , Fcrls , Tmem119 , Siglech , Gpr34 , P2ry12 , Mef2c ) , phagocytic ( Tyrobp , Trem2 ) , and inflammatory ( Il1β , Tnf , Ccl2 , Mrc1 , Arg1 ) markers were analyzed by qPCR . Bars represent mean of relative expression ( % of saline ; Gapdh as housekeeping gene ) ± SEM ( * p < 0 . 05 ; * * p < 0 . 01 by two - tailed Student ' s test ; n = 4 ) . Three - four months old BL / 6 mice were treated with an acute dose of LPS ( 4 µg / g body ) or vehicle ( saline ) . Microglia ( pool of 2 mice per group per replicate ; 1 female and 1 male ) were FACS - sorted 24 hours later . Representative multicolour flow cytometry analysis of 5 independent experiments showing CD11b and CD45 positive populations in single viable cells in saline or LPS - injected mouse brains . Three - four months old BL / 6 mice were treated with an acute dose of LPS ( 4 µg / g body ) or vehicle ( saline ) . Microglia ( pool of 2 mice per group per replicate ; 1 female and 1 male ) were FACS - sorted 24 hours later . Representative multicolour flow cytometry analysis showing the percentage of the mean ± SEM of 5 independent experiments of Ly6C and CD206 expressing cells in CD11b + CD45int cells from saline or LPS - injected mice . Three - four months old BL / 6 mice were treated with an acute dose of LPS ( 4 µg / g body ) or vehicle ( saline ) . Microglia ( pool of 2 mice per group per replicate ; 1 female and 1 male ) were FACS - sorted 24 hours later . Gene expression levels of the monocytic markers Ly6c1 and Ccr2 in purified microglia ( n = 4 ) and isolated bone marrow monocytes ( n = 2 ) by qPCR . Bars represent mean of relative expression ( Gapdh as housekeeping gene ) ± SEM ( * * p < 0 . 01 by two - tailed Student ' s test ) . Primary adult microglia were cultivated in the presence of TGF - β ( 50 μg / ml ) and M - CSF ( 10 ng / ml ) , while neonatal cells were stimulated for 24 hours with TGF - β ( 50 μg / ml ) followed by 6 hours stimulation with LPS ( 1 ng / mL ) or left untreated . Expression levels of microglia homeostatic ( Olfml3 , Tmem119 , Gpr34 ) and inflammatory ( Il1β , Tnf , Ccl2 ) genes were analysed by qPCR . Bars represent mean of relative expression ( Gapdh as housekeeping gene ) ± SEM ( * * p < 0 . 01 by two - tailed Student ' s test ; 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Three-four months old BL/6 mice were treated with an acute dose of LPS (4 µg/g body) or vehicle (saline). Microglia (pool of 2 mice per group per replicate; 1 female and 1 male) were FACS-sorted 24 hours later. Gene expression levels of microglia homeostatic (Olfml3, Fcrls, Tmem119, Siglech, Gpr34, P2ry12, Mef2c), phagocytic (Tyrobp, Trem2), and inflammatory (Il1β, Tnf, Ccl2, Mrc1, Arg1) markers were analyzed by qPCR. Bars represent mean of relative expression (% of saline; Gapdh as housekeeping gene) ± SEM (*p < 0.05; **p < 0.01 by two-tailed Student's test; n=4).Three-four months old BL/6 mice were treated with an acute dose of LPS (4 µg/g body) or vehicle (saline). Microglia (pool of 2 mice per group per replicate; 1 female and 1 male) were FACS-sorted 24 hours later. Representative multicolour flow cytometry analysis of 5 independent experiments showing CD11b and CD45 positive populations in single viable cells in saline or LPS-injected mouse brains.Three-four months old BL/6 mice were treated with an acute dose of LPS (4 µg/g body) or vehicle (saline). Microglia (pool of 2 mice per group per replicate; 1 female and 1 male) were FACS-sorted 24 hours later. Representative multicolour flow cytometry analysis showing the percentage of the mean ± SEM of 5 independent experiments of Ly6C and CD206 expressing cells in CD11b+CD45int cells from saline or LPS-injected mice.Three-four months old BL/6 mice were treated with an acute dose of LPS (4 µg/g body) or vehicle (saline). Microglia (pool of 2 mice per group per replicate; 1 female and 1 male) were FACS-sorted 24 hours later. Gene expression levels of the monocytic markers Ly6c1 and Ccr2 in purified microglia (n=4) and isolated bone marrow monocytes (n=2) by qPCR. Bars represent mean of relative expression (Gapdh as housekeeping gene) ± SEM (**p < 0.01 by two-tailed Student's test).Primary adult microglia were cultivated in the presence of TGF-β (50 μg/ml) and M-CSF (10 ng/ml), while neonatal cells were stimulated for 24 hours with TGF-β (50 μg/ml) followed by 6 hours stimulation with LPS (1 ng/mL) or left untreated. Expression levels of microglia homeostatic (Olfml3, Tmem119, Gpr34) and inflammatory (Il1β, Tnf, Ccl2) genes were analysed by qPCR. Bars represent mean of relative expression (Gapdh as housekeeping gene) ± SEM (**p < 0.01 by two-tailed Student's test; n=3).


Figure 3Heatmap showing clustering analysis of 1 , 247 single cells , featuring 100 most variable genes ( FDR < 0 . 05 ) . Single - cell RNA - seq results are obtained from 2 mice per group ( 1 female and 1 male each ) . Expression of specific homeostatic ( Tmem119 , P2ry12 , Siglech ) and inflammatory ( Ccl2 , Gpr84 ) genes overlaid on the 2D - tSNE space . Bars represent log2 ( Count + 1 ) . Representative multicolour flow cytometry analysis of 2 independent experiments showing TMEM119 and P2RY12 expression levels in CD11b + CD45int microglia of saline or LPS - injected mouse brains . For the unconjugated TMEM119 antibody , negative stands for primary antibody without secondary antibody . For P2RY12 antibody , negative represents isotype PE control .

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Figure 3Heatmap showing clustering analysis of 1,247 single cells, featuring 100 most variable genes (FDR<0.05). Single-cell RNA-seq results are obtained from 2 mice per group (1 female and 1 male each).Expression of specific homeostatic (Tmem119, P2ry12, Siglech) and inflammatory (Ccl2, Gpr84) genes overlaid on the 2D-tSNE space. Bars represent log2 (Count+1).Representative multicolour flow cytometry analysis of 2 independent experiments showing TMEM119 and P2RY12 expression levels in CD11b+CD45int microglia of saline or LPS-injected mouse brains. For the unconjugated TMEM119 antibody, negative stands for primary antibody without secondary antibody. For P2RY12 antibody, negative represents isotype PE control.


Figure 4Heatmap showing examples of specific genes mainly up - regulated in " main LPS " ( Manf ) or " subset LPS " ( Ash1l ) and down - regulated in " main LPS " ( Mef2c ) or " subset LPS " ( Lamp1 ) overlaid on the 2D - tSNE space . Bars represent log2 ( Count + 1 ) .

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Figure 4Heatmap showing examples of specific genes mainly up-regulated in "main LPS" (Manf) or "subset LPS" (Ash1l) and down-regulated in "main LPS" (Mef2c) or "subset LPS" (Lamp1) overlaid on the 2D-tSNE space. Bars represent log2 (Count+1).


Figure 5Pseudotime dynamics of inflammatory ( Ccl12 , Ccl2 , Gpr84 ) and homeostatic ( Mef2c , P2ry12 , Siglech ) genes in dependence on inferred cell states .

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Figure 5Pseudotime dynamics of inflammatory (Ccl12, Ccl2, Gpr84) and homeostatic (Mef2c, P2ry12, Siglech) genes in dependence on inferred cell states.


Figure 1 ( A ) HeLa cells stably expressing GFP - Parkin ( HeLa - GFP - Parkin cells ) were treated with 1 µM valinomycin and immunostained with anti - NDP52 and anti - Tom20 antibodies . Boxed areas in the images are shown in the next panels on the right . Colocalization of GFP - Parkin , NDP52 , and Tom20 were determined using Line scan . Fluorescence intensities of each channels were measured along the dotted white arrow . Scale bars , 10 µm . Images are representative of three independent experiments ( B ) HeLa - GFP - Parkin cells were transfected with siNDP52 or scrambled siRNA . Seventy - two hours after transfection , cells were incubated with 1 µM valinomycin for the indicated times . Western blotting was performed using the indicated antibodies . Data are representative of three independent experiments ( C ) Mitophagy flux assay using Mtphagy dye . Increase in Mtphagy dye fluorescence intensity indicates the recruitment of mitochondria to lysosomes during valinomycin treatment . Results are a summary of three independent experiments . Values are the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 compared with scrambled oligo - treated cells , determined with one - way ANOVA followed by the Student ' s t test ( D ) Fluorescence images of NDP52 KD and control cells expressing mCherry - LC3 and MTS - TagBFP . Arrows indicate mCherry - LC3 puncta colocalized with MTS - TagBFP . Scale bars , 10 µm . Boxed areas in images are shown in the next panels on the right . Images are representative of four independent experiments ( E ) The number of mCherry - LC3 puncta colocalized with MTS - TagBFP ( representing mitophagosomes ) per cell was counted . Results were from at least six microscopic fields of three independent experiments . Values are the means ± SEM . * * p < 0 . 01 compared with scrambled oligo - treated cells , determined with one - way ANOVA followed by the Student ' s t test ( F , G ) Electron micrographs of NDP52 KD and control cells . Asterisks indicate mitochondria sequestered by autophagosomes ( mitophagosomes , yellow dotted line ) . The ratios of mitophagosomes per mitochondrial area are shown in ( G ) . Ten cells from three independent experiments were counted . Values are the means ± SD . * p < 0 . 05 compared with scrambled - oligo treated cells , determined with one - way ANOVA followed by the Student ' s t

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Figure 1 (A) HeLa cells stably expressing GFP-Parkin (HeLa-GFP-Parkin cells) were treated with 1 µM valinomycin and immunostained with anti-NDP52 and anti-Tom20 antibodies. Boxed areas in the images are shown in the next panels on the right. Colocalization of GFP-Parkin, NDP52, and Tom20 were determined using Line scan. Fluorescence intensities of each channels were measured along the dotted white arrow. Scale bars, 10 µm. Images are representative of three independent experiments (B) HeLa-GFP-Parkin cells were transfected with siNDP52 or scrambled siRNA. Seventy-two hours after transfection, cells were incubated with 1 µM valinomycin for the indicated times. Western blotting was performed using the indicated antibodies. Data are representative of three independent experiments  (C) Mitophagy flux assay using Mtphagy dye. Increase in Mtphagy dye fluorescence intensity indicates the recruitment of mitochondria to lysosomes during valinomycin treatment. Results are a summary of three independent experiments. Values are the means ± SEM. *p < 0.05, **p < 0.01 compared with scrambled oligo-treated cells, determined with one-way ANOVA followed by the Student's t test  (D) Fluorescence images of NDP52 KD and control cells expressing mCherry-LC3 and MTS-TagBFP. Arrows indicate mCherry-LC3 puncta colocalized with MTS-TagBFP. Scale bars, 10 µm. Boxed areas in images are shown in the next panels on the right. Images are representative of four independent experiments  (E) The number of mCherry-LC3 puncta colocalized with MTS-TagBFP (representing mitophagosomes) per cell was counted. Results were from at least six microscopic fields of three independent experiments. Values are the means ± SEM. **p < 0.01 compared with scrambled oligo-treated cells, determined with one-way ANOVA followed by the Student's t test  (F, G) Electron micrographs of NDP52 KD and control cells. Asterisks indicate mitochondria sequestered by autophagosomes (mitophagosomes, yellow dotted line). The ratios of mitophagosomes per mitochondrial area are shown in (G). Ten cells from three independent experiments were counted. Values are the means ± SD. *p < 0.05 compared with scrambled-oligo treated cells, determined with one-way ANOVA followed by the Student's t test


Figure 2 ( Images of HeLa GFP - Parkin cells expressing mCherry - NDP52 ( A under valinomycin treatment . The images are from Movie EV 2 and 3 , respectively . Cyan , GFP - Parkin ; magenta , mCherry - NDP5 Scale bars , 10 µm . Images are representative of five independent experiments ( B ) Time - lapse images of HeLa GFP - Parkin cells expressing mCherry - NDP52 under valinomycin treatment . The images are representative of five independent experiments and correspond to the boxed regions of Figure 2A and Movie EV 2 . Cyan , GFP - Parkin ; magenta , mCherry - NDP52 . The white arrowheads indicate colocalization of GFP - Parkin and mCherry - NDP52 . The yellow arrowheads indicate mCherry - NDP existing inside GFP - Parkin ring structuresImages o HeLa GFP - Parkin cells expressin mCherry - OPTN ( C ) under valinomycin treatment . The images are from Movie EV 2 and 3 , respectively . Cyan , GFP - Parkin ; magenta OPTN . Scale bars , 10 µm . Images are representative of five independent experiments ( D ) mCherry - NDP52 or OPTN signals colocalized with Parkin - ring structures and existing inside of Parkin - ring structures ( irrupted ) were quantified . Data were from five independent experiments in cells incubated with valinomycin for 2 h . Values are the means ± SEM . * * p < 0 . 01 compared with mCherry - OPTN expressing cells , determined with one - way ANOVA followed by the Student " s t ( E ) Mitophagic flux in NDP52 KD cells expressing siRNA - resistant NDP52 or NDP52 mutants . Cells were incubated with valinomycin for 3 h . Results are the summary of three independent experiments . Values are the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 vs scrambled oligo - transfected cells ; † p < 0 . 05 , † † p < 0 . 01 vs empty vector - transfected cells , determined with one - way ANOVA followed by the Tukey - Kramer post hoc test ( F ) HeLa - GFP - Parkin cells expressing mCherry - NDP52 mutants were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h . Cell lysates were subjected to immunoprecipitation using anti - RFP magnetic beads . Immunoprecipitates were analyzed by western blotting . Data are representative of three independent experiments ( G ) Images of HeLa GFP - Parkin cells expressing mCherry - NDP52 ∆ LIM - L or mCherry - NDP52 ∆ SKICH treated with valinomycin for 2h . The images are from Movie EV 4 . Cyan , GFP - Parkin ; magenta , mCherry - NDP52 mutant . Scale bars , 10 µm . Images are representative of three independent

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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( Images of HeLa GFP-Parkin cells expressing mCherry-NDP52 (A under valinomycin treatment. The images are from Movie EV 2 and 3, respectively. Cyan, GFP-Parkin; magenta, mCherry-NDP5 Scale bars, 10 µm. Images are representative of five independent experiments(B) Time-lapse images of HeLa GFP-Parkin cells expressing mCherry-NDP52 under valinomycin treatment. The images are representative of five independent experiments and correspond to the boxed regions of Figure 2A and Movie EV 2. Cyan, GFP-Parkin; magenta, mCherry-NDP52. The white arrowheads indicate colocalization of GFP-Parkin and mCherry-NDP52. The yellow arrowheads indicate mCherry-NDP existing inside GFP-Parkin ring structuresImages o HeLa GFP-Parkin cells expressin mCherry-OPTN (C) under valinomycin treatment. The images are from Movie EV 2 and 3, respectively. Cyan, GFP-Parkin; magenta OPTN. Scale bars, 10 µm. Images are representative of five independent experiments(D) mCherry-NDP52 or OPTN signals colocalized with Parkin-ring structures and existing inside of Parkin-ring structures (irrupted) were quantified. Data were from five independent experiments in cells incubated with valinomycin for 2 h. Values are the means ± SEM. **p < 0.01 compared with mCherry-OPTN expressing cells, determined with one-way ANOVA followed by the Student"s t(E) Mitophagic flux in NDP52 KD cells expressing siRNA-resistant NDP52 or NDP52 mutants. Cells were incubated with valinomycin for 3 h. Results are the summary of three independent experiments. Values are the means ± SEM. *p < 0.05, **p < 0.01 vs scrambled oligo-transfected cells; †p < 0.05, ††p < 0.01 vs empty vector-transfected cells, determined with one-way ANOVA followed by the Tukey-Kramer post hoc test(F) HeLa-GFP-Parkin cells expressing mCherry-NDP52 mutants were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h. Cell lysates were subjected to immunoprecipitation using anti-RFP magnetic beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments(G) Images of HeLa GFP-Parkin cells expressing mCherry-NDP52∆LIM-L or mCherry-NDP52∆SKICH treated with valinomycin for 2h. The images are from Movie EV 4. Cyan, GFP-Parkin; magenta, mCherry-NDP52 mutant. Scale bars, 10 µm. Images are representative of three independent experiments


Figure 3 ( A ) Mitophagic flux in cells transfected with siNDP52 , siTBK1 , siOPTN or a combination of siRNAs . Results are the summary of three independent experiments . Values are the means ± SEM . * * p < 0 . 01 vs scrambled oligo - transfected cells ; † p < 0 . 05 vs siNDP52 - transfected cells ; § p < 0 . 05 , § § p < 0 . 01 vs siTBK1 ; NS : not significant , determined with one - way ANOVA followed by the Tukey - Kramer post hoc test ( B ) HeLa - GFP - Parkin cells transfected with siNDP52 , siOPTN , siTBK1 or scrambled siRNA were incubated with 1 µM valinomycin for the indicated times . Cell lysates were subjected to SDS - PAGE or Phos - tag - PAGE prior to western blotting using the indicated antibodies . Data are representative of two independent experiments . The position of phosphorylated forms of NDP52 or OPTN are indicated by arrowheads ( C ) Images of TBK1 KD cells expressing mCherry - NDP52 or mCherry - OPTN under valinomycin treatment from Movie EV 5 . Cyan , GFP - Parkin ; magenta , mCherry - NDP52 . Images are representative of two independent experiments . Scale bars , 10 µm ( D ) TBK1 KD and control cells expressed mCherry - NDP52 . Lysates were subjected to immunoprecipitation using anti - RFP beads . Immunoprecipitates were analyzed by western blotting . Data are representative of three independent

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Figure 3(A) Mitophagic flux in cells transfected with siNDP52, siTBK1, siOPTN or a combination of siRNAs. Results are the summary of three independent experiments. Values are the means ± SEM. **p < 0.01 vs scrambled oligo-transfected cells; †p < 0.05 vs siNDP52-transfected cells; §p < 0.05, §§p < 0.01 vs siTBK1; NS: not significant, determined with one-way ANOVA followed by the Tukey-Kramer post hoc test(B) HeLa-GFP-Parkin cells transfected with siNDP52, siOPTN, siTBK1 or scrambled siRNA were incubated with 1 µM valinomycin for the indicated times. Cell lysates were subjected to SDS-PAGE or Phos-tag-PAGE prior to western blotting using the indicated antibodies. Data are representative of two independent experiments. The position of phosphorylated forms of NDP52 or OPTN are indicated by arrowheads(C) Images of TBK1 KD cells expressing mCherry-NDP52 or mCherry-OPTN under valinomycin treatment from Movie EV 5. Cyan, GFP-Parkin; magenta, mCherry-NDP52. Images are representative of two independent experiments. Scale bars, 10 µm(D) TBK1 KD and control cells expressed mCherry-NDP52. Lysates were subjected to immunoprecipitation using anti-RFP beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments


Figure 4Anti - RFP immunoprecipitates from mCherry - NDP52 and mCherry - NDP52 ∆ SKICH - transfected cells were subjected to SDS - PAGE followed by silver staining . Four bands specific for mCherry - NDP52 are numbered and were identified by mass spectrometric analysis . Asterisk ( * ) and double asterisk ( * * ) indicate mCherry - NDP52 and mCherry - NDP52 ∆ SKICH bands , respectively ( C ) HeLa - GFP - Parkin cells expressing mCherry - NDP52 mutants were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h . Cell lysates were subjected to immunoprecipitation using anti - RFP magnetic beads . Immunoprecipitates were analyzed by western blotting . Data are representative of three independent experiments ( D ) HeLa - GFP - Parkin cells transfected with siMTPAP were incubated with 1 µM valinomycin for the indicated times . Western blotting was performed using the indicated antibodies . Data are representative of three independent experiments ( E ) Mitophagic flux in cells transfected with siNDP52 , siMTPAP and combinations of siRNAs . Cells were incubated with valinomycin for 3 h . Results are the summary of three independent experiments . Values are the means ± SEM . * * p < 0 . 01 vs scrambled oligo - transfected cells ; NS : not significant , determined with one - way ANOVA followed by the Tukey - Kramer post hoc test ( F ) Images of MTPAP KD cells expressing mCherry - NDP52 under valinomycin treatment . The images are from Movie EV 7 . Cyan , GFP - Parkin ; magenta , mCherry - NDP52 . Images are representative of two independent experiments . Scale bars , 10

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Figure 4Anti-RFP immunoprecipitates from mCherry-NDP52 and mCherry-NDP52 ∆SKICH-transfected cells were subjected to SDS-PAGE followed by silver staining. Four bands specific for mCherry-NDP52 are numbered and were identified by mass spectrometric analysis. Asterisk (*) and double asterisk (**) indicate mCherry-NDP52 and mCherry-NDP52∆SKICH bands, respectively(C) HeLa-GFP-Parkin cells expressing mCherry-NDP52 mutants were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h. Cell lysates were subjected to immunoprecipitation using anti-RFP magnetic beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments(D) HeLa-GFP-Parkin cells transfected with siMTPAP were incubated with 1 µM valinomycin for the indicated times. Western blotting was performed using the indicated antibodies. Data are representative of three independent experiments(E) Mitophagic flux in cells transfected with siNDP52, siMTPAP and combinations of siRNAs. Cells were incubated with valinomycin for 3 h. Results are the summary of three independent experiments. Values are the means ± SEM. **p < 0.01 vs scrambled oligo-transfected cells; NS: not significant, determined with one-way ANOVA followed by the Tukey-Kramer post hoc test(F) Images of MTPAP KD cells expressing mCherry-NDP52 under valinomycin treatment. The images are from Movie EV 7. Cyan, GFP-Parkin; magenta, mCherry-NDP52. Images are representative of two independent experiments. Scale bars, 10 µm


Figure 5 ( A ) HeLa - GFP - Parkin cells were incubated with valinomycin for 0 or 2 h . Immunostaining was performed with anti - MTPAP and anti - Tom20 antibodies . Images are representative of two independent experiments . Scale bar , 10 µm ( B ) Mitochondrial - rich fraction prepared from HeLa - GFP - Parkin cells treated with or without valinomycin and epoxomicin for 2 h , was incubated with various concentrations of trypsin ( 0 - 1000 µg / ml ) for 30 min on ice . Protease protection was analyzed by western blotting using antibodies for mitochondrial marker proteins ( Tom20 for OMM , Tim23 for intermembrane space , UQCRC1 and TFAM for mitochondrial matrix ) . Data are representative of five independent experiments ( C ) HeLa - GFP - Parkin cells were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 0 - 3 h in the presence or absence of 10 µM epoxomicin . Cell lysates were subjected to immunoprecipitation using anti - NDP52 antibody or control IgG . Immunoprecipitates were analyzed by western blotting . Data are representative of five independent experiments . ( D ) Quantification of MTPAP co - immunoprecipitated with NDP52 in ( C ) . Results are a summary of five independent experiments . Values are the means ± SEM . * * p < 0 . 01 compared to cells treated with valinomycin and bafilomycin A1 for 3 h , determined with one - way ANOVA followed by the Tukey - Kramer post hoc test ( E ) HeLa - GFP - Parkin cells were incubated with 100 µM DFO and 100 nM bafilomycin A1 for 0 , 4 and 8 h in the presence or absence of epoxomicin . Cell lysates were subjected to immunoprecipitation using anti - NDP52 antibody . Immunoprecipitates were analyzed by western blotting . Data are representative of two independent experiments ( F ) HeLa cells and HEK293T cells were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h or 100 µM DFO and 100 nM bafilomycin A1 for 8 h . Cell lysates were subjected to immunoprecipitation using anti - NDP52 antibody . Immunoprecipitates were analyzed by western blotting . Data are representative of two independent experiments ( G ) Immunoelectron micrographs of HeLa - GFP - Parkin cells expressing FLAG - NDP52 . Cells treated for 2 h with valinomycin and bafilomycin A1 with or without epoxomicin , were processed for immune - electron microscopy with anti - FLAG antibody . Images are representative of ten cells . Arrow , immunogold labeled FLAG - NDP52 . Mito , mitochondrion ; MP , mitophagosome . Scale bars , 200 nm ( H ) Selected frames from time - lapse images of HeLa GFP - Parkin cells expressing mCherry - NDP52 under valinomycin and bafilomycin A1 treatment . The images are representative of two independent experiments and correspond to the boxed regions of Movie EV 6 . Cyan , GFP - Parkin ; magenta , mCherry - NDP52 . The white arrowheads indicate colocalization of GFP - Parkin and mCherry - NDP52 . The yellow arrowheads indicate mCherry - NDP existing inside GFP - Parkin ring structures . Scale bars , 1

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa-GFP-Parkin cells were incubated with valinomycin for 0 or 2 h. Immunostaining was performed with anti-MTPAP and anti-Tom20 antibodies. Images are representative of two independent experiments. Scale bar, 10 µm(B) Mitochondrial-rich fraction prepared from HeLa-GFP-Parkin cells treated with or without valinomycin and epoxomicin for 2 h, was incubated with various concentrations of trypsin (0-1000 µg/ml) for 30 min on ice. Protease protection was analyzed by western blotting using antibodies for mitochondrial marker proteins (Tom20 for OMM, Tim23 for intermembrane space, UQCRC1 and TFAM for mitochondrial matrix). Data are representative of five independent experiments(C) HeLa-GFP-Parkin cells were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 0-3 h in the presence or absence of 10 µM epoxomicin. Cell lysates were subjected to immunoprecipitation using anti-NDP52 antibody or control IgG. Immunoprecipitates were analyzed by western blotting. Data are representative of five independent experiments. (D) Quantification of MTPAP co-immunoprecipitated with NDP52 in (C). Results are a summary of five independent experiments. Values are the means ± SEM. **p < 0.01 compared to cells treated with valinomycin and bafilomycin A1 for 3 h, determined with one-way ANOVA followed by the Tukey-Kramer post hoc test(E) HeLa-GFP-Parkin cells were incubated with 100 µM DFO and 100 nM bafilomycin A1 for 0, 4 and 8 h in the presence or absence of epoxomicin. Cell lysates were subjected to immunoprecipitation using anti-NDP52 antibody. Immunoprecipitates were analyzed by western blotting. Data are representative of two independent experiments(F) HeLa cells and HEK293T cells were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h or 100 µM DFO and 100 nM bafilomycin A1 for 8 h. Cell lysates were subjected to immunoprecipitation using anti-NDP52 antibody. Immunoprecipitates were analyzed by western blotting. Data are representative of two independent experiments(G) Immunoelectron micrographs of HeLa-GFP-Parkin cells expressing FLAG-NDP52. Cells treated for 2 h with valinomycin and bafilomycin A1 with or without epoxomicin, were processed for immune-electron microscopy with anti-FLAG antibody. Images are representative of ten cells. Arrow, immunogold labeled FLAG-NDP52. Mito, mitochondrion; MP, mitophagosome. Scale bars, 200 nm(H) Selected frames from time-lapse images of HeLa GFP-Parkin cells expressing mCherry-NDP52 under valinomycin and bafilomycin A1 treatment. The images are representative of two independent experiments and correspond to the boxed regions of Movie EV 6. Cyan, GFP-Parkin; magenta, mCherry-NDP52. The white arrowheads indicate colocalization of GFP-Parkin and mCherry-NDP52. The yellow arrowheads indicate mCherry-NDP existing inside GFP-Parkin ring structures. Scale bars, 1 µm


Figure 6 ( A , B ) mCherry - LC3 and MTS - TagBFP were expressed in MTPAP KD and control cells . Cells were incubated with valinomycin for the indicated times and immunostained with anti - NDP52 antibody . Images are representative of two independent experiments . Yellow arrows indicate co - localization of MTS - TagBFP , mCherry - LC3 and NDP52 . White arrows indicate co - localization of MTS - TagBFP and NDP52 ( not mCherry - LC3 ) . Scale bars , 10 µm . Co - localization of mCherry - LC3 and NDP52 on mitochondria is quantified in ( B ) . At least ten cells from two independent experiments were quantified . Values are the means ± SEM . * * p < 0 . 01 vs control , determined with one - way ANOVA followed by the Student " s tMTPAP KD and control cells expressed mCherry - NDP52 or empty vector ( C Lysates from cells treated with 1 µM valinomycin and 100 nM bafilomycin A1 for 2 h were subjected to immunoprecipitation using anti - RFP beads . Immunoprecipitates were analyzed by western blotting . Data are representative of three independent experimentsMTPAP KD and control cells expresse mCherry - LC3 ( D ) . Lysates from cells treated with 1 µM valinomycin and 100 nM bafilomycin A1 for 2 h were subjected to immunoprecipitation using anti - RFP beads . Immunoprecipitates were analyzed by western blotting . Data are representative of three independent experiments ( E ) MTPAP KD and control cells were incubated with valinomycin and bafilomycin A1 for 2 h . Cell lysates were subjected to immunoprecipitation using an anti - NDP52 antibody or control IgG . Immunoprecipitates were analyzed by western blotting . Data are representative of three independent experiments ( F ) GST - NDP52 conjugated with glutathione - Sepharose resin was incubated with purified recombinant LC3 and MTPAP ∆ MTS ∆ C . The pulled down complex was subjected to western blotting . Data are representative of three independent

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O 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, B) mCherry-LC3 and MTS-TagBFP were expressed in MTPAP KD and control cells. Cells were incubated with valinomycin for the indicated times and immunostained with anti-NDP52 antibody. Images are representative of two independent experiments. Yellow arrows indicate co-localization of MTS-TagBFP, mCherry-LC3 and NDP52. White arrows indicate co-localization of MTS-TagBFP and NDP52 (not mCherry-LC3). Scale bars, 10 µm. Co-localization of mCherry-LC3 and NDP52 on mitochondria is quantified in (B). At least ten cells from two independent experiments were quantified. Values are the means ± SEM. **p < 0.01 vs control, determined with one-way ANOVA followed by the Student"s tMTPAP KD and control cells expressed mCherry-NDP52 or empty vector (C Lysates from cells treated with 1 µM valinomycin and 100 nM bafilomycin A1 for 2 h were subjected to immunoprecipitation using anti-RFP beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experimentsMTPAP KD and control cells expresse mCherry-LC3 (D). Lysates from cells treated with 1 µM valinomycin and 100 nM bafilomycin A1 for 2 h were subjected to immunoprecipitation using anti-RFP beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments(E) MTPAP KD and control cells were incubated with valinomycin and bafilomycin A1 for 2 h. Cell lysates were subjected to immunoprecipitation using an anti-NDP52 antibody or control IgG. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments(F) GST-NDP52 conjugated with glutathione-Sepharose resin was incubated with purified recombinant LC3 and MTPAP∆MTS∆C. The pulled down complex was subjected to western blotting. Data are representative of three independent experiments


Figure 7 ( A ) HeLa - GFP - Parkin cells expressing mCherry - NDP52 D439R / C443K mutant or WT NDP52 were incubated with valinomycin and nM bafilomycin A1 for 3 h . Cell lysates were subjected to immunoprecipitation using anti - RFP magnetic beads . Immunoprecipitates were analyzed by western blotting . Data are representative of two independent experiments ( B ) Selected frames from time - lapse images of HeLa GFP - Parkin cells expressing mCherry - NDP52 D439R / C443K mutant under valinomycin and bafilomycin A1 treatment . The images are representative of two independent experiments and correspond to the boxed regions of Movie EV 9 . Cyan , GFP - Parkin ; magenta , mCherry - NDP52 D439R / C443K . The white arrowheads indicate colocalization of GFP - Parkin and mCherry - NDP52 . The yellow arrowheads indicate mCherry - NDP existing inside GFP - Parkin ring structures . Scale bars , 1

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa-GFP-Parkin cells expressing mCherry-NDP52 D439R/C443K mutant or WT NDP52 were incubated with valinomycin and nM bafilomycin A1 for 3 h. Cell lysates were subjected to immunoprecipitation using anti-RFP magnetic beads. Immunoprecipitates were analyzed by western blotting. Data are representative of two independent experiments  (B) Selected frames from time-lapse images of HeLa GFP-Parkin cells expressing mCherry-NDP52 D439R/C443K mutant under valinomycin and bafilomycin A1 treatment. The images are representative of two independent experiments and correspond to the boxed regions of Movie EV 9. Cyan, GFP-Parkin; magenta, mCherry-NDP52 D439R/C443K. The white arrowheads indicate colocalization of GFP-Parkin and mCherry-NDP52. The yellow arrowheads indicate mCherry-NDP existing inside GFP-Parkin ring structures. Scale bars, 1 µm


Figure 2 ( A ) Confocal images of ALIX ( red ) , Asl ( white ) , α - tubulin ( green ) and Hoechst ( blue ) in wild type ( w1118 ) , Sas - 6c02901 , DSpdG02143 , cnnHK21 and γ - Tub23CPI NBs ( scale bars , 5 μm ) . The insets show higher magnification of the boxed centrosomes ( B ) The centrosomal fluorescence intensity of ALIX was measured and the graph shows the average intensity ( relative to w1118 intensity ) of three separate experiments ( ± S . E ) , where in total 78 w1118 , 73 Sas - 6c02901 , 45 DSpdG02143 , 61 cnnHK21 and 26 γ - Tub23CPI NBs were analyzed . Statistically significant differences are indicated ( * p < 0 . 05 and * * p < 0 . 01 , Student ' s t - test ) ( C ) GST or GST - ALIX were incubated with in vitro translated 35S - labelled Cnn or DSpd - 2 proteins . Binding was detected by autoradiography ( upper panel ) , and expression of GST and GST - ALIX was visualized by Ponceau S - staining ( lower panel ) ( D ) HeLa cells were transfected with control , Cep192 or Cep215 siRNA . Efficient depletion of Cep192 and Cep215 was confirmed by Western blotting analysis , which also showed unchanged expression levels of ALIX . Unspecific bands , below Cep192 and above Cep215 , are indicated with * * . ( E ) Control , Cep192 or Cep215 siRNA - transfected HeLa cells were immunostained with anti - ALIX ( red ) , anti - α - tubulin ( green ) and Hoechst ( blue ) . Confocal micrographs are presented and the insets show the indicated centrosomes ( arrows ) at higher magnification ( scale bars , 5 μm ) ( F ) The centrosomal ALIX fluorescence intensity was measured and the graph shows the average intensity ( relative to control siRNA cells ) of three independent experiments ( ± S . E ) , where in total 41 control siRNA , 40 Cep192 siRNA and 39 Cep215 siRNA - transfected cells were analyzed , * * p < 0 . 01 , Student ' s t -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal images of ALIX (red), Asl (white), α-tubulin (green) and Hoechst (blue) in wild type (w1118), Sas-6c02901, DSpdG02143, cnnHK21 and γ-Tub23CPI NBs (scale bars, 5 μm). The insets show higher magnification of the boxed centrosomes(B) The centrosomal fluorescence intensity of ALIX was measured and the graph shows the average intensity (relative to w1118 intensity) of three separate experiments (±S.E), where in total 78 w1118, 73 Sas-6c02901, 45 DSpdG02143, 61 cnnHK21 and 26 γ-Tub23CPI NBs were analyzed. Statistically significant differences are indicated (*p<0.05 and **p<0.01, Student's t-test)(C) GST or GST-ALIX were incubated with in vitro translated 35S-labelled Cnn or DSpd-2 proteins. Binding was detected by autoradiography (upper panel), and expression of GST and GST-ALIX was visualized by Ponceau S-staining (lower panel)(D) HeLa cells were transfected with control, Cep192 or Cep215 siRNA. Efficient depletion of Cep192 and Cep215 was confirmed by Western blotting analysis, which also showed unchanged expression levels of ALIX. Unspecific bands, below Cep192 and above Cep215, are indicated with **. (E) Control, Cep192 or Cep215 siRNA-transfected HeLa cells were immunostained with anti-ALIX (red), anti-α-tubulin (green) and Hoechst (blue). Confocal micrographs are presented and the insets show the indicated centrosomes (arrows) at higher magnification (scale bars, 5 μm)(F) The centrosomal ALIX fluorescence intensity was measured and the graph shows the average intensity (relative to control siRNA cells) of three independent experiments (±S.E), where in total 41 control siRNA, 40 Cep192 siRNA and 39 Cep215 siRNA-transfected cells were analyzed, **p< 0.01, Student's t-test


Figure 3 ( A ) Brains were dissected from w1118 , alix1 , alix - l / CyO ; alix1 and mud4 larvae and stained with anti - Bazooka ( red ) , anti - Cnn ( white ) and Hoechst ( blue ) . Representative confocal images are shown . Scale bars , 5 μm . The graph summarizes the average variation of the relative spindle angles in w1118 ( n = 74 ) , alix1 ( n = 96 ) , alix - l / CyO ; alix1 ( n = 78 ) and mud4 ( n = 32 ) NBs ( four separate experiments ) ( ± S . E ) . Compared to w1118 or alix - l / CyO ; alix1 NBs both alix1 and mud4 NBs showed greater variation in MS orientation ( * * * p < 0 . 001 , ANOVA ) ( C ) Brain lysates of w1118 , alix1 and alix - l / CyO ; alix1 larvae were immunoblotted for ALIX , α - tubulin and GAPDH ( loading control ) ( D ) Time - lapse imaging of Asl - YFP , α - tubulin - GFP in control ( left panel ) and alix3 ( right panel ) mutant SOPs of the pupal notum . SOPs were identified by the apical meshwork of MTs ( not shown ) . Time is indicated in min : sec . Mitotic cells are indicated with arrows . Scale bar represents 5 μm ( E ) Quantification of the rotation angle ( ± S . E ) in control ( n = 26 ) and alix3 ( n = 45 ) mutant SOPs ( at least three separate experiments ) . * * p < 0 . 01 ( Student ' s t - test ) compared to control ( G ) Western blotting analysis of lysates from control Asl - YFP , α - tubulin - GFP and Asl - YFP , α - tubulin - GFP ; alix3 pupae showed absence of ALIX in the alix3 homozygous mutant and unchanged expression of α - tubulin and GAPDH ( loading control )

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 B-PANEL_START O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Brains were dissected from w1118, alix1, alix-l/CyO; alix1 and mud4 larvae and stained with anti-Bazooka (red), anti-Cnn (white) and Hoechst (blue). Representative confocal images are shown. Scale bars, 5 μm. The graph summarizes the average variation of the relative spindle angles in w1118 (n=74), alix1 (n= 96), alix-l/CyO; alix1 (n= 78) and mud4 (n=32) NBs (four separate experiments) (±S.E). Compared to w1118 or alix-l/CyO; alix1 NBs both alix1 and mud4 NBs showed greater variation in MS orientation (***p<0.001, ANOVA) (C) Brain lysates of w1118, alix1 and alix-l/CyO; alix1 larvae were immunoblotted for ALIX, α-tubulin and GAPDH (loading control)(D) Time-lapse imaging of Asl-YFP, α-tubulin-GFP in control (left panel) and alix3 (right panel) mutant SOPs of the pupal notum. SOPs were identified by the apical meshwork of MTs (not shown). Time is indicated in min:sec. Mitotic cells are indicated with arrows. Scale bar represents 5 μm(E) Quantification of the rotation angle (±S.E) in control (n=26) and alix3 (n=45) mutant SOPs (at least three separate experiments). **p<0.01 (Student's t-test) compared to control(G) Western blotting analysis of lysates from control Asl-YFP, α-tubulin-GFP and Asl-YFP, α-tubulin-GFP; alix3 pupae showed absence of ALIX in the alix3 homozygous mutant and unchanged expression of α-tubulin and GAPDH (loading control)


Figure 4 ( A ) Ovaries of adult female flies ( w1118 , alix1 and alix - l / CyO ; alix1 ) were stained with rhodamine - phalloidin ( red ) , anti - α - tubulin ( green ) , anti - Cnn ( white ) and Hoechst ( blue ) . Typical confocal micrographs of metaphase FECs are presented and the apical membrane facing the egg chamber lumen is indicated with * . Scale bar , 5 μm . The MS orientation was measured and the graph shows the average percentage of FECs ( w1118 , n = 45 , alix1 , n = 45 , alix - l / CyO ; alix1 , n = 39 , from three independent experiments ) with the indicated relative spindle angle ( ± S . E ) . Statistically significant difference is indicated as * * * p < 0 . 001 ( ANOVA ) ( C ) Lysates of ovaries from w1118 , alix1 and alix - l / CyO ; alix1 flies were immunoblotted for ALIX , α - tubulin and GAPDH ( loading control ) ( D ) The MS orientation was determined in metaphase cells in organoids of Caco - 2 cells transfected with control ( n = 75 ) , ALIX ( n = 88 ) or NuMA ( n = 79 ) siRNA ( from three independent experiments ) . The graph shows the average percentage of Caco - 2 cells with the indicated relative spindle angle ( ± S . E ) . Greater spindle angle variation was observed in ALIX - and NuMA - depleted cells compared to control cells ( * * * p < 0 . 001 , ANOVA ) . Representative confocal images are shown where the cells are stained with rhodamine - phalloidin ( red ) , α - tubulin ( green ) and Hoechst ( blue ) . The apical membrane facing the lumen of the organoid is marked ( * ) . Scale bar , 5 μm ( E ) Lysates of control , ALIX or NuMA - depleted Caco - 2 cells were immunoblotted for NuMA , ALIX , α - tubulin and GAPDH ( loading control )

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

Figure 4 (A) Ovaries of adult female flies (w1118, alix1 and alix-l/CyO; alix1) were stained with rhodamine-phalloidin (red), anti-α-tubulin (green), anti-Cnn (white) and Hoechst (blue). Typical confocal micrographs of metaphase FECs are presented and the apical membrane facing the egg chamber lumen is indicated with *. Scale bar, 5 μm. The MS orientation was measured and the graph shows the average percentage of FECs (w1118, n=45, alix1, n=45, alix-l/CyO; alix1, n=39, from three independent experiments) with the indicated relative spindle angle (±S.E). Statistically significant difference is indicated as ***p<0.001 (ANOVA)(C) Lysates of ovaries from w1118, alix1 and alix-l/CyO; alix1 flies were immunoblotted for ALIX, α-tubulin and GAPDH (loading control)(D) The MS orientation was determined in metaphase cells in organoids of Caco-2 cells transfected with control (n=75), ALIX (n=88) or NuMA (n=79) siRNA (from three independent experiments). The graph shows the average percentage of Caco-2 cells with the indicated relative spindle angle (±S.E). Greater spindle angle variation was observed in ALIX- and NuMA-depleted cells compared to control cells (***p<0.001, ANOVA). Representative confocal images are shown where the cells are stained with rhodamine-phalloidin (red), α-tubulin (green) and Hoechst (blue). The apical membrane facing the lumen of the organoid is marked (*). Scale bar, 5 μm(E) Lysates of control, ALIX or NuMA-depleted Caco-2 cells were immunoblotted for NuMA, ALIX, α-tubulin and GAPDH (loading control)


Figure 5 ( A ) The MARCM strategy was used to generate alix mutant NBs clones ( FRT82B , alix1 ) or genetic equivalent control clones ( FRT82B con ) in the larval brain . Dissected brains were immunostained with anti - α - tubulin ( red ) and Hoechst ( blue ) . Recombinant NBs are positively marked with GFP ( green ) and outlined . The presence or lack of astral MTs is indicated with arrows or asterisks , respectively . Scale bars , 5 μm . The average intensity of astral , total and astral / total MT ( ± S . E ) in GFP - positive relative to GFP - negative control cells ( set to 100 % , indicated as dotted line in the graph ) was calculated from three separate experiments ( including 13 GFP - negative FRT82B con NBs , 11 GFP - positive FRT82B con NBs , 10 GFP - negative FRT82B , alix1 NBs and 8 GFP - positive FRT82B , alix1 NBs ) ( * p < 0 . 05 and * * p < 0 . 01 , Student ' s t - test ) ( B ) HeLa cells depleted of ALIX or not were immunostained with anti - α - tubulin ( red ) , anti - Pericentrin ( green ) and Hoechst ( blue ) . Representative confocal images are presented . Scale bars , 5 μm . The graph shows MT intensities as percentage of control cells ( ± S . E ) obtained from 99 control and 90 ALIX siRNA transfected cells ( data obtained from six separate experiments ) . Significant differences are noted as * p < 0 . 05 and * * * p < 0 . 001 ( Student ' s t - test ) ( C , D ) Regrowth of MTs after cold - induced depolymerization was performed using brains of w1118 and alix1 larvae , stained with anti - α - tubulin ( red ) , anti - Bazooka ( green ) and Hoechst ( blue ) ( C Typical confocal images showing MT - regrowth at given time points are shown ( scale bars , 5 μm ) . The graphs show the time - dependent MT intensities relative to the respective 0 ' ' - samples ( ± S . E ) calculated from three independent experiments . In ( C ) 46 , 64 , 65 and 48 w1118 NBs and 73 , 37 , 44 and 49 alix1 NBs from 0 , 30 , 60 and 300 ' ' were analyzed , respectively ( * p < 0 . 05 ) ( C , D ) Regrowth of MTs after cold - induced depolymerization was performed usin HeLa cells depleted of ALIX or not stained with anti - α - tubulin ( white ) and anti - Pericentrin ( green ) ( D ) Typical confocal images showing MT - regrowth at given time points are shown ( scale bars , 5 μm ) . The graphs show the time - dependent MT intensities relative to the respective 0 ' ' - samples ( ± S . E ) calculated from three independent experiments In ( D ) 47 , 40 and 42 control cells and 48 , 47 and 47 ALIX - depleted cells were calculated 0 , 30 ' ' and 420 ' ' after regrowth , respectively ( * p < 0 . 05 ) ( E ) Brains dissected from control ( FRT82B con ) or alix1 ( FRT82B , alix1 ) larvae were immunostained with anti - γ - tubulin ( red ) and Cnn ( white , left panel ) or anti - Dgrip91 ( red ) and α - tubulin ( white , right panel ) , as well as Hoechst ( blue ) . Recombinant NBs are positively marked with GFP ( green ) . The confocal images show typical accumulation of centrosomal γ - tubulin or Dgrip91 , and the insets zoom in at the indicated centrosomes . Scale bars , 5 μm . The intensities of γ - tubulin , Dgrip91 and Cnn in control NBs ( GFP - negative , set to 100 % ) and GFP - positive , recombinant NBs are presented in the table ( ± S . E ) , also indicating the number of NBs quantified ( n ) from three separate experiments ( * p < 0 . 05 ) ( F ) GST - ALIX ( lane 3 ) , but not GST ( lane 2 ) pulled out Dgrip91 and γ - tubulin from larval brain lysates ( G ) GST or GST - ALIX were incubated with in vitro translated 35S - methionine - labelled γ − tubulin or Dgrip91 . The upper panel shows the autoradiography indicating interaction between GST - ALIX and radiolabelled γ - tubulin or Dgrip91 . The lower panel shows the Ponceau S - stained membrane visualizing the expression of GST and GST - ALIX . The experiment was performed at least three times and a representative result is presented ( H ) HeLa cells depleted of ALIX or not grown on coverslips were stained with anti - γ − tubulin ( white ) , anti - Pericentrin ( red ) and Hoechst ( blue ) before being examined by confocal fluorescence microscopy . The confocal images and insets show the centrosomal accumulation of γ - tubulin and Pericentrin . Scale bars , 5 μm . The graph presents the average centrosomal intensities of γ - tubulin and Pericentrin relative to control cells ( set to 100 % ) calculated from three independent experiments ( 10 cells per condition / experiment ) ( ± S . E ) . Significant difference is noted as * * p < 0 . 01 ( Student ' s t - test ) ( I ) Lysates of control or ALIX - depleted HeLa cells were immunoblotted for ALIX , γ - tubulin , α - tubulin and GAPDH ( loading control )

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O 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) The MARCM strategy was used to generate alix mutant NBs clones (FRT82B, alix1) or genetic equivalent control clones (FRT82B con) in the larval brain. Dissected brains were immunostained with anti-α-tubulin (red) and Hoechst (blue). Recombinant NBs are positively marked with GFP (green) and outlined. The presence or lack of astral MTs is indicated with arrows or asterisks, respectively. Scale bars, 5 μm. The average intensity of astral, total and astral/total MT (±S.E) in GFP-positive relative to GFP-negative control cells (set to 100%, indicated as dotted line in the graph) was calculated from three separate experiments (including 13 GFP-negative FRT82B con NBs, 11 GFP-positive FRT82B con NBs, 10 GFP-negative FRT82B, alix1 NBs and 8 GFP-positive FRT82B, alix1 NBs) (*p<0.05 and **p<0.01, Student's t-test) (B) HeLa cells depleted of ALIX or not were immunostained with anti-α-tubulin (red), anti-Pericentrin (green) and Hoechst (blue). Representative confocal images are presented. Scale bars, 5 μm. The graph shows MT intensities as percentage of control cells (±S.E) obtained from 99 control and 90 ALIX siRNA transfected cells (data obtained from six separate experiments). Significant differences are noted as *p<0.05 and ***p<0.001 (Student's t-test)(C, D) Regrowth of MTs after cold-induced depolymerization was performed using brains of w1118 and alix1 larvae, stained with anti-α-tubulin (red), anti-Bazooka (green) and Hoechst (blue) (C Typical confocal images showing MT-regrowth at given time points are shown (scale bars, 5 μm). The graphs show the time-dependent MT intensities relative to the respective 0''-samples (±S.E) calculated from three independent experiments. In (C) 46, 64, 65 and 48 w1118 NBs and 73, 37, 44 and 49 alix1 NBs from 0, 30, 60 and 300'' were analyzed, respectively (*p<0.05)(C, D) Regrowth of MTs after cold-induced depolymerization was performed usin HeLa cells depleted of ALIX or not stained with anti-α-tubulin (white) and anti-Pericentrin (green) (D) Typical confocal images showing MT-regrowth at given time points are shown (scale bars, 5 μm). The graphs show the time-dependent MT intensities relative to the respective 0''-samples (±S.E) calculated from three independent experiments In (D) 47, 40 and 42 control cells and 48, 47 and 47 ALIX-depleted cells were calculated 0, 30'' and 420'' after regrowth, respectively (*p<0.05)(E) Brains dissected from control (FRT82B con) or alix1 (FRT82B, alix1) larvae were immunostained with anti-γ-tubulin (red) and Cnn (white, left panel) or anti-Dgrip91 (red) and α-tubulin (white, right panel), as well as Hoechst (blue). Recombinant NBs are positively marked with GFP (green). The confocal images show typical accumulation of centrosomal γ-tubulin or Dgrip91, and the insets zoom in at the indicated centrosomes. Scale bars, 5 μm. The intensities of γ-tubulin, Dgrip91 and Cnn in control NBs (GFP-negative, set to 100 %) and GFP-positive, recombinant NBs are presented in the table (±S.E), also indicating the number of NBs quantified (n) from three separate experiments (*p<0.05)(F) GST-ALIX (lane 3), but not GST (lane 2) pulled out Dgrip91 and γ-tubulin from larval brain lysates(G) GST or GST-ALIX were incubated with in vitro translated 35S-methionine-labelled γ−tubulin or Dgrip91. The upper panel shows the autoradiography indicating interaction between GST-ALIX and radiolabelled γ-tubulin or Dgrip91. The lower panel shows the Ponceau S-stained membrane visualizing the expression of GST and GST-ALIX. The experiment was performed at least three times and a representative result is presented(H) HeLa cells depleted of ALIX or not grown on coverslips were stained with anti-γ−tubulin (white), anti-Pericentrin (red) and Hoechst (blue) before being examined by confocal fluorescence microscopy. The confocal images and insets show the centrosomal accumulation of γ-tubulin and Pericentrin. Scale bars, 5 μm. The graph presents the average centrosomal intensities of γ-tubulin and Pericentrin relative to control cells (set to 100%) calculated from three independent experiments (10 cells per condition/experiment) (±S.E). Significant difference is noted as **p<0.01 (Student's t-test)(I) Lysates of control or ALIX-depleted HeLa cells were immunoblotted for ALIX, γ-tubulin, α-tubulin and GAPDH (loading control)


Figure 6 ( A ) The MS orientation was determined in metaphase NBs from brains dissected from wild type ( w1118 ) , alix1 , γ − Tub23CPI or γ − Tub23CPI ; alix1 larvae . Compared to w1118 , significantly greater variation in the relative spindle angles was observed in alix1 NBs and γ − Tub23CPI ; alix1 NBs ( * p < 0 . 05 , ANOVA ) . The γ − Tub23CPI ; alix1 NBs displayed significantly greater variation of their spindle orientations than either alix1 or γ − Tub23CPI NBs ( * p < 0 . 05 , ANOVA ) . The graph shows the average percentage of NBs with the indicated relative spindle angles ( ± S . E ) from in total 57 w1118 NBs , 55 alix1 NBs , 76 γ − Tub23CPI NBs and 62 γ − Tub23CPI ; alix1 NBs ( three independent experiments ) ( B ) Lysates of the respective genotypes were immunoblotted for ALIX , γ - tubulin and GAPDH ( loading control ) . The average expression of ALIX or γ - tubulin ( relative to GAPDH ) from three independent experiments is given below ( ± S . E ) and significant differences are indicated as * p < 0 . 05 , Student ' s t - test ( C ) Dissected brains of w1118 , alix1 , γ − Tub23CPI and γ − Tub23CPI ; alix1 larvae were subjected to MT regrowth assay and stained with anti - α - tubulin ( red ) , anti - Cnn ( white ) and Hoechst ( blue ) . Representative images of NBs after 0 and 60 seconds regrowth are presented . Scale bars , 5 μm ( D ) The MT - intensities after 0 and 60 seconds regrowth were determined , here presented as the average of three separate experiments ( ± S . E ) where in total 27 w1118 - 0 ' ' , 28 w1118 - 60 ' ' , 26 alix1 - 0 ' ' , 19 alix1 - 60 ' ' , 28 γ − Tub23CPI - 0 ' ' , 26 γ − Tub23CPI - 60 ' ' , 28 γ − Tub23CPI ; alix1 - 0 ' ' and 23 γ − Tub23CPI ; alix1 - 60 ' ' NBs were analyzed . Compared to w1118 - NBs , significantly less MT polymerized in alix1 , γ − Tub23CPI or γ − Tub23CPI ; alix1 NBs ( * p < 0 . 05 , ANOVA ) ( E ) The relative spindle angle between the polarity axis perpendicular to the apical phalloidin - stained F - actin surface and the MS was determined in metaphase Caco - 2 cysts cells transfected with control , ALIX and / or γ - tubulin siRNA ( 29 , 29 , 18 and 32 metaphase cells , respectively , were analyzed from three separate experiments ) . Compared to control cells , depletion of either ALIX or both ALIX and γ - tubulin , induced a significantly increased randomization of the relative spindle angles ( * * p < 0 . 01 and * * * p < 0 . 001 , ANOVA ) . Caco - 2 cells depleted of ALIX + γ - tubulin showed a greater variation in the spindle angles compared to depletion of ALIX or γ - tubulin ( * p < 0 . 05 , ANOVA ) . ALIX - depleted cells showed greater spindle angle - variation than γ - tubulin - depleted cells ( * p < 0 . 05 , ANOVA ) ( F ) Efficient knockdown of ALIX and γ - tubulin relative to GAPDH ( loading control ) was verified by Western blotting analysis , and the average expression of ALIX or γ - tubulin relative to GAPDH ( ± S . E ) is given below ( * p < 0 . 05 , Student ' s t - test )

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

Figure 6 (A) The MS orientation was determined in metaphase NBs from brains dissected from wild type (w1118), alix1, γ−Tub23CPI or γ−Tub23CPI; alix1 larvae. Compared to w1118, significantly greater variation in the relative spindle angles was observed in alix1 NBs and γ−Tub23CPI; alix1 NBs (*p<0.05, ANOVA). The γ−Tub23CPI;alix1 NBs displayed significantly greater variation of their spindle orientations than either alix1 or γ−Tub23CPI NBs (*p<0.05, ANOVA). The graph shows the average percentage of NBs with the indicated relative spindle angles (±S.E) from in total 57 w1118 NBs, 55 alix1 NBs, 76 γ−Tub23CPI NBs and 62 γ−Tub23CPI; alix1 NBs (three independent experiments) (B) Lysates of the respective genotypes were immunoblotted for ALIX, γ-tubulin and GAPDH (loading control). The average expression of ALIX or γ-tubulin (relative to GAPDH) from three independent experiments is given below (±S.E) and significant differences are indicated as *p<0.05, Student's t-test(C) Dissected brains of w1118, alix1, γ−Tub23CPI and γ−Tub23CPI;alix1 larvae were subjected to MT regrowth assay and stained with anti-α-tubulin (red), anti-Cnn (white) and Hoechst (blue). Representative images of NBs after 0 and 60 seconds regrowth are presented. Scale bars, 5 μm(D) The MT-intensities after 0 and 60 seconds regrowth were determined, here presented as the average of three separate experiments (± S.E) where in total 27 w1118-0'', 28 w1118-60'', 26 alix1-0'', 19 alix1-60'', 28 γ−Tub23CPI-0'', 26 γ−Tub23CPI-60'', 28 γ−Tub23CPI;alix1-0'' and 23 γ−Tub23CPI;alix1-60'' NBs were analyzed. Compared to w1118-NBs, significantly less MT polymerized in alix1, γ−Tub23CPI or γ−Tub23CPI;alix1 NBs (*p<0.05, ANOVA)(E) The relative spindle angle between the polarity axis perpendicular to the apical phalloidin-stained F-actin surface and the MS was determined in metaphase Caco-2 cysts cells transfected with control, ALIX and/or γ-tubulin siRNA (29, 29, 18 and 32 metaphase cells, respectively, were analyzed from three separate experiments). Compared to control cells, depletion of either ALIX or both ALIX and γ-tubulin, induced a significantly increased randomization of the relative spindle angles (**p<0.01 and ***p<0.001, ANOVA). Caco-2 cells depleted of ALIX+γ-tubulin showed a greater variation in the spindle angles compared to depletion of ALIX or γ-tubulin (*p<0.05, ANOVA). ALIX-depleted cells showed greater spindle angle-variation than γ-tubulin-depleted cells (*p<0.05, ANOVA)(F) Efficient knockdown of ALIX and γ-tubulin relative to GAPDH (loading control) was verified by Western blotting analysis, and the average expression of ALIX or γ-tubulin relative to GAPDH (±S.E) is given below (*p<0.05, Student's t-test)