Fig. 7: The p97-mediated disintegration of aggresomes is required for their packing into autophagosomes.

a HeLa cells treated with Btz (1 μM, 8 h) were fixed after 15 h recovery, or recovered for six additional hours in the absence or presence of 1 μM CB-5083, 5 μM NMS-873, or 100 nM Baf prior to fixation and analysis by confocal microscopy using antibodies against ubiquitin and LC3. Scale bars, 50 μm. b Quantification of the percentage of cells with an aggresome in a. Shown is the mean ± SD from n = 4 biological replicates with ≥60 cells per condition and replicate. One-way ANOVA. c Quantification of the number of ubiquitin- and LC3-positive structures per cell in a. Shown is the mean ± SD from n = 4 biological replicates with ≥60 cells per condition and replicate. One-way ANOVA, corrected for multiple comparisons by Holm–Šidák’s multiple comparison test. d HeLa cells treated with Btz (1 μM, 8 h) were fixed after 15 h recovery, or recovered for six additional hours in the absence or presence of 1 μM CB-5083 prior to fixation and analysis by confocal microscopy using antibodies against p62 and TAX1BP1. Scale bars, 50 μm. Representative result from two independent experiments. e HeLa cells treated with Btz (1 μM, 8 h) were lysed after 15 h recovery, or recovered for six additional hours in the absence or presence of 1 μM Btz, 1 μM CB-5083, 10 μM VER, or 100 nM Baf prior to lysis in SDS sample buffer and immunoblotting for p-p62 S403 and S349. Control samples were taken without prior Btz treatment. AGM, aggresome. Representative result from three independent experiments. f HeLa cells treated with Btz (1 μM, 8 h) and recovered for 21 h in the absence or presence of 100 nM Baf, 1 μM CB-5083, or 5 μM NMS-873 for the last 6 h were immunoblotted for LC3 and GAPDH. Control samples were taken without prior Btz treatment. AGM, aggresome. Representative result from two independent experiments. g HeLa cells treated with Btz (1 μM, 8 h) and recovered for 21 h in the absence or presence of 1 μM CB-5083, 100 nM Baf, 2 μM Wortmannin, or combinations of these inhibitors in the last 6 h were immunostained against LC3. Control samples without prior Btz treatment were included. The number of LC3 puncta per cell was quantified. Shown in the mean ± SD from n = 3 biological replicates with ≥60 cells per condition and replicate. Unpaired, two-tailed Student’s t-tests. h HeLa cells treated with Btz (1 μM, 8 h) and fixed after 15 h recovery, or recovered for six additional hours in the absence or presence of 1 μM CB-5083 were analyzed by confocal microscopy using antibodies against WIPI2 and TAX1BP1. Scale bars, 50 μm. i Quantification of the number of WIPI2 and TAX1BP1 double-positive structures per cell in h. Shown is the mean ± SD from n = 3 replicates with ≥55 cells per condition and replicate. One-way ANOVA. j HeLa cells treated with Btz (1 μM, 8 h) and fixed after 15 h recovery, or recovered for six additional hours in the absence or presence of 1 μM CB-5083 or 100 nM Baf were analyzed by 3D-SIM using antibodies against ubiquitin and LC3. Shown are representative 3D renderings of image stacks. Scale bars, 10 μm. k Box-and-whisker plot showing the mean area of the ubiquitin foci from a total of 29–36 cells per condition, of which 12 representative cells per condition are shown in Supplementary Fig. 8f. The box shows the 25th and 75th percentiles and the median, and the whiskers reach from the minimum to the maximum value; each individual value is plotted as a point. One-way ANOVA was performed over three independent experiments (n = 30 cells for +6 h Ctrl, n = 33 cells for +6 h CB, n = 29 cells for + 6 h Baf), corrected for multiple comparisons by Dunnett’s multiple comparison test.