Fig. 7: PKC inhibition recapitulates Ror2 depletion in regulation of HFSC maintenance.

a Immunoblotting analyses of FACS-purified HFSCs from 3dpd Ror2 Ctrl and cKO HFs. b Whole-mount immunofluorescence staining of late telogen Ror2 Ctrl and cKO HFs for ROR2 (red) and PKCα (green). Quantification analyses of fluorescence intensity for ROR2 and PKCα staining are shown at right. Data are reported as the median (the line within the box), the 25th to 75th percentiles (bottom and top lines of the box) and the 10th to 90th percentiles (bottom and top whiskers); n = 18 (Ctrl) or 20 (cKO) regions over 7 (Ctrl) or 8 (cKO) independent HFs; ***p < 0.0001. Unpaired two-sided t-test. c Immunoblotting analyses for indicated proteins with Ror2^+/+ and Ror2^−/− HFSCs that were treated with DMSO (vehicle) or GF109203X (PKCi) for 24 h. d PKC inhibition in β-cat cKO HFs leads to the loss of HFSCs and wrong fate differentiation. (Top) Schematic diagram illustrating the hair cycle progression of Ror2/β-cat Ctrl, dKO mice, and β-cat cKO mice treated with PKC inhibitor at the 2nd telogen phase. (Bottom) Oil Red O staining of sebocytes on 1-week post-treated whole-mount skins (left) and quantification of HFs (right) are shown. e Real-time PCR analyses of FACS-purified HFSCs from 24 h acetone (vehicle)- or GF109203X (PKCi)-treated β-cat Ctrl or cKO mice. Values of GF109203X-treated β-cat Ctrl and cKO HFSCs were normalized to mRNA of vehicle-treated β-cat Ctrl and cKO HFSCs, respectively. Data are reported as average ± s.d.; n = 3 biological independent animals; *p < 0.05, **p < 0.005. Unpaired two-sided t-test. f An illustrating model summarizing our results in this study. In HFSCs, ROR2 not only transduces Wnt5a-induced non-canonical Wnt signaling via activation of PKC, JNK, and small GTPases, but also modulates Wnt3a-induced canonical Wnt signaling by controlling GSK3β stability. In parallel, ROR2 also plays a role in regulation of ATM/ATR-dependent DDR. These ROR2-dependent regulations modulate cell proliferation, motility, and DNA repair, which are essential for the long-term maintenance of HFSCs. All immunoblotting results are representative of at least two independent experiments. Scale bars in b, d represent 50 μm. Source data are provided as a Source Data file.