Fig 2: 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 3: 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 4: The CD82 scaffold regulates PKCa expression and activation.(A) Cartoon depicting mutated palmitoylation sites within CD82 and mCherry fusion. Flow cytometry analysis of (B) total and (C) surface CD82 expression using CD82KD, control, CD82OE, and Palm-CD82OE KG1a cells (Biolegend, ASL-24). (n = 3 experiments; error bars indicate SD; mean fluorescence intensity normalized to control levels). (D) Western blot analysis for total PKCa (Cell Signaling #2056, polyclonal) and phospho-PKCa (Thr638) expression. Densitometric analysis of (E) total and (F) phosphorylated PKCa expression from Western blot analyses (n = 4 experiments; error bars indicate SD). (G) Real-time PCR analysis of KG1a cells. (H) Immunofluorescence imaging of CD82 (Biolegend, ASL-24) and PKCa-488 (primary, abcam, Y124; secondary, Invitrogen, rabbit-488) under resting and 1 hr of PMA treatment with corresponding line scan plots for both channels. All channels were scaled equally across conditions. (I) Western blot analysis of total and phosphorylated PKCa expression following PMA stimulation (n = 4 experiments; error bars indicate SD). (J) Cells were treated with DMSO, PMA or PMA + MG132 (10 uM) for 4 hrs and total and phospho-PKCa were quantified using Western blot analysis and densitometry. (n = 4 independent experiments; error bars indicate SD; post-hoc unpaired t-test).

Fig 5: PRKCA overexpression reverses the effect of tsRNA-04002 mimics on NPCs. A qRT-PCR was applied for mRNA validation. B Protein expression of PRKCA was validated by western blot. C The mRNA expression of IL-1ß and TNF-a was determined by qRT-PCR. D The protein expression of COL2A1 was determined by western blot analysis. E The protein expression of Sox-9 was determined by western blot analysis. F The apoptosis rate of NPCs was assessed by flow cytometry. G The protein expression of Cleaved caspase3 was determined by western blot analysis. *p value < 0.05, **p value < 0.01, n = 3