Figure 7(A) Linear ubiquitination reduces the fraction of cells with Htt-Q97 aggregates. SH-SY5Y cells were transfected with Htt-Q97 and either control siRNA, HOIP siRNA or OTULIN siRNA. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 9. Expression levels of HOIP and OTULIN were analyzed by Western blotting. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. (B) Linear ubiquitination reduces the fraction of cells with Htt-Q97 aggregates. SH-SY5Y cells were transfected with Htt-Q97 and either WT HOIP or the catalytically inactive HOIP mutant C885A. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey s Multiple Comparison Test, n = 5. Expression levels of WT and mutant HOIP and OTULIN were analyzed by Western blotting. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. (C) The effect of HOIP on Htt-Q97 aggregation is independent of NF-κB signaling. SH-SY5Y cells transiently transfected with Htt-Q97 and either control vector (CO) or WT HOIP plus the NF-κB super-repressor IκB-2S/A were analyzed by immunocytochemistry. Data are mean ± SD with n=5 from an unpaired two-tailed Student's t-test. Expression levels of WT HOIP and IκB-2S/A were analyzed by Western blotting. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. (D) Catalytically active HOIP decreases the number of cells with Htt-Q97 aggregates independently of autophagy. WT and ATG5 KO MEFs were transiently transfected with Htt-Q97-GFP and either control vector (CO), HOIP or HOIP C885A. Cells were treated 24 h after transfection with the proteasomal inhibitor MG132 (1 µM, 16 h). Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 7. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. (E) Linear ubiquitination reduces the fraction of cells with TDP-43 Q331K aggregates. SH-SY5Y cells were transfected with TDP-43 Q331K and either control siRNA, HOIP siRNA or OTULIN siRNA. Data are displayed as mean ± SD and were analyzed by Kruskal-Wallis test, followed by Dunn's Multiple Comparison Test, n = 7. Expression levels of HOIP and OTULIN were analyzed by Western blotting. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. (F) Catalytically active HOIP decreases the number of cells with TDP-43 aggregates dependent on proteasomal degradation. SH-SY5Y cells were transfected with TDP-43 Q331K and either control vector (CO), HOIP or HOIP C885A. Cells were treated 48 h after transfection with the proteasomal inhibitor MG132 (1 µM) or the p97/VCP inhibitor NSM-873 (1 µM) for 3 h. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Dunnett's Multiple Comparison Test, n = 5. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. (G) Inhibition of the proetasome and p97/VCP do not have additive effects on the degradation of misfolded TDP-43 species. SH-SY5Y cells expressing TDP-43 Q331K were treated 48 h after transfection with MG132 (1 µM) and/or NSM-873 (1 µM) for 3 h. All data are mean ± SD, data were analyzed by one-way ANOVA followed by Bonferroni's Multiple Comparison Test, n = 5. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.