Fig. 3: PKCδ controls the onset of FGFR2b-triggered keratinocyte differentiation.

Confluent HaCaT pBp and HaCaT pBp-FGFR2b clones were left untreated or stimulated with FGF7 in presence or not of the PKCδ inhibitor as reported in Materials and methods section (a, c). Alternatively, HaCaT clones were transfected with PKCδ siRNA or with an unrelated siRNA as control, and then left untreated or stimulated with FGF7 (b, d). a Western blot analysis confirms that PKCδ inhibitor interferes with the basal, as well as the FGF7-induced, phosphorylation of PKCδ at its autophosphorylation site Ser645 but not with that of PKCα at its autophosphorylation site Ser657 (left panel). PKCδ inhibition induces a repressive effect on the K1, DSG1, and INV increase induced by FGF7, which is particularly evident in FGFR2b overexpressing clones (right panel). b Western blot analysis confirms that PKCδ silencing specifically dampened the basal, as well as the FGF7-induced, PKCδ protein level but not that of PKCα (left panel). PKCδ silencing also strongly counteracts the increase of K1, DSG1, and INV in response to FGF7 (right panel). The equal loading was assessed with anti-PKCδ or anti-PKCα and anti-ACTIN or anti-TUBULIN antibodies. For densitometric analysis the values from a representative of three independent experiments are reported. c, d Real time RT-PCR analysis confirms that PKCδ inhibition (c) or depletion (d) decreases FGF7-induced, as well as basal mRNA transcript levels, of K10 DSG1 and INV. Results are expressed as mean values ± SD. Student’s t test was performed and significance levels have been defined as above: c *p < 0.05 vs the corresponding FGF7-unstimulated cells; **p < 0.05 vs the corresponding pBp cells; ***p < 0.05 vs the corresponding PKCδ inhibitor-untreated cells; d *p < 0.05 vs the corresponding FGF7-unstimulated cells; **p < 0.05 vs the corresponding pBp cells; ***p < 0.05 and ****p < 0.005 vs the corresponding control siRNA cells