Figure 3.

USP29 regulates EMT, CSC self‐renewal, metastasis, and cellular sensitivity to chemotherapy through TWIST1. A) MDA‐MB‐231 cells stably expressing control (Ctrl) or USP29 shRNAs (#1 and #2) were generated and western blot was performed with indicated antibodies. B,C) The migration and invasion abilities of cells as in (A) were measured by Transwell migration and invasion assays and results were quantified in (B,C). The results represent mean ± s.d. from three independent experiments; **p < 0.01. D,E) Graphic representation of mammosphere formation assay from cells described in (A). The results represent mean ± s.d. from three independent experiments; **p < 0.01. F) Graphic representation of the CD44⁺/CD24⁻ population from cells described in (A) was examined by FACS analysis. The results represent mean ± s.d. from three independent experiments; **p < 0.01. Cells as in (A) were treated with G) cisplatin or H) paclitaxel and cell survival was determined. The results represent mean ± s.d. from three independent experiments. I,J) Cells as in (A) were subcutaneously implanted into nude mice and mice were treated with saline or cisplatin (2 mg kg⁻¹). I) Xenograft tumors were dissected and J) tumor weights were measured. The results represent the mean ± s.d. from six mice; **p < 0.01. K,L). Cells as in (A) were injected into the mammary fat pads of female NOD‐SCID mice. The primary tumors were surgically removed when tumor volume reached 400 mm³. After 8 weeks, mice were sacrificed and lung metastatic nodules were examined macroscopically. The results represent the mean ± s.d. from six mice; **P < 0.01.