Fig 2: PS VII reduces the expression of MMP-2 and -9 of U2OS osteosarcoma cells. (A) MMP-2 and -9 mRNA levels were assessed by qPCR following treatment of U2OS cells with 0 (control), 0.5, 1 and 2.5 μM PS VII for 24 h. (B) MMP-2 and -9 mRNA levels were assessed by qPCR following treatment of U2OS cells with 1 μM PS VII for 0 (control), 6, 12 and 24 h. (C) MMP-2 and -9 protein expression was assessed by quantitative western blot analysis following stimulation of U2OS cells with 0 (control), 0.5, 1 and 2.5 μM PS VII for 24 h. (D) MMP-2 and -9 protein expression was assessed by quantitative western blot analysis following stimulation of U2OS cells with 1 μM PS VII for 0 (control), 6, 12 and 24 h. Values are presented as the mean ± standard deviation from three independent experiments. *P<0.05, **P<0.01 vs. control. PS VII, Paris saponin VII; MMP, matrix metalloproteinase; qPCR, quantitative polymerase chain reaction; GADPH, glyceraldehyde 3-phosphate dehydrogenase.

Fig 3: Overexpression of SOX4 promotes melanoma cell migration and invasion and upregulates the expression of MMP2, MMP9 and nuclear factor-κB (NF-κB) p65. Melanoma A375 cells were transfected with SOX4 overexpression lentivirus transfection for 72 h. (A) Real-time PCR assay for SOX4, p65, MMP2 and MMP9 mRNA expression. (B) Western blot analysis for SOX4, p65, MMP2 and MMP9 protein expression. (C) Migration and invasion assay. The invading cells were stained with 0.5% crystal violet, and counted in 5 random squares by microscopic. Each experiment was performed in triplicate. **P<0.01 compared to the control. SOX4-OE represents SOX4 overexpression.

Fig 4: Downregulation of SOX4 induced by SOX4 siRNA suppresses melanoma cell migration through the inhibition of p65, MMP2 and MMP9 mRNA expression. A375, A2058, and A875 cells were transfected with si-SOX4 (5 and 10 nM) or scrambled siRNA. (A) Real-time PCR assay for SOX4 mRNA expression following transfection with siRNA for 24 h. (B) Transwell migration assay: the invading cells were stained with 0.5% crystal violet, and counted in 5 random squares using a microscope. Each experiment was performed in triplicate. (C) Real-time PCR assay for p65, MMP2 and MMP9 mRNA expression following transfection with SOX4 siRNA transfection for 24 h. *P<0.05 and **P<0.01 compared to the control.

Fig 5: Expression of proteins involved in epithelial-mesencchymal transition. Western blots show downregulated N-cadherin, MMP-2, β-catenin, Slug and ZEB1 and upregulated E-cadherin following KIFC1 knockdown. *P<0.05. KIFC1, kinesin family member C1; MMP2, matrix metalloproteinase 2; ZEB1, Zinc finger E-box-binding homeobox 1; NC, negative control; si-1/S1, small interfering RNA 1; si-2/S2, small interfering RNA 2.