Figure 3A . Cell lysates of LN229 cells were subjected to IP using Axin antibody or control IgG , followed by IB with indicated antibodies . B . Axin siRNA or control siRNA were transfected into LN229 cells , after 36 hours , lysate of the cells was performed with co - IP using FoxM1 antibody followed by IB with indicated antibodies . C . Axin siRNA or control siRNA were transfected into LN229 cells for 36 hours . Lysates of the cells were subjected to IB with FoxM1 phospho - S474 antibody . D . Cell lysates of LN229 cells with or without Wnt - 3a treatment for 4 hours were subjected to IP using Axin antibody or control IgG , followed by IB with FoxM1 and Axin antibodies . . E . Axin siRNA or control siRNA with HA - Ubiquitin were transfected into 293T cells , after 36 hours , cells were treated with MG132 with or without Wnt - 3a ( 50 ng / ml ) for six hours . Then lysate of the cells was performed with IP using FoxM1 antibody followed by IB with indicated antibodies . F . Wild type Flag - FoxM1 or S474A mutant was transfected into 293T cells after 36 hours , cells were treated with MG132 for six hours . Then lysate of the cells was performed with IP using Flag antibody followed by IB with indicated antibodies . G . HA - Ubiquitin and / or FBXW7 , GSK3β CA , Flag - FoxM1 were co - transfected into 293T cells , after 36 hours , cells were treated with MG132 for six hours . Then lysate of the cells was performed with IP using Flag antibody followed by IB with indicated antibodies . H . FBXW7 siRNA or control siRNA and HA - Ubiquitin were transfected into 293T cells , after 36 hours , cells were treated with MG132 with or without Wnt - 3a ( 50 ng / ml ) for six hours . Then lysate of the cells was performed with IP using FoxM1 antibody followed by IB with indicated antibodies .

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Figure 3A. Cell lysates of LN229 cells were subjected to IP using Axin antibody or control IgG, followed by IB with indicated antibodies.B. Axin siRNA or control siRNA were transfected into LN229 cells, after 36 hours, lysate of the cells was performed with co-IP using FoxM1 antibody followed by IB with indicated antibodies.C. Axin siRNA or control siRNA were transfected into LN229 cells for 36 hours. Lysates of the cells were subjected to IB with FoxM1 phospho-S474 antibody.D. Cell lysates of LN229 cells with or without Wnt-3a treatment for 4 hours were subjected to IP using Axin antibody or control IgG, followed by IB with FoxM1 and Axin antibodies..E. Axin siRNA or control siRNA with HA-Ubiquitin were transfected into 293T cells, after 36 hours, cells were treated with MG132 with or without Wnt-3a (50 ng/ml) for six hours. Then lysate of the cells was performed with IP using FoxM1 antibody followed by IB with indicated antibodies.F. Wild type Flag-FoxM1 or S474A mutant was transfected into 293T cells after 36 hours, cells were treated with MG132 for six hours. Then lysate of the cells was performed with IP using Flag antibody followed by IB with indicated antibodies.G. HA-Ubiquitin and/or FBXW7, GSK3β CA, Flag-FoxM1 were co-transfected into 293T cells, after 36 hours, cells were treated with MG132 for six hours. Then lysate of the cells was performed with IP using Flag antibody followed by IB with indicated antibodies.H. FBXW7 siRNA or control siRNA and HA-Ubiquitin were transfected into 293T cells, after 36 hours, cells were treated with MG132 with or without Wnt-3a (50 ng/ml) for six hours. Then lysate of the cells was performed with IP using FoxM1 antibody followed by IB with indicated antibodies.


Figure 4A . 293T cells were transfected with USPs cDNA or control plasmid . After 36 hr of transfection , cells were treated with 50 ng / ml Wnt - 3a for 4 hr . FoxM1 protein levels in the cells were then determined by Western blotting . B . 293T cells were transfected with 1 µg control plasmid or USP5 cDNA in 6 - well plates . After 36 hr of transfection , cells were treated with 100 µg / ml CHX for 0 , 2 , 4 , or 6 hr . FoxM1 protein levels in the cells were then determined by Western blotting . C . GSK3 - β CA , USP5 , or control plasmid was co - transfected with Flag - FoxM1 into 293T cells . After 36 hr of transfection , cells were treated with 50 ng / ml Wnt - 3a or control PBS for 4 hr . Cell lysates were subjected to IP with USP5 antibody and followed by IB with Flag or USP5 antibody . D . GSK3βCA , USP5 , or control plasmid was co - transfected with Flag - FoxM1 into 293T cells . After 36 hr of transfection , cells were treated with 50 ng / ml Wnt - 3a or control PBS for 6 hr . Cell lysates were subjected to USP5 with Flag antibody and followed by USP5 with Flag or USP5 antibody . E . USP5 and Flag - FoxM1 ( WT ) or mutants Flag - FoxM1S474A or Flag - FoxM1S474E plasmids were co - transfected into 293T cells for 36 hr . Cell lysates were subjected to IP with Flag antibody and followed by IB with Flag or USP5 antibody . F . GSK3β CA , USP5 , or control plasmid was co - transfected with Flag - FoxM1 ( WT ) or mutants Flag - FoxM1S474A into 293T cells for 36 hr . Cell lysates were subjected to IP with Flag antibody and followed by IB with Flag or USP5 antibody . G . HA - ubiquitin and Flag - FoxM1 were co - transfected with or without GSK3β CA into 293T cells . After 36 hr , cells were treated with 25 nM MG132 for 6 hr . Cell lysates were subjected to IP with Flag followed by IB with HA or FoxM1 antibody . H . HA - ubiquitin and Flag - FoxM1 were co - transfected with or without siUSP5 into U87 cells . After 36 hr , cells were treated with 50 ng / ml Wnt - 3a and 25 nM MG132 for 6 hr . Cell lysates were subjected to IP with Flag antibody followed by IB with HA or FoxM1 antibody .

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Figure 4A. 293T cells were transfected with USPs cDNA or control plasmid. After 36 hr of transfection, cells were treated with 50 ng/ml Wnt-3a for 4 hr. FoxM1 protein levels in the cells were then determined by Western blotting.B. 293T cells were transfected with 1 µg control plasmid or USP5 cDNA in 6-well plates. After 36 hr of transfection, cells were treated with 100 µg/ml CHX for 0, 2, 4, or 6 hr. FoxM1 protein levels in the cells were then determined by Western blotting.C. GSK3-β CA, USP5, or control plasmid was co-transfected with Flag-FoxM1 into 293T cells. After 36 hr of transfection, cells were treated with 50 ng/ml Wnt-3a or control PBS for 4 hr. Cell lysates were subjected to IP with USP5 antibody and followed by IB with Flag or USP5 antibody.D. GSK3βCA, USP5, or control plasmid was co-transfected with Flag-FoxM1 into 293T cells. After 36 hr of transfection, cells were treated with 50 ng/ml Wnt-3a or control PBS for 6 hr. Cell lysates were subjected to USP5 with Flag antibody and followed by USP5 with Flag or USP5 antibody.E. USP5 and Flag-FoxM1 (WT) or mutants Flag-FoxM1S474A or Flag-FoxM1S474E plasmids were co-transfected into 293T cells for 36 hr. Cell lysates were subjected to IP with Flag antibody and followed by IB with Flag or USP5 antibody.F. GSK3β CA, USP5, or control plasmid was co-transfected with Flag-FoxM1 (WT) or mutants Flag-FoxM1S474A into 293T cells for 36 hr. Cell lysates were subjected to IP with Flag antibody and followed by IB with Flag or USP5 antibody.G. HA-ubiquitin and Flag-FoxM1 were co-transfected with or without GSK3β CA into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with Flag followed by IB with HA or FoxM1 antibody.H. HA-ubiquitin and Flag-FoxM1 were co-transfected with or without siUSP5 into U87 cells. After 36 hr, cells were treated with 50 ng/ml Wnt-3a and 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with Flag antibody followed by IB with HA or FoxM1 antibody.


Figure 5A . Expression of FoxM1 and ICAT was examined via IHC staining in 30 GBM and paired adjacent normal tissues . Representative ICAT and FoxM1 expressions are shown in a section of a GBM tumor with adjacent normal brain . Scale bar : 50 µmB . ICAT stainings were scored as 1 - 12 according to the intensity and percentage in the 30 GBM and paired adjacent normal tissues . Boxes indicate interquartile range . Bars from each box extend to largest and smallest observations . C . FoxM1 stainings were scored as 1 - 12 according to the intensity and percentage in the 30 GBM and paired adjacent normal tissues . Boxes indicate interquartile range . Bars from each box extend to largest and smallest observations . D . Increased amounts of FoxM1 plasmids were transfected into 293T cells . After 36 hr , cells were treated with 50 ng / ml Wnt - 3a for 6 hr . Cell lysates were then subjected to IP with β - catenin antibody , followed by IB with β - catenin , ICAT , FoxM1 or IgG antibodies . E . Increased amounts of ICAT plasmids were transfected into 293T cells . After 36 hr , cells were treated with 50 ng / ml Wnt - 3a for 6 hr . Cell lysates were then subjected to IP with β - catenin antibody , followed by IB with β - catenin , ICAT , FoxM1 or IgG antibodies . F . FoxM1 siRNA or control siRNA was transfected into U87 cells , lysates were subjected to IP with β - catenin antibody , followed by IB with β - catenin , ICAT , FoxM1 or IgG antibodies . G . ICAT siRNA or control siRNA was transfected into LN229 cells , lysates were subjected to IP with β - catenin antibody , followed by IB with β - catenin , ICAT , FoxM1 or IgG antibodies . H . ChIP analyses of endogenous β - catenin binding at the TCF - binding site of the Axin2 promoter were performed in vector - , FoxM1 - or / and ICAT plasmid - transfected U87 cells . Fold was calculated relative to that in cells transfected with the vector , which was set as 1 . I . ChIP analyses of endogenous β - catenin binding at the TCF - binding site of the Axin2 promoter were performed in FoxM1 siRNA - , ICAT siRNA - , control siRNA - or combination of FoxM1 siRNA and ICAT siRNA - transfected U87 cells as described in ( H ) . J . ChIP analyses of endogenous β - catenin binding at the TCF - binding site of the Axin2 promoter were performed in vector - , β - catenin - NLS - , sh - FoxM1 - , FoxM1 - shR - and / or ICAT plasmid - transfected U87 cells as described in ( H ) .

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Figure 5A. Expression of FoxM1 and ICAT was examined via IHC staining in 30 GBM and paired adjacent normal tissues. Representative ICAT and FoxM1 expressions are shown in a section of a GBM tumor with adjacent normal brain. Scale bar: 50 µmB. ICAT stainings were scored as 1-12 according to the intensity and percentage in the 30 GBM and paired adjacent normal tissues. Boxes indicate interquartile range. Bars from each box extend to largest and smallest observations.C. FoxM1 stainings were scored as 1-12 according to the intensity and percentage in the 30 GBM and paired adjacent normal tissues. Boxes indicate interquartile range. Bars from each box extend to largest and smallest observations.D. Increased amounts of FoxM1 plasmids were transfected into 293T cells. After 36 hr, cells were treated with 50 ng/ml Wnt-3a for 6 hr. Cell lysates were then subjected to IP with β-catenin antibody, followed by IB with β-catenin, ICAT, FoxM1 or IgG antibodies.E. Increased amounts of ICAT plasmids were transfected into 293T cells. After 36 hr, cells were treated with 50 ng/ml Wnt-3a for 6 hr. Cell lysates were then subjected to IP with β-catenin antibody, followed by IB with β-catenin, ICAT, FoxM1 or IgG antibodies.F. FoxM1 siRNA or control siRNA was transfected into U87 cells, lysates were subjected to IP with β-catenin antibody, followed by IB with β-catenin, ICAT, FoxM1 or IgG antibodies.G. ICAT siRNA or control siRNA was transfected into LN229 cells, lysates were subjected to IP with β-catenin antibody, followed by IB with β-catenin, ICAT, FoxM1 or IgG antibodies.H. ChIP analyses of endogenous β-catenin binding at the TCF-binding site of the Axin2 promoter were performed in vector-, FoxM1- or/and ICAT plasmid-transfected U87 cells. Fold was calculated relative to that in cells transfected with the vector, which was set as 1.I. ChIP analyses of endogenous β-catenin binding at the TCF-binding site of the Axin2 promoter were performed in FoxM1 siRNA-, ICAT siRNA-, control siRNA- or combination of FoxM1 siRNA and ICAT siRNA-transfected U87 cells as described in (H).J. ChIP analyses of endogenous β-catenin binding at the TCF-binding site of the Axin2 promoter were performed in vector-, β-catenin-NLS-, sh-FoxM1-, FoxM1-shR- and/or ICAT plasmid-transfected U87 cells as described in (H).


Figure 6A . FoxM1 or / and ICAT plasmids were co - transfected with Axin - 2 promoter luciferase and TK - Renilla plasmid into U87 cells . Luciferase activity was measured with the Dural Luciferase reporter assay , and relative luciferase activity was normalized to that of the vector group . Values are mean ± SD for triplicate samples . B . FoxM1 siRNA # 1 , # 2 or control siRNA were co - transfected to U87 cells with Axin - 2 promoter reporter . Luciferase activity was measured with the Dural Luciferase reporter assay , and relative luciferase activity was normalized to that of the control siRNA group . Values are mean ± SD for triplicate samples . C . ICAT siRNA or control siRNA were co - transfected into LN229 cells with Axin - 2 promoter reporter . Luciferase activity was measured as in ( C ) . D . FoxM1 or / and ICAT plasmids were co - transfected with TOP - Flash reporter and TK - Renilla plasmids into U87 cells . Luciferase activity of TOP - Flash in the cells was measured as in A . Values are mean ± SD for triplicate samples . E . ICAT , FoxM1 or FoxM1 - ∆ FB ( FoxM1 mutant lacking the forkhead box domain ) plasmid or their combinations were co - transfected with TOP - Flash reporter and TK - Renilla plasmids into LN229 cells . Luciferase activity of TOP - Flash in the cells was measured as in A . Values are mean ± SD for triplicate samples . F . FoxM1 siRNA and / or ICAT siRNA were co - transfected with TOP - Flash reporter and TK - Renilla plasmids into LN229 cells , and the cells were treated with Wnt3a ( 50 ng / ml ) or control PBS . Luciferase activity of TOP - Flash in the cells was measured as in A . Values are mean ± SD for triplicate samples . G . Vector - , β - catenin - NLS - , sh - FoxM1 - , FoxM1 - shR - and / or ICAT plasmids were co - transfected with TOP - Flash reporter and TK - Renilla plasmids into U87 cells . Luciferase activity of TOP - Flash in the cells was measured as in ( A ) . Values are mean ± SD triplicate samples . H . Levels of Axin - 2 and cyclin D1 protein in LN229 cells transfected with FoxM1 siRNA # 1 or # 2 were determined by Western blotting . I . Levels of Axin - 2 and cyclin D1 protein in LN229 cells transfected with ICAT siRNA were determined by Western blotting . J . Levels of Axin - 2 and cyclin D1 protein in LN229 cells transfected with FoxM1 or / and ICAT plasmids were determined by Western blotting .

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Figure 6A. FoxM1 or/and ICAT plasmids were co-transfected with Axin-2 promoter luciferase and TK-Renilla plasmid into U87 cells. Luciferase activity was measured with the Dural Luciferase reporter assay, and relative luciferase activity was normalized to that of the vector group. Values are mean ± SD for triplicate samples.B. FoxM1 siRNA#1, #2 or control siRNA were co-transfected to U87 cells with Axin-2 promoter reporter. Luciferase activity was measured with the Dural Luciferase reporter assay, and relative luciferase activity was normalized to that of the control siRNA group. Values are mean ± SD for triplicate samples.C. ICAT siRNA or control siRNA were co-transfected into LN229 cells with Axin-2 promoter reporter. Luciferase activity was measured as in (C).D. FoxM1 or/and ICAT plasmids were co-transfected with TOP-Flash reporter and TK-Renilla plasmids into U87 cells. Luciferase activity of TOP-Flash in the cells was measured as in A. Values are mean ± SD for triplicate samples.E. ICAT, FoxM1 or FoxM1-∆FB (FoxM1 mutant lacking the forkhead box domain) plasmid or their combinations were co-transfected with TOP-Flash reporter and TK-Renilla plasmids into LN229 cells. Luciferase activity of TOP-Flash in the cells was measured as in A. Values are mean ± SD for triplicate samples.F. FoxM1 siRNA and/or ICAT siRNA were co-transfected with TOP-Flash reporter and TK-Renilla plasmids into LN229 cells, and the cells were treated with Wnt3a (50 ng/ml) or control PBS. Luciferase activity of TOP-Flash in the cells was measured as in A. Values are mean ± SD for triplicate samples.G. Vector-, β-catenin-NLS-, sh-FoxM1-, FoxM1-shR- and/or ICAT plasmids were co-transfected with TOP-Flash reporter and TK-Renilla plasmids into U87 cells. Luciferase activity of TOP-Flash in the cells was measured as in (A). Values are mean ± SD triplicate samples.H. Levels of Axin-2 and cyclin D1 protein in LN229 cells transfected with FoxM1 siRNA#1 or #2 were determined by Western blotting.I. Levels of Axin-2 and cyclin D1 protein in LN229 cells transfected with ICAT siRNA were determined by Western blotting.J. Levels of Axin-2 and cyclin D1 protein in LN229 cells transfected with FoxM1 or/and ICAT plasmids were determined by Western blotting.


Figure 7A . A total of 1 × 104 LN229 cells were transfected with control vector , FoxM1 , and / or ICAT plasmids and treated with Wnt3a ( 50 ng / ml ) and were then plated and counted 48 hr after seeding . The proliferation percentage was calculated relative to that in cells transfected with the control vector , which was set as 100 % . Data represent the mean ± SD of three independent experiments . B . LN229 cells were transfected with vector control , FoxM1 , FoxM1 plus sh - β - catenin or FoxM1 plus ICAT plasmid . The proliferation percentage was determined as described in ( A ) . C . LN229 - shFoxM1 cells were transfected with FoxM1 - shR ( siRNA - resistant FoxM1 ) , or FoxM1 - shR plus ICAT plasmid . The cells were then treated with Wnt3a ( 50 ng / ml ) . The proliferation percentage was determined as described in ( A ) . D . LN229 cells were transfected with USP5 and / or FoxM1 siRNA . The proliferation percentage was determined as described in ( A ) . E . LN229 cells were transfected with USP5 siRNA and / or FoxM1 plasmid . The cells were then treated with Wnt3a ( 50 ng / ml ) . The proliferation percentage was determined as described in ( A ) . Data shown in ( A ) - ( E ) represent the mean ± SD of three independent experiments . F - H . Expression of FoxM1 , Cyclin D1 and USP5 proteins was examined via IHC staining in 50 GBM specimens . Staining of FoxM1 , Cyclin D1 , or USP5 was scored as 1 - 12 according to the intensity and percentage . The significance of the correlations was determined by Pearson ' s correlation test . I - J . Axin2 mRNA levels in the above 50 GBM specimens were examined through RNAscope technology . The staining was scored as 1 - 12 according to the intensity and percentage . The significance of the correlations was determined by Pearson ' s correlation test .

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Figure 7A. A total of 1 × 104 LN229 cells were transfected with control vector, FoxM1, and/or ICAT plasmids and treated with Wnt3a (50 ng/ml) and were then plated and counted 48 hr after seeding. The proliferation percentage was calculated relative to that in cells transfected with the control vector, which was set as 100%. Data represent the mean ± SD of three independent experiments.B. LN229 cells were transfected with vector control, FoxM1, FoxM1 plus sh-β-catenin or FoxM1 plus ICAT plasmid. The proliferation percentage was determined as described in (A).C. LN229-shFoxM1 cells were transfected with FoxM1-shR (siRNA-resistant FoxM1), or FoxM1-shR plus ICAT plasmid. The cells were then treated with Wnt3a (50 ng/ml). The proliferation percentage was determined as described in (A).D. LN229 cells were transfected with USP5 and/or FoxM1 siRNA. The proliferation percentage was determined as described in (A).E. LN229 cells were transfected with USP5 siRNA and/or FoxM1 plasmid. The cells were then treated with Wnt3a (50 ng/ml). The proliferation percentage was determined as described in (A). Data shown in (A)-(E) represent the mean ± SD of three independent experiments.F-H. Expression of FoxM1, Cyclin D1 and USP5 proteins was examined via IHC staining in 50 GBM specimens. Staining of FoxM1, Cyclin D1, or USP5 was scored as 1-12 according to the intensity and percentage. The significance of the correlations was determined by Pearson's correlation test.I-J. Axin2 mRNA levels in the above 50 GBM specimens were examined through RNAscope technology. The staining was scored as 1-12 according to the intensity and percentage. The significance of the correlations was determined by Pearson's correlation test.


Figure 1B Equilibrium titration of Hsf1 binding to Alexa Fluor ® 488 - labeled HSE - DNA . Fluorescence polarization plotted versus the Hsf1 trimer concentration . KD = 4 . 7 ± 1 . 9 nM ( n = 4 ) . C Hsf1 rapidly switches between different HSE - containing double stranded DNA oligonucleotides . Trimeric Hsf1 was pre - incubated with Alexa Fluor ® 488 - labeled HSE - DNA in MgCl2 - free buffer . At timepoint 0 ( arrow ) buffer , 4 mM MgCl2 , 2 mM ATP + 4 mM MgCl2 , control DNA ( DNACtrl at 10 - fold molar excess over HSE - DNA ) , and HSE - containing DNA was added as indicated and fluorescence polarization followed over time . D Trimeric Hsf1 were bound to HSE - DNA and the indicated components added at timepoint 0 . E Hsp70 dissociates Hsf1 from DNA with similar rates as Hsc70 . Rates were determinedThe rate of Hsc70 / DnaJB1 - mediated dissociation of Hsf1 from HSE - DNA depends on temperature ( for 25 ° C n = 6 , for 37 ° C n = 5 , T - test , p = 0 . 011 ) ( F ) The rate of Hsc70 / DnaJB1 - mediated dissociation of Hsf1 from HSE - DNA depends on concentration of Hsc70 and DnaJB1 ( n = 3 ) ( G ) The rate of Hsc70 / DnaJB1 - mediated dissociation of Hsf1 from HSE - DNA depends on concentration of ATP ( n = 3 ) ( H ) The rate of Hsc70 / DnaJB1 - mediated dissociation of Hsf1 from HSE - DNA depends on concentration of the nucleotide exchange factor Apg2 ( HSPA4 ) ( n = 3 , ANOVA , * * , p < 0 . 01 ; * * * * , p < 0 . 0001 ) ( I ) . Shown are mean ± SD .

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Figure 1B Equilibrium titration of Hsf1 binding to Alexa Fluor® 488-labeled HSE-DNA. Fluorescence polarization plotted versus the Hsf1 trimer concentration. KD = 4.7 ± 1.9 nM (n = 4).C Hsf1 rapidly switches between different HSE-containing double stranded DNA oligonucleotides. Trimeric Hsf1 was pre-incubated with Alexa Fluor® 488-labeled HSE-DNA in MgCl2-free buffer. At timepoint 0 (arrow) buffer, 4 mM MgCl2, 2 mM ATP+4 mM MgCl2, control DNA (DNACtrl at 10-fold molar excess over HSE-DNA), and HSE-containing DNA was added as indicated and fluorescence polarization followed over time.D Trimeric Hsf1 were bound to HSE-DNA and the indicated components added at timepoint 0.E Hsp70 dissociates Hsf1 from DNA with similar rates as Hsc70. Rates were determinedThe rate of Hsc70/DnaJB1-mediated dissociation of Hsf1 from HSE-DNA depends on temperature (for 25°C n = 6, for 37°C n = 5, T-test, p = 0.011) (F)The rate of Hsc70/DnaJB1-mediated dissociation of Hsf1 from HSE-DNA depends on concentration of Hsc70 and DnaJB1 (n = 3) (G)The rate of Hsc70/DnaJB1-mediated dissociation of Hsf1 from HSE-DNA depends on concentration of ATP (n = 3) (H)The rate of Hsc70/DnaJB1-mediated dissociation of Hsf1 from HSE-DNA depends on concentration of the nucleotide exchange factor Apg2 (HSPA4) (n = 3, ANOVA, **, p < 0.01; ****, p < 0.0001) (I). Shown are mean ± SD.


Figure 2A , B Standard Hsc70 / DnaJB1 - mediated dissociation reaction of Hsf1 from Alexa Fluor ® 488 - labeled HSE - DNA monitored by fluorescence polarization ( A ) . The same reaction mixture was incubated for different time intervals as indicated by the arrows below the x - axis , the reaction stopped by addition of blue - native loading buffer and stored on ice until separation by blue - native PAGE , blotted onto a PVDF membrane and detected with an Hsf1 - specific antiserum ( B ) . Lanes 1 , purified Hsf1 trimer ( T ) ; 2 , purified Hsf1 monomer ( M ) ; 3 - 11 , samples from the Hsc70 / DnaJB1 - mediated Hsf1 dissociation reaction ( 0 to 80 min ) ; 12 - 15 , Dissociation reaction incubated for 80 min missing individual components as indicated . HO , higher order oligomers ; T , trimer ; D , dimer ; M , mono mer . C Quantification of the amounts of Hsf1 species of the blot shown in ( B ) and five similar blots . For each lane the intensities of the two areas indicated by the brackets to the right were integrated ; upper bracket , DNA bound timers and higher order oligomers ( Hsf1b ) ; lower bracket , monomers and Hsf1 - species possibly bound by Hsc70 or DnaJB1 or both dissociated from DNA ( Hsf1d ) . Shown are means ± SD ( n = 3 ) . Inset , deoligomerization rate as determined by fitting an exponential decay function to the data . Shown are means ± SD ( n = 3 )

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Figure 2A,B Standard Hsc70/DnaJB1-mediated dissociation reaction of Hsf1 from Alexa Fluor® 488-labeled HSE-DNA monitored by fluorescence polarization (A). The same reaction mixture was incubated for different time intervals as indicated by the arrows below the x-axis, the reaction stopped by addition of blue-native loading buffer and stored on ice until separation by blue-native PAGE, blotted onto a PVDF membrane and detected with an Hsf1-specific antiserum (B). Lanes 1, purified Hsf1 trimer (T); 2, purified Hsf1 monomer (M); 3-11, samples from the Hsc70/DnaJB1-mediated Hsf1 dissociation reaction (0 to 80 min); 12-15, Dissociation reaction incubated for 80 min missing individual components as indicated. HO, higher order oligomers; T, trimer; D, dimer; M, monomer. C Quantification of the amounts of Hsf1 species of the blot shown in (B) and five similar blots. For each lane the intensities of the two areas indicated by the brackets to the right were integrated; upper bracket, DNA bound timers and higher order oligomers (Hsf1b); lower bracket, monomers and Hsf1-species possibly bound by Hsc70 or DnaJB1 or both dissociated from DNA (Hsf1d). Shown are means ± SD (n = 3). Inset, deoligomerization rate as determined by fitting an exponential decay function to the data. Shown are means ± SD (n = 3)


Figure 3C Fit of two Gaussian distributions to the peak intensities for peptic peptides 159 - 168 ( left ) , 169 - 175 ( middle ) , and 176 - 189 ( right ) . Representative plots of 3 independent experiments . E Relative deuteron incorporation of the low ( solid bars ) and high ( open bars ) exchanging subpopulations .

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Figure 3C Fit of two Gaussian distributions to the peak intensities for peptic peptides 159-168 (left), 169-175 (middle), and 176-189 (right). Representative plots of 3 independent experiments.E Relative deuteron incorporation of the low (solid bars) and high (open bars) exchanging subpopulations.


Figure 4B KD values for the indicated peptides as determined by fluorescence polarization . Peptides N74 - Q86 - C ( NMYGFRKVVHIEQC ) , P223 - H235 - C ( PKYSRQFSLEHVHC ) and A343 - R352 - C ( ASVTALTDARC ) represent the most hydrophobic part of HX protected segments 78 - 98 , 229 - 252 , and 347 - 375 , respectively ; N200 - K208 - C ( NRILGVKRKC ) , N200 - K208 - C _ SSS ( NRSSGSKRKC ) , G204 - N214 - C ( GVKRKIPLMLNC ) , and G204 - N214 - C _ SSS ( GVKRKSPSMSNC ) represent the two potential Hsc70 binding sites in segment 202 - 213 in original sequence or with hydrophobic residues replaced by serine ; S461 - Q471 - C ( SGKQLVHYTAQC ) and Y468 - S480 - C ( YTAQPLFLLDPGSC ) represent the two potential Hsc70 binding sites in segment 442 - 474 . C Hsf1 ( 1 - 408 ) with a deleted TAD is dissociated from HSE - DNA by Hsc70 , DnaJB1 and ATP with similar rates as Hsf1wt . Fluorescence polarization assays containing Alexa Fluor ® 488 - HSE - DNA bound Hsf1 ( 1 - 408 ) trimers and the indicated components . D The HR - B proximal Hsc70 binding site is essential for Hsc70 / DnaJB1 - mediated dissociation of HSE - DNA bound Hsf1 .

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Figure 4B KD values for the indicated peptides as determined by fluorescence polarization. Peptides N74-Q86-C (NMYGFRKVVHIEQC), P223-H235-C (PKYSRQFSLEHVHC) and A343-R352-C (ASVTALTDARC) represent the most hydrophobic part of HX protected segments 78-98, 229-252, and 347-375, respectively; N200-K208-C (NRILGVKRKC), N200-K208-C_SSS (NRSSGSKRKC), G204-N214-C (GVKRKIPLMLNC), and G204-N214-C_SSS (GVKRKSPSMSNC) represent the two potential Hsc70 binding sites in segment 202-213 in original sequence or with hydrophobic residues replaced by serine; S461-Q471-C (SGKQLVHYTAQC) and Y468-S480-C (YTAQPLFLLDPGSC) represent the two potential Hsc70 binding sites in segment 442-474.C Hsf1(1-408) with a deleted TAD is dissociated from HSE-DNA by Hsc70, DnaJB1 and ATP with similar rates as Hsf1wt. Fluorescence polarization assays containing Alexa Fluor® 488-HSE-DNA bound Hsf1(1-408) trimers and the indicated components.D The HR-B proximal Hsc70 binding site is essential for Hsc70/DnaJB1-mediated dissociation of HSE-DNA bound Hsf1.


Figure 5A The rate of Hsc70 / DnaJB1 - mediated dissociation of HSE - DNA bound Hsf1 depends on the number of HR - B proximal Hsc70 binding sites in the Hsf1 trimer . Hsf1wt ( wt ) and Hsf1 ∆ ( 202 - 213 ) ( ∆ ) monomers were mixed in the indicated ratios and heat shocked at 42 ° C for 10 min to form mixtures of different homo - and heterotrimers as indicated with the cartoons ( the red line and the arrow head indicate the Hsc70 binding site ) . Numbers indicate the relative fraction of the different species . B Moving the Hsc70 binding site away from the trimerization domain reduces the rate of Hsc70 / DnaJB1 - mediated dissociation of HSE - DNA - bound Hsf1 . Hsf1 _ t10 / 20 / 30 , region 202 - 213 moved by 10 , 20 , or 30 residues towards the C - terminus . C Anti - FLAG antibodies are not able to dissociate HSE - DNA - bound Hsf1 . Red lines in the cartoon indicate the Hsc70 binding site ; blue lines and arrow head indicate the inserted FLAG epitope DYKDDDDK . Hsf1 _ i201 / 211 / 221 , FLAG - epitope inserted after residue 201 , 211 or 221 . Anti - FLAG antibodies were split in halfmers by incubation with 2 mM DTTAnti - FLAG antibody halfmers bound to FLAG - epitope containing Hsf1 variants . Hsf1wt and FLAG - insertion variants were analyzed by blue - native gel electrophoresis in the absence or presence of DTT - treated anti - FLAG antibodies as indicated and Hsf1 detected by immunoblotting ; t - bound , FLAG antibody - bound Hsf1 trimers ; m - bound , FLAG antibody bound Hsf1 monomer . F Trimer - to - monomer ratio of freshly purified Hsf1wt and Hsf1 - I190S , I194S in the absence of heat shock , determined by gel filtration and BN - GelG Rate of Hsc70 / DnaJB1 - mediated dissociation of HSE - DNA bound Hsf1wt and Hsf1 - I190S ,

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Figure 5A The rate of Hsc70/DnaJB1-mediated dissociation of HSE-DNA bound Hsf1 depends on the number of HR-B proximal Hsc70 binding sites in the Hsf1 trimer. Hsf1wt (wt) and Hsf1∆(202-213) (∆) monomers were mixed in the indicated ratios and heat shocked at 42°C for 10 min to form mixtures of different homo- and heterotrimers as indicated with the cartoons (the red line and the arrow head indicate the Hsc70 binding site). Numbers indicate the relative fraction of the different species.B Moving the Hsc70 binding site away from the trimerization domain reduces the rate of Hsc70/DnaJB1-mediated dissociation of HSE-DNA-bound Hsf1. Hsf1_t10/20/30, region 202-213 moved by 10, 20, or 30 residues towards the C-terminus.C Anti-FLAG antibodies are not able to dissociate HSE-DNA-bound Hsf1. Red lines in the cartoon indicate the Hsc70 binding site; blue lines and arrow head indicate the inserted FLAG epitope DYKDDDDK. Hsf1_i201/211/221, FLAG-epitope inserted after residue 201, 211 or 221. Anti-FLAG antibodies were split in halfmers by incubation with 2 mM DTTAnti-FLAG antibody halfmers bound to FLAG-epitope containing Hsf1 variants. Hsf1wt and FLAG-insertion variants were analyzed by blue-native gel electrophoresis in the absence or presence of DTT-treated anti-FLAG antibodies as indicated and Hsf1 detected by immunoblotting; t-bound, FLAG antibody-bound Hsf1 trimers; m-bound, FLAG antibody bound Hsf1 monomer.F Trimer-to-monomer ratio of freshly purified Hsf1wt and Hsf1-I190S,I194S in the absence of heat shock, determined by gel filtration and BN-GelG Rate of Hsc70/DnaJB1-mediated dissociation of HSE-DNA bound Hsf1wt and Hsf1-I190S,I194S


Figure 6A Schematics of the used system . MEFs , in which HSF1 on chromosome 8 was deleted , were stably transfected with mTag - BFP expressed under the control of the HSPA6 promoter and subsequently transiently transfected with plasmids that expressed HSF1wt or mutant variants in an artificial operon with hrGFP , which is translated from an IRES element . B Exemplary flow cytometry data plotting BFP fluorescence versus GFP fluorescence , indicating the gates used for analysis . Numbers indicate the fraction ( % ) of cells ( forward scatter singlets ) in each gate . Color indicates the cell density from dark blue ( low ) to red ( high ) . C - H Geometric mean of BFP ( C , E ) and GFP fluorescence ( D , F , H ) of cells grown at 37 ° C ( non - stress , NS ) or incubated for 1 h at 43 ° C with a recovery period of 3 h at 37 ° C ( heat shocked , HS ) in the GFPhigh ( C , D ) , GFPlow ( E , F ) , and GFPlow - BFPhigh ( H ) gates . ( G ) Geometric mean of BFP fluorescence weighted by the relative number of cells in the GFPlow - BFPhigh gate ( fluorescence * fraction forward scatter singlets ) . Note that the GFPlow gate also comprises the GFPlow - BFPhigh gate . Hsf1 - 202SSS , Hsf1 - I202S , L203S , V205S ; Hsf1 - 209SSS , Hsf1 - I209S , L211S , L213S ; Hsf1 - 427SSSS , Hsf1 - M427S , L429S , L432S , L436S ; Hsf1 - 465SSS , Hsf1 - L465S , V466S , Y468S ; Hsf1 - 473SSSS , Hsf1 - L473S , F474S , L475S ,

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Figure 6A Schematics of the used system. MEFs, in which HSF1 on chromosome 8 was deleted, were stably transfected with mTag-BFP expressed under the control of the HSPA6 promoter and subsequently transiently transfected with plasmids that expressed HSF1wt or mutant variants in an artificial operon with hrGFP, which is translated from an IRES element. B Exemplary flow cytometry data plotting BFP fluorescence versus GFP fluorescence, indicating the gates used for analysis. Numbers indicate the fraction (%) of cells (forward scatter singlets) in each gate. Color indicates the cell density from dark blue (low) to red (high).C-H Geometric mean of BFP (C, E) and GFP fluorescence (D, F, H) of cells grown at 37°C (non-stress, NS) or incubated for 1 h at 43°C with a recovery period of 3 h at 37°C (heat shocked, HS) in the GFPhigh (C, D), GFPlow (E, F), and GFPlow-BFPhigh (H) gates. (G) Geometric mean of BFP fluorescence weighted by the relative number of cells in the GFPlow-BFPhigh gate (fluorescence * fraction forward scatter singlets). Note that the GFPlow gate also comprises the GFPlow-BFPhigh gate. Hsf1-202SSS, Hsf1-I202S,L203S,V205S; Hsf1-209SSS, Hsf1-I209S,L211S,L213S; Hsf1-427SSSS, Hsf1-M427S,L429S,L432S,L436S; Hsf1-465SSS, Hsf1-L465S,V466S,Y468S; Hsf1-473SSSS, Hsf1-L473S,F474S,L475S,L476S


Figure 7A Schematics of the used system . HeLa cells were stably transfected with mTag - BFP expressed under the control of the HSPA6 promoter and subsequently transiently transfected with plasmids that expressed HSF1wt or mutant variants in an artificial operon with hrGFP , which is translated from an IRES element . Exemplary flow cytometry data plotting BFP fluorescence versus GFP fluorescence , indicating the gates used for analysis . Numbers indicate the fraction ( % ) of cells ( forward scatter singlets ) in each gate . Color indicates the cell density from dark blue ( low ) to red ( high ) . NS , cells grown at 37 ° C ; HS , cells heat shocked at 43 ° C for 1 h with a recovery period of 3 h at 37 ° C . C - H Geometric mean of BFP ( C , E ) and GFP fluorescence ( D , F , H ) in the GFPhigh ( C , D ) , GFPlow ( E , F ) , and GFPlow - BFPhigh43 ( H ) gates . Of note , the number of data points show the number of experiments in which a significant number of cells was found in this gate and missing bars indicate that cells did not fall within this gate in more than two out of four experiments . ( G ) Geometric mean of BFP fluorescence weighted by the relative number of cells in the GFPlow - BFPhigh43 gate ( fluorescence * fraction forward scatter singlets ) . Note that the GFPlow gate also comprises the GFPlow - BFPhigh37 and that the GFPlow - BFPhigh37 gate also comprises the GFPlow - BFPhigh43 gate . Hsf1 - 202SSS , Hsf1 - I202S , L203S , V205S ; Hsf1 - 209SSS , Hsf1 - I209S , L211S , L213S ; Hsf1 - 427SSSS , Hsf1 - M427S , L429S , L432S , L436S ; Hsf1 - 465SSS , Hsf1 - L465S , V466S , Y468S ; Hsf1 - 473SSSS , Hsf1 - L473S , F474S , L475S ,

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Figure 7A Schematics of the used system. HeLa cells were stably transfected with mTag-BFP expressed under the control of the HSPA6 promoter and subsequently transiently transfected with plasmids that expressed HSF1wt or mutant variants in an artificial operon with hrGFP, which is translated from an IRES element. Exemplary flow cytometry data plotting BFP fluorescence versus GFP fluorescence, indicating the gates used for analysis. Numbers indicate the fraction (%) of cells (forward scatter singlets) in each gate. Color indicates the cell density from dark blue (low) to red (high). NS, cells grown at 37°C; HS, cells heat shocked at 43°C for 1 h with a recovery period of 3 h at 37°C.C-H Geometric mean of BFP (C, E) and GFP fluorescence (D, F, H) in the GFPhigh (C, D), GFPlow (E, F), and GFPlow-BFPhigh43 (H) gates. Of note, the number of data points show the number of experiments in which a significant number of cells was found in this gate and missing bars indicate that cells did not fall within this gate in more than two out of four experiments. (G) Geometric mean of BFP fluorescence weighted by the relative number of cells in the GFPlow-BFPhigh43 gate (fluorescence * fraction forward scatter singlets). Note that the GFPlow gate also comprises the GFPlow-BFPhigh37 and that the GFPlow-BFPhigh37 gate also comprises the GFPlow-BFPhigh43 gate. Hsf1-202SSS, Hsf1-I202S,L203S,V205S; Hsf1-209SSS, Hsf1-I209S,L211S,L213S; Hsf1-427SSSS, Hsf1-M427S,L429S,L432S,L436S; Hsf1-465SSS, Hsf1-L465S,V466S,Y468S; Hsf1-473SSSS, Hsf1-L473S,F474S,L475S,L476S


Figure 1Micrographs of S2 cells stained with PicoGreen , following 5 days of treatment with the dsRNA indicated . GFP and tamas ( Polg , CG8987 ) were used as negative and positive controls , respectively . In rescreening , CG5924 was also used as a positive control . Both were detected in the blinded screen as positives . Other images show a typical negative ( CG31380 ) , a typical positive ( CG6413 ) and a typical case of a target classed as abnormal , in this case CG2028 ( CkIα ) , which showed a decreased number of nucleoid signals per cell . Images are optimized for brightness and contrast but with no other manipulations . Scale bar indicates 50 μm .

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Figure 1Micrographs of S2 cells stained with PicoGreen, following 5 days of treatment with the dsRNA indicated. GFP and tamas (Polg, CG8987) were used as negative and positive controls, respectively. In rescreening, CG5924 was also used as a positive control. Both were detected in the blinded screen as positives. Other images show a typical negative (CG31380), a typical positive (CG6413) and a typical case of a target classed as abnormal, in this case CG2028 (CkIα), which showed a decreased number of nucleoid signals per cell. Images are optimized for brightness and contrast but with no other manipulations. Scale bar indicates 50 μm.


Figure 3A - C Relative mtDNA level ( A ) , TMRM ( B ) and MitoSox ( C ) following treatment for 5 days with dsRNA against the genes indicated . D MitoSox fluorescence following treatment for 1 - 3 days with dsRNA against the genes indicated . E Whole cell respiration following treatment for 5 days with dsRNA against the genes indicated . F , G Relative mtDNA level ( F ) and MitoSox fluorescence ( G ) after 5 days of treatment with oligomycin .

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Figure 3A-C Relative mtDNA level (A), TMRM (B) and MitoSox (C) following treatment for 5 days with dsRNA against the genes indicated.D MitoSox fluorescence following treatment for 1-3 days with dsRNA against the genes indicated.E Whole cell respiration following treatment for 5 days with dsRNA against the genes indicated.F, G Relative mtDNA level (F) and MitoSox fluorescence (G) after 5 days of treatment with oligomycin.


Figure 4A ‐ D Relative mtDNA level ( A , B ) and MitoSox fluorescence ( C , D ) following 5 days of treatment with dsRNA against the genes indicated . E ‐ G Relative mtDNA level ( E ) , MitoSox fluorescence ( F ) and TMRM fluorescence ( G ) of cells stably expressing AOX or empty vector , as indicated , after 5 days of treatment with dsRNA against CG2968 , as shown . Note that these cells were cultured in the presence of hygromycin to maintain the AOX ‐ expressing or control plasmid . H Indicated parameters , following 5 days of treatment with dsRNA against CG2968 , in cells grown in galactose ‐ containing ( Gal ) medium .

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Figure 4A‐D Relative mtDNA level (A, B) and MitoSox fluorescence (C, D) following 5 days of treatment with dsRNA against the genes indicated.E‐G Relative mtDNA level (E), MitoSox fluorescence (F) and TMRM fluorescence (G) of cells stably expressing AOX or empty vector, as indicated, after 5 days of treatment with dsRNA against CG2968, as shown. Note that these cells were cultured in the presence of hygromycin to maintain the AOX‐expressing or control plasmid.H Indicated parameters, following 5 days of treatment with dsRNA against CG2968, in cells grown in galactose‐containing (Gal) medium.


Figure 5A . LysoTracker Red and NAO fluorescence of cells treated as indicated for 5 days with dsRNA against the indicated genes , normalized against the values for untreated cells . Mean ± SD for four independent experiments ; asterisks indicate significant differences from untreated , P < 0 . 05 . B . Representative confocal microscopy images of living cells treated for 5 days with dsRNA against the indicated genes and stained with both MitoTracker Green and LysoTracker Red . The scale bar is 10 μm . C . Mean areas of LysoTracker red staining in cells treated for 5 days with dsRNA against the indicated genes , or with chloroquine . Mean ± SD for three independent experiments , in each of which ≥ 50 cells were analyzed by confocal microscopy in a single plane . Analysis of z − stacks in ≥ 30 cells gave similar results . Note that the increase in lysosome content was intermediate between that of untreated cells and cells treated with chloroquine to block lysosomal turnover of autophagosomes . Asterisks indicate significant differences from untreated , P < 0 . 01 . D . Western blot of total protein extracts from control S2 cells and cells treated with dsRNAs against GFP or CG2968 ( cV ) , probed for NDUFS3 ( cI ) , ATP synthase subunit α ( cV ) and GAPDH ( loading control ) . E . Relative mtDNA level after 5 days of treatment with dsRNAs against the indicated genes . Mean ± SD for five independent experiments ; other data classes were significantly different from untreated , P < 0 . 05 .

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Figure 5A. LysoTracker Red and NAO fluorescence of cells treated as indicated for 5 days with dsRNA against the indicated genes, normalized against the values for untreated cells. Mean ± SD for four independent experiments; asterisks indicate significant differences from untreated, P < 0.05.B. Representative confocal microscopy images of living cells treated for 5 days with dsRNA against the indicated genes and stained with both MitoTracker Green and LysoTracker Red. The scale bar is 10 μm.C. Mean areas of LysoTracker red staining in cells treated for 5 days with dsRNA against the indicated genes, or with chloroquine. Mean ± SD for three independent experiments, in each of which ≥ 50 cells were analyzed by confocal microscopy in a single plane. Analysis of z−stacks in ≥ 30 cells gave similar results. Note that the increase in lysosome content was intermediate between that of untreated cells and cells treated with chloroquine to block lysosomal turnover of autophagosomes. Asterisks indicate significant differences from untreated, P < 0.01.D. Western blot of total protein extracts from control S2 cells and cells treated with dsRNAs against GFP or CG2968 (cV), probed for NDUFS3 (cI), ATP synthase subunit α (cV) and GAPDH (loading control).E. Relative mtDNA level after 5 days of treatment with dsRNAs against the indicated genes. Mean ± SD for five independent experiments; other data classes were significantly different from untreated, P < 0.05.


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 )

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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)


Figure 8 ( 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 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 - 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 ) ( 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8 (A) 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 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-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)(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 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), respectively


Figure 1 ( C ) Mass spectra of recombinant HTTex1 23Q after 16 hour of co - incubation with by TBK1 showing complete phosphorylation of S13 and S16 . The lower panel is a representative western blot of the same phosphorylation reaction ( upper panel ) using anti - pT3 ( CHDI - 90001528 - 1 ) , pS13 HTT ( CHDI - 90001039 - 1 ) and pS16 ( ZCH11020 - generated in house ) , antibodies ( ab ) after the indicated phosphorylation reaction times . ( D ) Representative western blot of HTT ( ab - MAB5492 ) , HTT pS13 ( ab - CHDI - 90001039 - 1 ) and HTT pT3 ( ab - CHDI - 90001528 - 1 ) upon coexpression of HTTex1 16Q and 72Q eGFP with the indicated kinases for 48 hour in HEK293T cells . ( E ) Quantification of the fold changes in the HTT pS13 and pT3 ratios to total HTT compared to empty vector ( EV ) control upon coexpression of the indicated kinase from the experiments like in D .

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Figure 1(C) Mass spectra of recombinant HTTex1 23Q after 16 hour of co-incubation with by TBK1 showing complete phosphorylation of S13 and S16. The lower panel is a representative western blot of the same phosphorylation reaction (upper panel) using anti-pT3 (CHDI-90001528-1), pS13 HTT (CHDI-90001039-1) and pS16 (ZCH11020 -generated in house), antibodies (ab) after the indicated phosphorylation reaction times.(D) Representative western blot of HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) and HTT pT3 (ab-CHDI-90001528-1) upon coexpression of HTTex1 16Q and 72Q eGFP with the indicated kinases for 48 hour in HEK293T cells.(E) Quantification of the fold changes in the HTT pS13 and pT3 ratios to total HTT compared to empty vector (EV) control upon coexpression of the indicated kinase from the experiments like in D.


Figure 2 ( A ) Representative western blot of HTT ( ab - MAB5492 ) , HTT pS13 ( ab - CHDI - 90001039 - 1 ) and HTT pT3 ( ab - CHDI - 90001528 - 1 ) upon coexpression of HTTex1 16Q eGFP , mutants with S to A substitutions at T3 or S13 / S16 or HTTex1 72Q eGFP with TBK1 or TBK1 kinase - dead ( KD ) for 48 hour in HEK293T cells ( the additional high molecular weight HTTex1 and HTT pS13 band is possibly due to the co - occurrence of phosphorylation at both S13 and T3 ) . ( B ) Quantification of the fold changes in the HTT pS13 ( upper panel ) and pT3 ( lower panel ) ratio to total HTT compared to those of the kinase - dead mutant from the experiments like in A ( n = 3 ) . ( C ) Representative western blot of HTT ( ab - MAB2166 ) and HTT pS13 ( ab - CHDI - 90001039 - 1 ) upon coexpression of HTT N543 55Q with the indicated kinase for 24 hour in HEK293T cells . ( D ) Quantification of HTT pS13 using the Singulex assay ( using ab - MW1 as capture and CHDI - 90001039 or 2B7 as detection antibodies for pS13 or total HTT ) upon coexpression of HTT N543 55Q with the indicated kinase for 24 hour in HEK293T cells from the experiment in C ( representative of 3 repeats ) . ( E ) Representative western blot of HTT ( ab - MAB2166 ) and HTT pS13 ( ab - CHDI - 90001039 - 1 ) upon coexpression of HTT - FL 48Q or HTT - FL 48Q S13A / S16A with the indicated kinase for 24 hour in HEK293T cells . ( F ) Quantification of HTT pS13 using the Singulex assay ( using ab - MW1 as capture and CHDI - 90001039 or 2B7 as detection antibodies for pS13 or total HTT ) upon coexpression of HTT - FL 48Q or HTT - FL 48Q S13A / S16A with the indicated kinase for 24 hour in HEK293T cells from the experiments likein E ( n = 3 ) . ( G ) Representative western blot of immunoprecipitated ( IP ) HTT ( ab - MAB2166 ) and HTT pS13 ( ab - CHDI - 90001039 - 1 ) after lentivirus ( LV ) - mediated overexpression of TBK1 or TBK1 KD for 72 hour in rat primary striatal neuronal cells . ( H ) Quantification of the fold changes in the HTT pS13 ratio to total HTT compared to those of the kinase - dead mutant from the experiments like in G ( n = 3 ) .

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

Figure 2(A) Representative western blot of HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) and HTT pT3 (ab-CHDI-90001528-1) upon coexpression of HTTex1 16Q eGFP, mutants with S to A substitutions at T3 or S13/S16 or HTTex1 72Q eGFP with TBK1 or TBK1 kinase-dead (KD) for 48 hour in HEK293T cells (the additional high molecular weight HTTex1 and HTT pS13 band is possibly due to the co-occurrence of phosphorylation at both S13 and T3). (B) Quantification of the fold changes in the HTT pS13 (upper panel) and pT3 (lower panel) ratio to total HTT compared to those of the kinase-dead mutant from the experiments like in A (n=3). (C) Representative western blot of HTT (ab-MAB2166) and HTT pS13 (ab-CHDI-90001039-1) upon coexpression of HTT N543 55Q with the indicated kinase for 24 hour in HEK293T cells. (D) Quantification of HTT pS13 using the Singulex assay (using ab-MW1 as capture and CHDI-90001039 or 2B7 as detection antibodies for pS13 or total HTT) upon coexpression of HTT N543 55Q with the indicated kinase for 24 hour in HEK293T cells from the experiment in C (representative of 3 repeats). (E) Representative western blot of HTT (ab-MAB2166) and HTT pS13 (ab-CHDI-90001039-1) upon coexpression of HTT-FL 48Q or HTT-FL 48Q S13A/S16A with the indicated kinase for 24 hour in HEK293T cells. (F) Quantification of HTT pS13 using the Singulex assay (using ab-MW1 as capture and CHDI-90001039 or 2B7 as detection antibodies for pS13 or total HTT) upon coexpression of HTT-FL 48Q or HTT-FL 48Q S13A/S16A with the indicated kinase for 24 hour in HEK293T cells from the experiments likein E (n=3). (G) Representative western blot of immunoprecipitated (IP) HTT (ab-MAB2166) and HTT pS13 (ab-CHDI-90001039-1) after lentivirus (LV)-mediated overexpression of TBK1 or TBK1 KD for 72 hour in rat primary striatal neuronal cells. (H) Quantification of the fold changes in the HTT pS13 ratio to total HTT compared to those of the kinase-dead mutant from the experiments like in G (n=3).