( 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

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(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 structures


Images 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

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Images 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

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(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

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(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

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(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

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(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


( 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

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(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

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(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

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(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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(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


Anti - 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 ,

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Anti-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

(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

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(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

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(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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(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


( 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

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(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

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(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

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(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

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(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

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(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

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(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

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(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


( 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

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(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 t


MTPAP 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

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MTPAP 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 experiments


MTPAP 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

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MTPAP 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

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(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

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(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


( 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

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(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

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(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


( 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

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(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 )

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(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 )

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(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 *

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(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 )

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*. (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 -

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(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


( 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 )

O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

(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 )

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(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

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(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

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(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 )

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(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)


( 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 )

O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 )

O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O

(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

O O O O 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-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 )

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(E) Lysates of control, ALIX or NuMA-depleted Caco-2 cells were immunoblotted for NuMA, ALIX, α-tubulin and GAPDH (loading control)


( 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 )

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 )

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O 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) 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 )

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(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 )

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 )

O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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

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(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

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

(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 )

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O

(I) Lysates of control or ALIX-depleted HeLa cells were immunoblotted for ALIX, γ-tubulin, α-tubulin and GAPDH (loading control)


( 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 )

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O

(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 -

O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O 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) 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

O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 )

O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O

(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 )

O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O 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-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O

(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 B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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)


( A ) HeLa cells transfected with control or ALIX siRNA were fixed , permeabilized and stained with anti - MAP1S ( red ) , anti - γ - tubulin ( white ) and Hoechst ( blue ) . Interphase cells are shown and the insets show close ups of the centrosomes . Scale bars , 5 μm . ( B ) The fluorescence intensity of centrosomal MAP1S was determined and the graph shows the average of three independent experiments ( ± S . E ) , in which in total 52 control and 36 ALIX siRNA cells were analyzed . Significant difference is indicated as * p < 0 . 05 ( Student ' s t - test )

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(A) HeLa cells transfected with control or ALIX siRNA were fixed, permeabilized and stained with anti-MAP1S (red), anti-γ-tubulin (white) and Hoechst (blue). Interphase cells are shown and the insets show close ups of the centrosomes. Scale bars, 5 μm. (B) The fluorescence intensity of centrosomal MAP1S was determined and the graph shows the average of three independent experiments (± S.E), in which in total 52 control and 36 ALIX siRNA cells were analyzed. Significant difference is indicated as *p<0.05 (Student's t-test)


( C ) HeLa cells were transfected with control , ALIX , MAP1S or ALIX + MAP1S siRNA and the expression of ALIX , MAP1S and GAPDH ( loading control ) was determined by Western blotting analysis . Representative results are

O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O

(C) HeLa cells were transfected with control, ALIX, MAP1S or ALIX+MAP1S siRNA and the expression of ALIX, MAP1S and GAPDH (loading control) was determined by Western blotting analysis. Representative results are presented


( D , E ) Cells treated as in C were immunostained with anti - α - tubulin ( white ) , Hoechst ( blue ) ( D Scale bars , 5 μm . The intensities of astral MTs relative to total MTs were determined from confocal microscopy images and the graph in ( D ) shows the average of four separate experiments ( 10 cells per condition / experiment ) ( ± S . E ) . * p < 0 . 05 and * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D, E) Cells treated as in C were immunostained with anti-α-tubulin (white), Hoechst (blue) (D Scale bars, 5 μm. The intensities of astral MTs relative to total MTs were determined from confocal microscopy images and the graph in (D) shows the average of four separate experiments (10 cells per condition/experiment) (±S.E). *p<0.05 and **p<0.01, Student's t-test


( D , E ) Cells treated as in C were immunostained wit anti - acetylated tubulin ( green ) ( E ) The relative intensity of acetylated MT relative to total MT was determined in ( E ) The average of four separate experiments ( 10 cells per condition / experiment ) is graphically presented ( ± S . E ) . Significant differences are indicated as * p < 0 . 05 ( Student ' s t - test )

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D, E) Cells treated as in C were immunostained wit anti-acetylated tubulin (green) (E) The relative intensity of acetylated MT relative to total MT was determined in (E) The average of four separate experiments (10 cells per condition/experiment) is graphically presented (±S.E). Significant differences are indicated as *p<0.05 (Student's t-test)


( F ) The regrowth of MTs ( i . e total tubulin intensity ) was assessed in cells incubated at 37 ° C for 0 , 60 and 150 seconds after ice - induced depolymerization ( for 30 minutes ) . The graph represents the average of three independent experiments ( calculating 10 cells per time point for each condition ) ( ± S . E ) . Significant differences are noted as * p < 0 . 05 , Student ' s t -

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(F) The regrowth of MTs (i.e total tubulin intensity) was assessed in cells incubated at 37°C for 0, 60 and 150 seconds after ice-induced depolymerization (for 30 minutes). The graph represents the average of three independent experiments (calculating 10 cells per time point for each condition) (±S.E). Significant differences are noted as *p<0.05, Student's t-test


( G ) HeLa cells transfected with control or ALIX siRNA grown on coverslips were transiently transfected with pEYFP - C1 ( green ) , pEYFP - MAP1S ( green ) , pEGFP - C1 ( green ) or pEGFP - CLASP2α and immunostained with anti - α - tubulin ( white ) , anti - Pericentrin ( red ) and Hoechst ( blue ) . Scale bars , 5

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O

(G) HeLa cells transfected with control or ALIX siRNA grown on coverslips were transiently transfected with pEYFP-C1 (green), pEYFP-MAP1S (green), pEGFP-C1 (green) or pEGFP-CLASP2α and immunostained with anti-α-tubulin (white), anti-Pericentrin (red) and Hoechst (blue). Scale bars, 5 μm


( H ) The lengths and numbers of astral MTs of HeLa cells treated as in ( G ) were measured ( LSM software , version 3 . 2 ) . The graphs show the average numbers ( ± S . E ) based on calculating 30 control siRNA + YFP cells , 23 control siRNA + YFP - MAP1S cells , 30 ALIX siRNA + YFP cells , 30 ALIX siRNA + YFP - MAP1S cells , 28 control siRNA + GFP cells , 30 control siRNA + GFP - CLASP2α cells , 30 ALIX siRNA + GFP cells and 29 ALIX siRNA + GFP - CLASP2α cells from three separate experiments ( * 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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

(H) The lengths and numbers of astral MTs of HeLa cells treated as in (G) were measured (LSM software, version 3.2). The graphs show the average numbers (±S.E) based on calculating 30 control siRNA+YFP cells, 23 control siRNA+YFP-MAP1S cells, 30 ALIX siRNA+YFP cells, 30 ALIX siRNA+YFP-MAP1S cells, 28 control siRNA+GFP cells, 30 control siRNA+GFP-CLASP2α cells, 30 ALIX siRNA+GFP cells and 29 ALIX siRNA+GFP-CLASP2α cells from three separate experiments (*p<0.05, Student's t-test), respectively


A : Immunoblot analysis of wild type ( WT ) , FAM83D - / - knockout ( KO ) and FAM83DGFP / GFP knockin ( KI ) U2OS cell lines . Data information : All blots are representative of at least 3 independent experiments .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

A: Immunoblot analysis of wild type (WT), FAM83D-/- knockout (KO) and FAM83DGFP/GFP knockin (KI) U2OS cell lines. Data information: All blots are representative of at least 3 independent experiments.


D : AS or nocodazole - synchronised M KI cells were lysed and subjected to GFP TRAP immunoprecipitations ( IP ) . Extracts ( input ) and IP samples were analysed by immunoblotting ( IB ) with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D: AS or nocodazole-synchronised M KI cells were lysed and subjected to GFP TRAP immunoprecipitations (IP). Extracts (input) and IP samples were analysed by immunoblotting (IB) with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


E : KI cells synchronised in mitosis with either nocodazole ( M Noc . ) or STLC ( M STLC ) were collected by shake - off , and drug - treated cells that remained adherent after shake - off ( AS Noc . ; AS STLC ) were lysed and subjected to GFP TRAP IP . AS cells and free - GFP - expressing 2G - PAI1 U2OS cells were included as controls . Input and IP samples were analysed by IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E: KI cells synchronised in mitosis with either nocodazole (M Noc.) or STLC (M STLC) were collected by shake-off, and drug-treated cells that remained adherent after shake-off (AS Noc.; AS STLC) were lysed and subjected to GFP TRAP IP. AS cells and free-GFP-expressing 2G-PAI1 U2OS cells were included as controls. Input and IP samples were analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


F : Propidium iodide staining analyses revealing cell cycle distribution profiles for the

F: Propidium iodide staining analyses revealing cell cycle distribution profiles for the samples


G , H : AS or nocodazole - synchronised ( M ) WT U2OS cells were subjected to IP with IgG and either anti - FAM83D - coupled sepharose beads ( G ) , or anti - CK1α - coupled sepharose beads ( H ) . Input and IP samples were analysed by IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G, H: AS or nocodazole-synchronised (M) WT U2OS cells were subjected to IP with IgG and either anti-FAM83D-coupled sepharose beads (G), or anti-CK1α-coupled sepharose beads (H). Input and IP samples were analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


I : KI cells were synchronised in G2 with RO - 3306 , or arrested in mitosis ( M ) using STLC . STLC - treated shake - off cells were washed and re - plated , and cells lysed at the indicated time points after STLC wash - out . Cell lysates were subjected to GFP TRAP IP and input and IP extracts analysed by IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I: KI cells were synchronised in G2 with RO-3306, or arrested in mitosis (M) using STLC. STLC-treated shake-off cells were washed and re-plated, and cells lysed at the indicated time points after STLC wash-out. Cell lysates were subjected to GFP TRAP IP and input and IP extracts analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


J : Propidium iodide staining analyses revealing cell cycle distribution profiles for the

J: Propidium iodide staining analyses revealing cell cycle distribution profiles for the samples


K : AS or STLC - synchronised ( M ) HeLa , A549 and HaCaT cells were subjected to IP with either IgG - or anti - FAM83D - coupled sepharose beads . Input and IP samples were analysed by IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K: AS or STLC-synchronised (M) HeLa, A549 and HaCaT cells were subjected to IP with either IgG- or anti-FAM83D-coupled sepharose beads. Input and IP samples were analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


A : STLC - synchronised mitotic ( M ) wild - type ( WT ) , FAM83D - / - knockout ( KO ) and FAM83DGFP / GFP knockin ( KI ) U2OS cells were subjected to anti - CK1α immunofluorescence and GFP fluorescence microscopy . DNA is stained with DAPI . Scale bars , 20 µm .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O

A: STLC-synchronised mitotic (M) wild-type (WT), FAM83D-/- knockout (KO) and FAM83DGFP/GFP knockin (KI) U2OS cells were subjected to anti-CK1α immunofluorescence and GFP fluorescence microscopy. DNA is stained with DAPI. Scale bars, 20 µm.


B : STLC - synchronised mitotic ( M ) FAM83D - / - knockout ( KO ) , FAM83DGFP / GFP knockin ( KI ) and FAM83DGFP / GFP ( F283A ) knockin ( FA ) U2OS cells were subjected to anti - CK1α immunofluorescence and GFP fluorescence microscopy . DNA is stained with DAPI . Scale bars , 20

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B: STLC-synchronised mitotic (M) FAM83D-/- knockout (KO), FAM83DGFP/GFP knockin (KI) and FAM83DGFP/GFP(F283A) knockin (FA) U2OS cells were subjected to anti-CK1α immunofluorescence and GFP fluorescence microscopy. DNA is stained with DAPI. Scale bars, 20 µ


C : Quantification of CK1α spindle localisation for the cells Cell images denote the measured regions used to calculate the ratios on the box plot . Box plot whiskers denote the minimum and maximum measured values . The middle line represents the median , and the box ranges depict the 25th / 75th percentiles . * * * , P < 0 . 0001 ; ANOVA . Analysis was performed on the indicated number of cells , n = 2 .

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C: Quantification of CK1α spindle localisation for the cells Cell images denote the measured regions used to calculate the ratios on the box plot. Box plot whiskers denote the minimum and maximum measured values. The middle line represents the median, and the box ranges depict the 25th/75th percentiles. ***, P<0.0001; ANOVA. Analysis was performed on the indicated number of cells, n=2.


D : The cell lines were STLC - synchronised and mitotic cells ( M ) isolated by shake off . Asynchronous ( AS ) cells were included as a control . Cells were lysed and subjected to GFP TRAP immunoprecipitation ( IP ) and subsequent immunoblotting ( IB ) with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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D: The cell lines were STLC-synchronised and mitotic cells (M) isolated by shake off. Asynchronous (AS) cells were included as a control. Cells were lysed and subjected to GFP TRAP immunoprecipitation (IP) and subsequent immunoblotting (IB) with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


F : KI cells were infected with retroviruses encoding either VHL , aGFP . 16 , or VHL - aGFP . 16 . Uninfected cells were used as a control . Cells were lysed and subjected to IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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F: KI cells were infected with retroviruses encoding either VHL, aGFP.16, or VHL-aGFP.16. Uninfected cells were used as a control. Cells were lysed and subjected to IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


G : The cell lines were subjected to anti - CK1α immunofluorescence and GFP fluorescence microscopy . DNA is stained with DAPI . Scale bars , 20 µm .

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G: The cell lines were subjected to anti-CK1α immunofluorescence and GFP fluorescence microscopy. DNA is stained with DAPI. Scale bars, 20 µm.


B : FAM83D - / - ( KO ) , wild - type ( WT ) , and two independent clones from a CRISPR / Cas9 - mediated knockin rescue of FAM83D cDNA into FAM83D - / - cells ( c . 6 and c . 11 ) , were synchronized in mitosis ( M ) with STLC . Asynchronous ( AS ) cells were included as a control . Cells were lysed and subjected to immunoblotting ( IB ) with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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B: FAM83D-/- (KO), wild-type (WT), and two independent clones from a CRISPR/Cas9-mediated knockin rescue of FAM83D cDNA into FAM83D-/- cells (c. 6 and c. 11), were synchronized in mitosis (M) with STLC. Asynchronous (AS) cells were included as a control. Cells were lysed and subjected to immunoblotting (IB) with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


The cell lines were STLC - synchronized in mitosis , fixed , and stained with antibodies recognizing FAM83D ( C ) Representative images of mitotic cells are included . Scale bars , 20 µm .

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The cell lines were STLC-synchronized in mitosis, fixed, and stained with antibodies recognizing FAM83D (C) Representative images of mitotic cells are included. Scale bars, 20 µm.


The cell lines were STLC - synchronized in mitosis , fixed , and stained with antibodies recognizing CK1 ⍺ ( D ) . Representative images of mitotic cells are included . Scale bars , 20 µm .

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The cell lines were STLC-synchronized in mitosis, fixed, and stained with antibodies recognizing CK1⍺ (D). Representative images of mitotic cells are included. Scale bars, 20 µm.


E : Quantification of CK1 ⍺ spindle localization Box plot whiskers denote the minimum and maximum measured values . The middle line represents the median , and the box ranges depict the 25th / 75th percentiles . * * * , P < 0 . 0001 ; ANOVA . Analysis was performed on the indicated number of cells , n = 1 .

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E: Quantification of CK1⍺ spindle localization Box plot whiskers denote the minimum and maximum measured values. The middle line represents the median, and the box ranges depict the 25th/75th percentiles. ***, P<0.0001; ANOVA. Analysis was performed on the indicated number of cells, n=1.


F : The cell lines were STLC - synchronized in mitosis ( M ) or left AS , lysed and subjected to immunoprecipitation ( IP ) with anti - FAM83D - coupled sepharose beads , before IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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F: The cell lines were STLC-synchronized in mitosis (M) or left AS, lysed and subjected to immunoprecipitation (IP) with anti-FAM83D-coupled sepharose beads, before IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


G : Asynchronous ( AS ) FAM83DGFP / GFP / mCherry / mCherryCSNK1A1 knockin U2OS cells were fixed and imaged . Representative images from the indicated cell cycle stages are included . Scale bars , 10 µm .

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G: Asynchronous (AS) FAM83DGFP/GFP/ mCherry/mCherryCSNK1A1 knockin U2OS cells were fixed and imaged. Representative images from the indicated cell cycle stages are included. Scale bars, 10 µm.


A : Nocodazole - synchronised mitotic FAM83DGFP / GFP knockin U2OS cells were lysed and subjected to GFP TRAP immunoprecipitation ( IP ) , followed by incubation ± λ - phosphatase . Asynchronous ( AS ) cells were used as a control . Whole cell extracts ( input ) and IP samples were subjected to SDS - PAGE and subsequent immunoblotting ( IB ) with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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A: Nocodazole-synchronised mitotic FAM83DGFP/GFP knockin U2OS cells were lysed and subjected to GFP TRAP immunoprecipitation (IP), followed by incubation ± λ-phosphatase. Asynchronous (AS) cells were used as a control. Whole cell extracts (input) and IP samples were subjected to SDS-PAGE and subsequent immunoblotting (IB) with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


STLC - synchronised mitotic wild - type U2OS cells were lysed at the indicated time points following STLC washout and release into medium ( B ) AS cells were used as a control . Lysed extracts were subjected to IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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STLC-synchronised mitotic wild-type U2OS cells were lysed at the indicated time points following STLC washout and release into medium (B) AS cells were used as a control. Lysed extracts were subjected to IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


STLC - synchronised mitotic wild - type U2OS cells were lysed at the indicated time points following STLC washout and release into medium ± MG132 ( C ) AS cells were used as a control . Lysed extracts were subjected to IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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STLC-synchronised mitotic wild-type U2OS cells were lysed at the indicated time points following STLC washout and release into medium ± MG132 (C) AS cells were used as a control. Lysed extracts were subjected to IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


STLC - synchronised mitotic wild - type U2OS cells were lysed at the indicated time points following STLC washout and release into medium ± ProTAME ( D ) . AS cells were used as a control . Lysed extracts were subjected to IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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STLC-synchronised mitotic wild-type U2OS cells were lysed at the indicated time points following STLC washout and release into medium ± ProTAME (D). AS cells were used as a control. Lysed extracts were subjected to IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


E : Wild - type U2OS cells were either left asynchronous ( AS ) , or arrested in mitosis with STLC and collected by shake - off ( M ) . AS and M cells were incubated in media containing combinations of RO - 3306 and MG132 as indicated , prior to lysis . MG132 was applied for 1 . 5 h , whereas RO - 3306 was applied for the last 1 h of incubation prior to lysis . Samples were lysed and extracts were subjected to SDS - PAGE , before IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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E: Wild-type U2OS cells were either left asynchronous (AS), or arrested in mitosis with STLC and collected by shake-off (M). AS and M cells were incubated in media containing combinations of RO-3306 and MG132 as indicated, prior to lysis. MG132 was applied for 1.5 h, whereas RO-3306 was applied for the last 1 h of incubation prior to lysis. Samples were lysed and extracts were subjected to SDS-PAGE, before IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


F : STLC - synchronised mitotic wild - type U2OS cells were subjected to qRT - PCR analysis using primers for FAM83D , HMMR , CSNK1A1 , and CCNB1 . AS cells were used as a control . Error bars , SEM ; * , P < 0 . 01 ; Student ' s T - test . n =

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F: STLC-synchronised mitotic wild-type U2OS cells were subjected to qRT-PCR analysis using primers for FAM83D, HMMR, CSNK1A1, and CCNB1. AS cells were used as a control. Error bars, SEM; *, P<0.01; Student's T-test. n=3


H : STLC - synchronised mitotic wild - type ( WT ) and HMMR knockout ( KO ) mouse embryonic fibroblasts ( MEFs ) were lysed and subjected to IB with the indicated antibodies . AS cells were included as a control . Data information : All blots are representative of at least 3 independent experiments .

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H: STLC-synchronised mitotic wild-type (WT) and HMMR knockout (KO) mouse embryonic fibroblasts (MEFs) were lysed and subjected to IB with the indicated antibodies. AS cells were included as a control. Data information: All blots are representative of at least 3 independent experiments.


The cells were STLC - synchronised in mitosis and subjected to immunofluorescence microscopy with an anti - HMMR antibody ( I ) DNA is stained with DAPI . Scale bars , 20 µm .

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The cells were STLC-synchronised in mitosis and subjected to immunofluorescence microscopy with an anti-HMMR antibody (I) DNA is stained with DAPI. Scale bars, 20 µm.


The cells were STLC - synchronised in mitosis and subjected to immunofluorescence microscopy with an anti - CK1α antibody ( J ) . DNA is stained with DAPI . Scale bars , 20 µm .

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The cells were STLC-synchronised in mitosis and subjected to immunofluorescence microscopy with an anti-CK1α antibody (J). DNA is stained with DAPI. Scale bars, 20 µm.


B : STLC - synchronised mitotic FAM83DGFP / GFP knockin ( KI ) , FAM83DGFP / GFP ( F283A ) ( FA ) , and FAM83DGFP / GFP ( F283A ) stably expressing aGFP . 16 - CK1α ( FA + aGFP . 16 - CK1α ) U2OS cells were subjected to GFP TRAP immunoprecipitation ( IP ) , followed by immunoblotting ( IB ) with the indicated antibodies . Asynchronous ( AS ) cells were used as controls . Data information : All blots are representative of at least 3 independent experiments .

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B: STLC-synchronised mitotic FAM83DGFP/GFP knockin (KI), FAM83DGFP/GFP (F283A) (FA), and FAM83DGFP/GFP(F283A) stably expressing aGFP.16-CK1α (FA + aGFP.16-CK1α) U2OS cells were subjected to GFP TRAP immunoprecipitation (IP), followed by immunoblotting (IB) with the indicated antibodies. Asynchronous (AS) cells were used as controls. Data information: All blots are representative of at least 3 independent experiments.


C : FA cells were infected with retroviruses encoding wild - type aGFP . 16 - CK1α ( WT ) , or aGFP . 16 - CK1α with one of two distinct CK1α kinase - inactive mutants ( D136N or K46D ) . Uninfected cells ( - ) were included as a control . Cells were lysed , subjected to anti - GFP IP and IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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C: FA cells were infected with retroviruses encoding wild-type aGFP.16-CK1α (WT), or aGFP.16-CK1α with one of two distinct CK1α kinase-inactive mutants (D136N or K46D). Uninfected cells (-) were included as a control. Cells were lysed, subjected to anti-GFP IP and IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


D : Immunofluorescence analysis for the cells described in ( B ) following synchronisation with STLC . Cells were stained using anti - CK1α antibody , and DNA is stained with DAPI . Scale bars , 20 µm .

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D: Immunofluorescence analysis for the cells described in (B) following synchronisation with STLC. Cells were stained using anti-CK1α antibody, and DNA is stained with DAPI. Scale bars, 20 µm.


F : FAM83D - / - knockout ( KO ) , KI , and FA cells were synchronised in mitosis ( M ) using STLC . Lysed extracts were subjected to anti - GFP IPs , followed by ATP [ γ - 32P ] kinase assays , using an optimised CK1 substrate peptide ( CK1tide ) . AS cells were used as controls . Error bars , SEM ; P < 0 . 01 ; ANOVA . n = 3 . Input and IP samples were analysed by IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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F: FAM83D-/- knockout (KO), KI, and FA cells were synchronised in mitosis (M) using STLC. Lysed extracts were subjected to anti-GFP IPs, followed by ATP [γ-32P] kinase assays, using an optimised CK1 substrate peptide (CK1tide). AS cells were used as controls. Error bars, SEM; P<0.01; ANOVA. n=3. Input and IP samples were analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


A . Representative images initiating at metaphase for mitotic U2OS cell lines stained with Hoechst , taken every 5 min as they progress through division . Mitotic stage was determined by chromosome condensation and is indicated by the coloured boxes . Scale bar , 20 μm .

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A. Representative images initiating at metaphase for mitotic U2OS cell lines stained with Hoechst, taken every 5 min as they progress through division. Mitotic stage was determined by chromosome condensation and is indicated by the coloured boxes. Scale bar, 20 μm.


B . Graphical representation for the kinetics of transition from metaphase alignment ( yellow ) to anaphase ( green ) , and cytokinesis ( blue ) . 100 mitotic cells per genotype are plotted ; n = 2 .

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B. Graphical representation for the kinetics of transition from metaphase alignment (yellow) to anaphase (green), and cytokinesis (blue). 100 mitotic cells per genotype are plotted; n=2.


C . Length of time needed to transition from metaphase to anaphase . Mean of 100 mitotic cells per genotype are plotted ; n = 2 . Error bars , SEM . * * * , P < 0 . 0001 ; ANOVA .

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C. Length of time needed to transition from metaphase to anaphase. Mean of 100 mitotic cells per genotype are plotted; n=2. Error bars, SEM. ***, P< 0.0001; ANOVA.


D . Representative bright field images for mitotic U2OS cells indicating the long axis during interphase ( white line ) preceding mitosis , and the division axis determined at anaphase ( yellow line ) . Scale bar , 20 μm .

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D. Representative bright field images for mitotic U2OS cells indicating the long axis during interphase (white line) preceding mitosis, and the division axis determined at anaphase (yellow line). Scale bar, 20 μm.


E . Percentage of oriented divisions for U2OS cells grown in subconfluent cultures . An oriented division axis was defined as being less than 30 ° removed from the long axis of the interphase cell . Mean of 50 cells per genotype are plotted , n = 2 . Error bars , SEM . * * * , P < 0 . 0001 ; ANOVA .

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E. Percentage of oriented divisions for U2OS cells grown in subconfluent cultures. An oriented division axis was defined as being less than 30° removed from the long axis of the interphase cell. Mean of 50 cells per genotype are plotted, n=2. Error bars, SEM. ***, P< 0.0001; ANOVA.


F . Representative images of mitotic U2OS cells stained with Hoechst and grown on L - shaped micropatterns previously coated with fibronectin . The position of metaphase chromosomes is indicated ( yellow line ) as is the presence of cortical blebbing ( red arrows ) . Scale bar , 20 μm .

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F. Representative images of mitotic U2OS cells stained with Hoechst and grown on L-shaped micropatterns previously coated with fibronectin. The position of metaphase chromosomes is indicated (yellow line) as is the presence of cortical blebbing (red arrows). Scale bar, 20 μm.


G . Circular graphs , superimposed on L - shaped micropattern , show the distribution of cell division angles measured at anaphase . Angles for 100 U2OS cells are plotted per genotype , n = 2 . Metaphase position is indicated ( yellow line ) and the percentages of division angles ± 15 ° from the expected axis ( red line ) are indicated .

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G. Circular graphs, superimposed on L-shaped micropattern, show the distribution of cell division angles measured at anaphase. Angles for 100 U2OS cells are plotted per genotype, n=2. Metaphase position is indicated (yellow line) and the percentages of division angles ± 15° from the expected axis (red line) are indicated.


H . Percentage of metaphase U2OS cells that align chromosomes outside of the expected axis ( angles ± 15 ° ) . Mean of 100 cells per genotype are plotted , n = 2 . Error bars , SEM . * * * , P < 0 . 0001 ; ANOVA .

H. Percentage of metaphase U2OS cells that align chromosomes outside of the expected axis (angles ± 15°). Mean of 100 cells per genotype are plotted, n=2. Error bars, SEM. ***, P< 0.0001; ANOVA.


I . Representative images of RFP - actin localization in mitotic U2OS cells grown on fibronectin - coated , L - shaped micropatterns , which is superimposed . Arrowheads indicate polarized cortical actin . Heatmap shows the intensity of RFP - actin localization ( Image J z - projection standard deviation ) as the cell progresses from prophase to metaphase ( 12 - 24 min ) . Scale bar , 20 μm .

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I. Representative images of RFP-actin localization in mitotic U2OS cells grown on fibronectin-coated, L-shaped micropatterns, which is superimposed. Arrowheads indicate polarized cortical actin. Heatmap shows the intensity of RFP-actin localization (Image J z-projection standard deviation) as the cell progresses from prophase to metaphase (12- 24 min). Scale bar, 20 μm.


J . Heatmap additive intensities of RFP - actin localization in two representative mitotic U2OS cells for each genotype grown on fibronectin - coated , L - shaped micropatterns . Arrowheads indicate polarized cortical actin .

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J. Heatmap additive intensities of RFP-actin localization in two representative mitotic U2OS cells for each genotype grown on fibronectin-coated, L-shaped micropatterns. Arrowheads indicate polarized cortical actin.


B . Pull - down assay of Sec62 domain with different biotin - labeled Sec63 C - terminal peptides . Sec63 - C2P denotes di - phosphorylated Sec63 - C2 .

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B. Pull-down assay of Sec62 domain with different biotin-labeled Sec63 C-terminal peptides. Sec63-C2P denotes di-phosphorylated Sec63-C2.


C . ITC - thermogram obtained from ITC after titration of Sec62 domain with increasing amounts of Sec63 - C2P .

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C. ITC-thermogram obtained from ITC after titration of Sec62 domain with increasing amounts of Sec63-C2P.