Figure 2

Growth factor-induced EMT in C-PVR cells show upregulation of EMT markers. (a) After stimulation with TGF-β2 or combination treatment (TGF-β2 + TNF-α + IL-6), significant changes in α-SMA staining were identified at day 3. These changes in α-SMA expression were more prominent 7 days after induction. (b) Increased α-SMA expression levels were detected after stimulation with TGF-β2 and combination when compared to control at day 3 (*p < 0.05, ***p < 0.001, one-way ANOVA; n = 6 represented as mean ± SEM) and day 7 (***p < 0.001, ****p < 0.0001, one-way ANOVA; n = 6 represented as mean ± SEM). (c) TGF-β2 and combination treated cells showed increased protein expression of mesenchymal markers (α-SMA and N-Cadherin) at days 3 and 7 after induction. RUNX1 protein levels were also upregulated after induction by TGF-β2 and combination treatment. (d) An increase in N-Cadherin was observed at day 3 and in α-SMA protein levels at day 3 and day 7 after induction by TGF-β2 and combination treatments (*p < 0.05, ***p < 0.001, two-way ANOVA; n = 3 represented as mean ± SEM). Similarly, increased protein expression of RUNX1 was observed at day 3 and day 7 when C-PVR cells were stimulated with TGF-β2, and at day 7 with the combination of growth factors (*p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA; n = 3 represented as mean ± SEM). (e) N-Cadherin expression is reduced by RUNX1 knockdown via siRUNX1 in untreated and TGF-β2-induced cells (*p < 0.05, ****p < 0.0001, two-way ANOVA; n = 3 represented as mean ± SEM). (f) TGF-β2-induced α-SMA expression is reduced by RUNX1 knockdown by siRUNX1 (*p < 0.05, one-way ANOVA; n = 12 represented as mean ± SEM). (g) siRUNX1 induced a 70% reduction of RUNX1 expression measured by qRT-PCR 48 h post-transfection (***p < 0.001, two-tailed unpaired T-test; n = 3 represented as mean ± SEM). (h) Validation of siRUNX1 effect on RUNX1 using mouse and rabbit anti-RUNX1 antibodies. Protein levels quantification of RUNX1 showed 60% and 50% reduction of RUNX1 (*p < 0.05, **p < 0.01, two-tailed unpaired T-test; n = 2 represented as mean ± SEM). Samples used for quantitative comparisons derive from the same experiment and blots were processed in parallel. Representative immunoblots showing cropped images of the same gel for separation of markers. Full-length blots/gels are presented in Supplementary Figures S7–S9.