Fig 2: siRNA-mediated silencing of CDH2, LEP, POSTN, TIMP1 and VEGFC increased the proportion of PI-positive colon cancer cells. After 5-FU treatment (1 µg/ml) and silencing of CDH2, LEP, POSTN, TIMP1 or VEGFC, PI-positive (A) HCT116 and (B) LoVo cells were measured. The data was analyzed using an unpaired Student's t-test and expressed as the mean ± SD. Each assay was performed independent 3 times. ***P<0.001. si, small interfering; 5-FU, 5-fluorouracil.

Fig 3: Depletion of RNF126 suppressed BCa cell proliferation and metastasis through upregulating PTEN.A The MTT assay evaluated the growth and viability of the T24 cell from day 1 until day 5. A lentiviral shRNF126 and a lentiviral control group shNC stably transfected T24 cell were established. The cells were transfected with siNC (red line: shNC and orange line: shRNF126) or siPTEN (olive line: shNC + siPTEN and green line: shRNF126 + siPTEN). B, C The flow cytometry analysis demonstrated the distribution of cells in different phases of cell cycle. The four groups were the same as above. D The colony formation assay showed the effect of RNF126 knockdown on the cell survival of UMUC3 and T24 cells after the indicated transfection. Scale bar = 1 cm. E Colony numbers were counted and plotted as indicated. F The transwell assay evaluated cell migration of the RNF126 or PTEN knockdown treated T24 cells. Scale bar = 100 µm. G The relative number of cell migration was statistically analyzed. H The western blotting indicated the protein level of RNF126, PTEN, PI3K, p-AKT, AKT, CCND1, p21, and N-Cad. Loading control was GAPDH. ***p < 0.001, **p < 0.01, *p < 0.05.

Fig 4: Upregulation of PD-L1 weakens the inhibitive role of TQ in migration, invasion of BC cells. (A) 5637 and T24 cells were transfected with lentivirus carrying CD274 transcript, then administrated with TQ (50 µM,24 h) or not, and qRT-PCR was conducted to monitor the level of PD-L1 mRNA. (B) For 5637 and T24 cells same treated as Figure 8A, expression levels of MMP2, MMP9, E-cad, Vimentin, N-cad and PD-L1 proteins were tested using WB assay. (C, D) Cell scratch test, transwell migration and invasion experiments of 5637 and T24 cells same treated as Figure 8A. WB: western blot; *p<0.05, **p<0.01.

Fig 5: STAT3 inhibitor HO-3867 reverses the positive effects of TAGLN2 overexpression on the migration, invasion and EMT of colorectal cancer cells. SW480 cell migration and invasion were measured by (A and B) wound healing and (C and D) Transwell assays, respectively (scale bar, 100 µm). (E) Expression of MMP2 and MMP9 was measured by western blotting. (F and G) Expression of proteins associated with EMT (N-cad, vimentin, ZEB2 and E-cad) was measured by reverse transcription-quantitative PCR and western blotting. *P<0.05, **P<0.01 and ***P<0.001. EMT, epithelial-mesenchymal transition; MMP, matrix metalloproteinase; TAGLN2, transgelin 2; OV, overexpressing plasmid; NC, negative control; STAT3, signal transducer and activator of transcription 3; N-cad, N-cadherin; E-cad, E-cadherin; ZEB2, zinc finger E-box binding homeobox 2.