Fig. 4

Bu-induced proliferation followed by Cy-induced apoptosis in organ-cultured HFs. a Schedule for Bu/– and Bu/Cy treatments and subsequent analysis at 2, 4, and 6 days after alkylating chemotherapy. b Hair shaft elongation and c, hair cycle score of organ-cultured human HFs after alkylating chemotherapy (n = 38 biological replicates/timepoint, 3 independent experiments). Note that the score of the Bu/Cy group was lower than that of the control group on day 6. d Representative images of organ-cultured HFs of experimental groups. Control HFs maintained anagen morphology, and Bu-treated HFs prematurely entered catagen stage (black arrow); however, Bu/Cy-treated HFs showed completely shrunken bulb morphology (black arrowhead). Representative images and quantification of e, Ki67⁺ cells among K15⁺ HFSCs (n = 5 biological replicates/group), f p53⁺ cells among integrin β4⁺ basal cells (n = 6 biological replicates/group), and g, p53⁺ c-caspase-3⁺ cells (n = 7 biological replicates/group) in the bulge after alkylating chemotherapy. HFSCs showed remarkable cellular proliferation after Bu treatment (white arrowhead) and were completely quenched with large-scale apoptosis after Bu/Cy treatment (white arrow; immunofluorescence; scale bar = 100 μm). Bu busulfan, Cy cyclophosphamide, Bu/Cy busulfan followed by cyclophosphamide, 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 < 0.05 (vs. Con); ***p < 0.001 (vs. Con, two-way analysis of variance with Dunnett’s multiple comparisons test) in b and c; ***p < 0.001 (vs. Con, unpaired t test) in e, f, and g