Fig. 2

PKCε signaling is responsible for FGFR2c-mediated modulation of EMT markers. HaCaT pBp and HaCaT pBp-FGFR2c clones were transfected with PKCε small interfering RNA (siRNA) or with an unrelated siRNA (Cx siRNA) as control, and then left untreated or stimulated with FGFR2 ligands as above. a Western blot shows that in untreated cells PKCε siRNA transfection induces an efficient depletion of PKCε. Equal loading was assessed with the anti-actin antibody. The densitometric analysis and Student t test were performed as reported in Fig. 1: *p < 0.05, ** p < 0.01. b Western blot analysis shows that the decrease of the epithelial markers E-cadherin and β4-integrin and the appearance of the mesenchymal marker N-cadherin, observed only in pBp-FGFR2c clones upon FGF2 stimulation, is reversed by PKCε silencing. Equal loading was assessed with the anti-actin antibody. The densitometric analysis and Student t test were performed as reported above: *p < 0.05, *** p < 0.001. c Real Time RT-PCR analysis shows that, in pBp-FGFR2c cultures, PKCε depletion is sufficient to counteract the FGF2-induced mRNA transcript modulation of E-cadherin, β4-integrin and the mesenchymal marker vimentin. Human Fibroblasts (HFs) are used as a positive control for mesenchymal marker expression. Results are expressed as mean value ± SE. Student’s t test was performed and significance levels were defined as p < 0.05: *p < 0.05, ** p < 0.01. d HaCaT clones were seeded on matrigel pre-coated transwell Boyden chamber filters. Cells were then serum starved and FGF2 was added in the bottom chamber to stimulate cell chemotaxis. An high number of invasive cells is induced by FGF2 stimulation only in pBp-FGFR2c control siRNA clones and appears strongly reduced in the corresponding PKCε siRNA cultures. Quantitative analysis was assessed as reported in materials and methods. Results are expressed as mean values ± standard deviation (SD). Student’s t test was performed as reported in materials and methods and significance level has been defined as p < 0.05: ***p < 0.001. Bar: 50 μm