Fig. 3

Priming proliferation precedes loss of stem cell reserve in the bulge. a Experimental models for transient loss after Cy only treatment vs. permanent loss after Bu/Cy treatment. b Representative images and quantification of Ki67⁺ cells among K15⁺ HFSCs in the bulge (n = 8 (D0 Con), 9 (D4 Cy only), 8 (D7 Cy only), 6 (D4 Bu), and 7 (D7 BuCy) biological replicates). Few Ki67⁺ cells were observed in the K15^‒ suprabasal layer in control HFs (white arrowhead). HFSCs showed large-scale proliferation after Bu treatment (white arrow), and this proliferation was completely quenched after Bu/Cy treatment. c Representative images and quantification of p53⁺ cells in the K14⁺ HF epithelium in the bulge (n = 6 (D0 Con), 5 (D4 Cy only), 4 (D7 Cy only), 5 (D4 Bu), and 6 (D7 BuCy) biological replicates). p53⁺ cells were observed in the suprabasal layer after Cy only treatment, but lining p53⁺ cells emerged in the basal layer after Bu/Cy treatment. d Representative images and quantification of c-caspase-3⁺ cells among K15⁺ HFSCs in the bulge (n = 10 biological replicates/timepoint). Several c-caspase-3⁺ cells were detected in the K15⁺ basal layer after Bu/Cy treatment. e Representative images for γ-H2AX⁺ cells among K15⁺ HFSCs in the bulge. γ-H2AX⁺ cells were massively detected and persistent in the basal and suprabasal layers after Bu/Cy treatment. f Representative images and quantification of Shh⁺ cells among K15⁺ HFSCs in the bulge (n = 9 biological replicates/timepoint). Shh⁺ cells disappeared after Bu treatment and then reappeared without K15 expression after Bu/Cy treatment in the basal layer. g Representative images of Lhx2⁺ cells in the bulge on day 13 after alkylating chemotherapy (immunofluorescence; scale bar = 100 μm in b, f, g; 50 μm in c, d, e). Bu busulfan, Cy cyclophosphamide, Bu/Cy busulfan followed by cyclophosphamide, SB suprabasal layer, B basal layer, HF hair follicle, HPF high-power field, HFSC hair follicle stem cell, c-caspase-3 cleaved caspase-3. Data are mean ± SEM. Source data are provided as a Source Data file. ***p < 0.001 (Cy only vs. Bu/Cy), ^†††p < 0.001 (SB vs. B, two-way analysis of variance with Sidak’s multiple comparisons test) in b and c; *p < 0.05 (vs. D0), ***p < 0.001 (vs. D0, two-way analysis of variance with Sidak’s multiple comparisons test) in d and f