Fig 2: 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 PKCa (green). Quantification analyses of fluorescence intensity for ROR2 and PKCa 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 ß-cat cKO HFs leads to the loss of HFSCs and wrong fate differentiation. (Top) Schematic diagram illustrating the hair cycle progression of Ror2/ß-cat Ctrl, dKO mice, and ß-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 ß-cat Ctrl or cKO mice. Values of GF109203X-treated ß-cat Ctrl and cKO HFSCs were normalized to mRNA of vehicle-treated ß-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 GSK3ß 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.

Fig 3: ATG5 deficiency limits tumor initiation and progression in vivo. (A) In vivo tumor growth after subcutaneous injection of 10,000 T18-derived TIC cells and subsequent intraperitoneal treatment with either CQ or PBS (n = 12 tumors per group). Data are presented as means tumor volumes (mm3) ± SEM. (B) T18 tumor weight after treatment with CQ or PBS, respectively. Data are presented as mean tumor weights (mg) ± SEM. (C) In vivo tumor growth after subcutaneous injection of 10,000 T20-derived TIC cells with/without stable knockdown of ATG5, n = 5 mice/group. (D-E) Tumor (D) growth and (E) weight in immune-deficient mice (NSG mice), 8 weeks after subcutaneous injection of 10,000 cells derived from primary T18 TICs, following a stable knockdown of ATG5 or respective control vector; n = 6/group. A representative ATG5 protein expression of extracted T18-derived xenografts is shown. All data are shown as mean ± SEM. Two-way ANOVA followed by the Tukey post-hoc test was used to test for statistical significance in A, C and D. Paired t-tests were used to assess significance in B, and E. *p < 0.05, ***p < 0.001 (F) p-EZR immunofluorescence staining in extracted xenograft tumors from (D). Scale bar: 100 µm. Representative images are shown (left panel) as well as the quantification of p-EZR-positive area (right panel). Four mice out of the six from (D) were used, as the remaining two were used in (G). Data are presented as mean ± SD, *p < 0.05. (G) Serial in vivo limiting dilution experiment with T18 TICs, following stable knockdown of ATG5 or the corresponding control vector. After an initial round of xenotransplantation, two extracted tumors (from mice appearing in [D]) were dissociated and different cell doses (100, 250 and 500 cells) were subcutaneously injected into secondary recipient NSG mice. Secondary tumor incidence was evaluated after 12 weeks. Statistical significance was assessed with a Chi-square test ** p < 0.01. (H) Potential mechanism of action. Hypoxia within a solid tumor leads to activation of autophagy, especially in TICs. Kinases, such as PRKCA, are activated and further induce phosphorylation of EZR on Thr567 in TICs. EZR, most likely through MAPK14/p38 activation, leads to increased self-renewal capacity of TICs in vitro and in vivo.

Fig 4: TANC2-PRKCA-expressing cells exhibit reduced levels of cleaved caspase-3 and decreased apoptosis.A, immunoblot analysis of lysates from parental, CRISPR-edited TANC2-PKCa fusion, and heterozygous PKCa knockout cells, probed for caspase-3, cleaved caspase-3, PKCa, and ß-actin. Data are representative of three independent experiments. B, apoptosis assay performed on MDA-MB-231 parental (gray), CRISPR-edited TANC2-PKCa fusion (purple), or heterozygous PKCa knockout (orange) cells. Data represent luminescence upon binding of annexin V-luciferase to cell-surface-exposed phosphatidylserine as a function of time following addition of reagents. Graph depicts mean ± SEM from three independent experiments. **p < 0.01, ***p < 0.001 (Student’s t-test). Asterisk indicates significance between parental and fusion-expressing cells, and # indicates significance between parental and heterozygous PKCa knockout cells. C, apoptosis assay described in (B) was performed on MDA-MB-231 parental (gray), CRISPR-edited TANC2-PKCa fusion (purple), or heterozygous PKCa knockout (orange) cells treated with etoposide (12.5 µM) or vehicle (DMSO) for 24 h. Graph depicts mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test). Asterisk indicates significance between vehicle and etoposide-treated conditions for each cell line. # and % indicate significance between the parental and CRISPR-edited fusion or heterozygous PKCa knockout cells in basal and etoposide-treated conditions, respectively.

Fig 5: The TANC2-PRKCA fusion is detected at the mRNA level but not the protein level in CRISPR-edited cells.A, schematic illustrating strategy to generate cells expressing the TANC2-PRKCA fusion via CRISPR/Cas9 gene editing. B, mRNA expression of the TANC2-PRKCA fusion and actin (ACTB) in the parental and CRISPR-edited clone analyzed by RT-PCR. Product indicated by black bar on schematic. Reactions analyzed on agarose gel, with each lane representing an individual replicate. C, TANC2 mRNA expression at the 5' (“N-Term”) and 3' (“C-Term”) ends in the parental and CRISPR-edited clone analyzed by RT-qPCR and normalized to actin (ACTB). Products indicated by black bars on schematic. Data represent means ± SEM from at least three independent experiments. **p < 0.01 (Student’s t-test). D, PRKCA mRNA expression at the 5' (“N-Term”) and 3' (“C-Term”) ends in the parental and CRISPR-edited clone analyzed by RT-qPCR and normalized to actin (ACTB). Products indicated by black bars on schematic. Data represent means ± SEM from at least three independent experiments. **p < 0.01, ***p < 0.001 (Student’s t-test). E, immunoblot analysis of lysates from parental and CRISPR-edited cells, probed for TANC2, PKCa, and a-tubulin. Arrow indicates predicted mobility of the fusion protein. Data are representative of three independent experiments.