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)