Fig 2: HIPK1 knockdown rescues the effects of miR-200c/141 inhibition on breast cancer cells. SUM149 and T47D cells were transfected with HIPK1-shRNA lentivirus and then treated with miR-200c/141 inhibitors. (A) Protein expression level of HIPK1, E-Cadherin (E-cad), Snail and Vimentin (VIM) were detected by Western Blotting. Samples were harvested after cells had been treated for 48 hours. (B-C) MTT assay was performed with treated SUM149 (B) and T47D (C) cells for cell proliferation ability. *p<0.05, **p<0.01. (D-E) The invasive ability was investigated with treated SUM149 (D) or T47D (E) cells by transwell assay. Quantitative analysis of the total invasive cells from three independent experiments. ***p<0.001 (F-G) The heterogeneity of BCSCs was analyzed by Flow cytometry with two sets of markers (CD24-CD44+ and ALDH+) in treated SUM149 (F) and T47D (G) cells.

Fig 3: TRIM59 drives tumour growth and metastasis in vivo. (A) Representative images of two xenograft groups. A549 cells tranfected with shCtrl or shTRIM59 plasmid were subcutaneously injected into the back of athymic mice to estimate carcinogenesis. (B) Tumour volumes were measured on the indicated days to assess the effects of TRIM59 on subcutaneous tumour growth. (C) Representative images of tumour samples stained with haematoxylin & eosin (H&E) (200× magnification). (D) The weight of the xenotransplanted tumours in two groups were compared at 30 days postimplantation (n = 8). (E) Representative photos of Ki-67 immunostaining and statistical analyses of the Ki-67-positive index in subcutaneous tumour sections. (F) Proteins of subcutaneous tumours were used for western blotting of CDK6 expression. (G) Western blot evaluations were used to evaluate E-cadherin, N-cadherin, Snail, Slug, and Vimentin expression in xenograft tumours. Results are expressed as means ± SEM. The significance was determined by Student's t test. *P < 0.05; **P < 0.01

Fig 4: ALDH2 is essential for EMT progression through the elevation of E-cadherin level and attenuation of vimentin expression. (A) Expression levels of the EMT-related proteins ZEB1, E-cadherin, N-cadherin, vimentin, and SNAIL in PLVX-Flag-ALDH2 and PLVX-Flag H1299 cells. (B) IHC was used to explore the expression of E-cadherin and vimentin in BM. (C) Expression levels of the EMT-related proteins ZEB1, E-cadherin, N-cadherin, vimentin, and SNAIL in shCK A549 and shALDH2 A549 cells.

Fig 5: Effect of chrysin on CsA- or TGF-ß1-induced EMT-signaling. LLC-PK1 cells were treated with vehicle (Veh), 4.2 µM CsA, 5 ng/mL TGF-ß1 or 25 µM chrysin (Chr), or combinations. (A) Protein expression for TGF-ß1, phospho-Smad3, total Smad 3 and Snail were determined by Western blot analysis. Phosphorylation of Akt (B) and GSK-3ß (C) was determined by ELISA. Representative Western blots are shown, and data are represented as mean ± SEM of three individual experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s test for multiple comparisons. * indicates p < 0.05 vs. control; † indicates p < 0.05 vs. chrysin co-treatment. The effects of chrysin on CsA- or TGF-ß1-induced localization of ß-catenin (panels D–I) were also visualized. Release of membrane-bound ß-catenin is indicated using white arrowheads. Treatments: (D) control, (E) 5 ng/mL TGF-ß1, (F) 5 ng/mL TGF-ß1 + 25 µM chrysin, (G) 4.2 µM CsA, (H) 4.2 µM CsA + 25 µM chrysin and (I) 25 µM chrysin. Scale bar indicates 100 µm.