PKCμ inhibition/reduction impairs wound healing in vitro
(A) HaCaT cells treated with PMA (10 nM) in the absence or presence of CRT (1 μM CRT) for 16 h. Western blots of cell lysates probed for pPKCμ-S916, PKCμ, and tubulin.
(B) Relative pPKCμ-S916 levels in treated samples compared to DMSO control based on densitometric analysis from three replicate experiments as shown in (A).
(C) Representative images of in vitro wound healing assay at 0 and 16 h on HaCaT cells treated with DMSO, PMA (10 nM), or PMA with CRT (1 μM CRT). Scale bar = 100 μm.
(D) Percentage of wound area remaining after 16 h from three replicate experiments as shown in (C).
(E) Representative images from immunofluorescence staining of pPKCμ-S916 in scratch wounded HaCaT cells at 30- and 120-min post-wounding. Scale bar = 40 μm.
(F) Relative pPKCμ-S916 levels based on fluorescence intensity from three images from 30- and 120-min post-wounding as shown in (E). Relative levels are based on 10 random fields taken from cells adjacent to the wound and cells interior to the wound for each time point.
(G) HaCaT cells transduced with control shRNA (shGFP) or two shRNAs directed against PKCμ. Cell lysates were probed by western blotting for PKCμ and tubulin.
(H) Quantification of PKCμ levels relative to shGFP from three replicate experiments as shown in (G).
(I) Representative images of in vitro wound healing assay at 0 and 16 h on HaCaT cells transduced with control shRNA (shGFP) or two shRNAs directed against PKCμ treated in the absence or presence of PMA (10 nM). Scale bar = 100 μm.
(J) Percentage of wound area remaining after 16 h from three replicate experiments as shown in (I). All calculations are based on three replicates −/+ S.D. ∗∗p < 0.01, ∗∗∗p < 0.001 (Student’s t test).