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. 2018 Jun 1;37(13):e97741. doi: 10.15252/embj.201797741

Figure EV2. ALIX regulates spindle orientation in mGSCs, Caco‐2, and HeLa cells.

Figure EV2

  1. Testes from wild‐type (w 1118) and alix 1 males were immunostained with anti‐FasIII (red), anti‐γ‐tubulin (red), anti‐phospho HistoneSer10 (PH3) (green), and Hoechst (blue). The MS axis spanning the centrosomes (indicated with arrows) was mislocalized in 23.4 ± 5.5% of the alix 1 mGSCs compared to 1.6 ± 1.6% of w 1118 mGSCs (± SE) (*P < 0.05, Student's t‐test). Scale bars, 5 μm.
  2. Western blot showing ALIX expression in w 1118 testes but not in alix 1 testes, and equal expression of GAPDH (loading control) in both genotypes.
  3. Quantification of the mitotic spindle rotation velocity (°/min) (± SE) of control (n = 26) and alix 3 mutant (n = 45) SOPs from centrosome separation until telophase as determined from the same live imaging movies as in Fig 3E. **P < 0.01 (Student's t‐test) compared to control. In the boxplot the dot is the mean, the line is the median and the box represents the values included between 25 and 75 percentile.
  4. Western blotting analysis determining the expression levels of ALIX and α‐tubulin in Caco‐2 cells transfected with control siRNA, ALIX siRNA#2, or ALIX siRNA#3.
  5. Caco‐2 cells described in (D) were grown in organoids, fixed, and stained with rhodamine‐phalloidin (red), anti‐α‐tubulin (green), and Hoechst (blue). Scale bars, 5 μm. The relative angle between the spindle axis and the polarity axis (perpendicular to the apical membrane indicated with an asterisk) was determined. The average distribution of relative angles (± SE) from at least three separate experiments (43 control, 41 ALIX siRNA#2, and 38 ALIX siRNA#3 cells) is shown. Both ALIX siRNA#2 and siRNA#3 cells displayed greater variation of the relative spindle angles than control siRNA cells (*P < 0.05, ANOVA with Dunnett's adjustment for multiple testing).
  6. HeLa cells transfected with control, ALIX, Hrs, or CHMP4B siRNA were immunostained with anti‐α‐tubulin (green), anti‐γ‐tubulin (red), and Hoechst (blue). Z‐stacks (of 0.32 μm per plane) through metaphase cells were acquired and the number of confocal planes between the two centrosomes was determined, reflecting the MS tilt. Scale bars, 5 μm. The graph illustrates the average percentage of cells displaying centrosomes with the indicated number of z‐planes apart (± SE). Approximately 50% of controls displayed the two centrosomes 0‐3 z‐planes apart (as shown), whereas in ALIX‐deprived cells, the distance in z‐plane between the two centrosomes was generally longer (shown is a cell displaying centrosomes separated by 7 z‐planes) (**P < 0.01, ANOVA with Dunnett's adjustment for multiple testing). Centrosomes in focus are indicated with arrows. In total, 50 control cells, 43 ALIX‐, 48 Hrs‐, and 47 CHMP4B‐depleted cells were scored (four separate experiments).
  7. Western blotting analysis determining the expression levels of ALIX, Hrs, CHMP4B, and GAPDH in HeLa cells transfected with control, ALIX, Hrs, or CHMP4B siRNA.
  8. Lysates of HeLa Kyoto‐GFP‐tubulin‐mCherry‐Histone2B cells transfected with control siRNA or ALIX siRNA were immunoblotted for ALIX and GAPDH (loading control).
  9. Live cell imaging of HeLa Kyoto‐GFP‐tubulin‐mCherry‐Histone2B cells depleted of ALIX or not was performed to assess cell cycle progression. Typical still images of control cell (upper panel) and ALIX‐depleted cells (lower panel). Time is indicated in min. The table below summarizes the duration of different cell cycle phases (± SE). Compared to control cells, ALIX‐depleted cells spend longer time in prometaphase (21.6 ± 1.4 versus 16.8 ± 0.9 min, **P < 0.01, Student's t‐test), metaphase (73.6 ± 14.8 versus 39 ± 4.2 min, **P < 0.01, Student's t‐test), and cytokinesis (it takes on average 60.4 ± 10.7 min before the intercellular bridge abscises in ALIX siRNA cells and only 41 ± 2.4 min in control cells, *P < 0.05, Student's t‐test).
  10. The table shows that ALIX‐depleted cells are slightly more frequently arrested in metaphase (10.9%) compared to control cells (6.7%, *P < 0.05, Student's t‐test). As much as 65.2% of ALIX siRNA cells died during metaphase in contrast to only 2.3% of control siRNA cells (***P < 0.001, Student's t‐test).
Data information: The tables in (I) and (J) show the average of 44 control cells and 46 ALIX knockdown cells (from three experiments).Source data are available online for this figure.