Fig. 2: USP9X mediates TGF-β function in promoting CSCs occurance and chemoresistance.

A IB analysis of USP9X, E-Cadherin, N-Cadherin, Slug, and Snail in USP9X knocked-down CAOV3 or ID8 cells treated with or without TGF-β1 (5 ng/mL), CAOV3 was treated for 96 h and ID8 was treated for 48 h from wound-healing assay (Fig. S2A). B Spheroid formation assay of USP9X knocked-down CAOV3 or ID8 cells treated with or without TGF-β1 (5 ng/mL) for 2 weeks. Scale bars, 100 µm. The right panels were the quantization chart. C FACS analysis of stem cell populations (ALDH1A1+/CD44+) in sphere-forming cells from USP9X knocked-down CAOV3 or ID8 cells treated with or without TGF-β1 (5 ng/mL) for 2 weeks. D Representative images of organoids formed in USP9X knocked-down primary HGSOC cells for 2 weeks. Organoids larger than 75 μm in diameter were used for analysis. The right panels were the quantization chart. Scale bars, 100 µm. E Luciferin bioluminescence signal intensities were monitored from orthotopic and intraperitoneal tumors in transplanted mice. n = 3 mice per group. The right panels were the quantization chart. F Orthotopic tumors from (E) were harvested and weighted, n = 4 mice per group. The right panels were the quantization chart. G Representative images of Hematoxylin and eosin (H&E), p-Smad3, Ki-67, ALDH1A1, and Snail IHC stainings on orthotopic tumors from (F). The right panels were the quantization chart. Scale bars, 50 µm. H Dose–response curve and IC50 values of ID8, ID8-rPTX (upper), ID8-rCDDP (below), USP9X knocked-down ID8-rPTXor ID8-rCDDP cells for 72 h. I Representative images of Cleaved-Caspase3 immunofluorescence (IF) stainings on ID8, ID8-rPTX, ID8-rCDDP, USP9X knocked-down ID8-rPTX or ID8-rCDDP cells. Scale bars, 50 µm. J KEGG pathway analysis based on USP9X knocked-down ID8 cells RNA-seq. Data are shown as the mean ± s.d (B–H). P values were calculated by unpaired two-tailed Student’s t test (B, D–G). n = 3 biological independent samples (A–I).