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. 2023 Feb 13;10(11):2205873. doi: 10.1002/advs.202205873

Figure 6.

Figure 6

CDK1‐mediated phosphorylation of USP29 regulates TWIST1 protein stability and subsequent EMT, CSC self‐renewal, metastasis, and cellular sensitivity to chemotherapy. A) Cells stably expressing control or USP29 shRNA were transfected with indicated plasmids and treated with vehicle or RO‐3306 and western blot was performed with indicated antibodies. B) MDA‐MB‐231 cells were transfected with indicated plasmids and treated with vehicle or RO‐3306. Cell lysates were subjected to immunoprecipitation with anti‐HA antibody and the ubiquitination of TWIST1 protein was examined by western blot. C,D) Cells were cotransfected with indicated plasmids. Ni‐NTA bead was used to pull down His‐tagged ubiquitin and the polyubiquitylated TWIST1 protein was examined by western blot. E) MDA‐MB‐231 cells stably expressing USP29 shRNA were transfected with indicated plasmids and western blot was performed with indicated antibodies. F) Graphic representation of the migration ability from cells described in (E) was examined by Transwell migration assay. The results represent mean ± s.d. from three independent experiments; **p < 0.01. G) Graphic representation of mammosphere formation assay from cells described in (E). The results represent mean ± s.d. from three independent experiments; **p < 0.01. Cells as in (E) were treated with H) cisplatin or I) paclitaxel and cell survival was determined. The results represent mean ± s.d. from three independent experiments. J,K) Cells as in (E) were subcutaneously implanted into nude mice, and mice were treated with saline or cisplatin (2 mg kg−1). J) Xenograft tumors were dissected and K) tumor weights were measured. The results represent the mean ± s.d. from six mice; **p < 0.01. L,M) Cells as in (E) 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 mean ± s.d. from six mice; **P < 0.01.