Fig. 4: PKCα is required for FGFR2b-induced terminal stages of keratinocyte differentiation.

Post-confluent HaCaT pBp and HaCaT pBp-FGFR2b cells were left untreated or stimulated with FGF7 in presence or not of the PKCα inhibitor as reported in Materials and methods section (a, c). Alternatively, HaCaT clones were transfected with PKCα siRNA or with an unrelated siRNA as control, transfected cells were grown until post-confluence and left untreated or stimulated with FGF7 as above (b, d). a Western blot analysis shows that PKCα inhibitor decreases the basal, as well as the FGF7-induced, PKCα phosphorylation, but not that of PKCδ phosphorylation (left panel). PKCα inhibitor does not interfere with the K1 expression, but it displays a repressive effect on the FGF7-induced up-modulation of LOR and FIL (right panel). b Western blot analysis confirms that PKCα silencing specifically dampened the basal, as well as the FGF7-induced, PKCα protein level but not that of PKCδ (left panel). PKCα silencing also strongly counteracts the increase of LOR and FIL but not that of K1 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 PKCα inhibition (c) or depletion (d) decreases FGF7-induced, as well as basal mRNA transcript levels, of LOR and FIL but it does not affect K10 expression. Results are expressed as mean values ± SD. Student’s t test was performed and significance levels have been defined as above: *p < 0.05 vs the corresponding FGF7-unstimulated cells; **p < 0.05 vs the corresponding pBp cells; NS and ***p < 0.05 vs the corresponding PKCα inhibitor-untreated cells; d *p < 0.05 vs the corresponding FGF7-unstimulated cells; **p < 0.05 vs the corresponding pBp cells; NS, ***p < 0.05 and ****p < 0.005 vs the corresponding control siRNA cells