Fig. 5: Aggresome formation of OTUD1 is critical for promoting OSCS maintenance.

1 × 10⁴, 1 × 10⁵ or 1 × 10⁶ SKOV3 cells were subcutaneously injected into BALB/c nude female mice (n = 6 mice per group). After 25, 32, or 54 days, the mice were sacrificed and the tumor tissues were collected as shown in a. The table shows the number of harvested tumors in each group (b). Tumor growth curve of indicated xenografts are created based on tumor volume of indicated time (c). EV empty vector. d Effect of WT OTUD1 or Δ105–164 mutant on CD44⁺ or CD133⁺ cells proportions were evaluated by flow cytometry analysis. EV empty vector. e Representative images of the effects of depletion of OTUD1 IDR region (105–164 aa) on sphere-formation in SKOV3 cells. Scale bar, 50 μm. IDR intrinsically disordered protein region. EV empty vector. f Representative images of the effect of WT (OTUD1^WT) or IDR depleted mutant OTUD1 (OTUD1^Δ105–164) on anchor-independent growth capacity in SKOV3 cells. Scale bar, 400 μm. EV empty vector. g qPCR was employed to confirm the expression of stemness-associated genes (ie, CD133, CD44, CD24, OCT4, SOX2, NOTCH1) in SKOV3 OTUD1^WT or OTUD1^Δ105-164 expressing cells (n = 3). EV empty vector. h Immunofluorescence analysis was performed to examine the correlation of OTUD1^WT or OTUD1^Δ105–164 expression and ASK1 protein level or distribution in vivo. Scale bar, 15 μm. i Statistical analysis showing the OTUD1-based aggresome average diameter of OTUD1 (WT) or mutant OTUD1 proteins (Δ105–164, M1) in SKOV3 xenografted tumor samples (n = 25 cells examined over 2 xenografted tumor samples). EV, empty vector. P values are calculated using two-way ANOVA (c), one-way ANOVA (g), two-tailed unpaired Student’s t test (h). Representative of n = 3 independent experiments (d–f). Source data are provided as a Source Data file.