Figure 4.

Sox9 ablation leads to a decrease in the number of DCT⁺(/Nestin⁺)/GFP⁺ cells in the HF

(A) Schematic demonstration of the applied tamoxifen protocol to specifically ablate Sox9 in Nestin⁺ cells in HF of adult mice in vivo; Either Nestin-CreER^T2; GFP mice (Ctrl) or Nestin-CreER^T2; GFP with floxed alleles of Sox9 (Sox9KO) were intraperitoneally injected with tamoxifen every 12 h for 5 consecutive days starting at P56 and sacrificed 7 dpi.

(B) Quantification of Sox9⁺/GFP⁺ cells over total GFP⁺ cells per HF in Sox9KO mice and Ctrl mice 7 dpi confirming successful knock out of Sox9; n = 4 mice per group; 4–10 HF per mice; data are presented as mean ± SEM.

(C) Quantification of the mean cell number of GFP⁺ or Nestin⁺/GFP⁺ cells per HF in Sox9KO mice and Ctrl mice 7 dpi; n = 4 mice per group; 12 HF per mice; data are presented as mean ± SEM.

(D) Immunohistochemical staining for GFP (green), Nestin (red) and DCT (white) in Sox9KO mice and Ctrl mice 7 dpi; dashed line marks the hair follicle; E = Epidermis, IF = Interfollicular area, B = Bulge, ORS = Outer root sheath, Bu = Bulb; Magnifications show signals in the bulge (upper panels), the ORS (middle panels) and the hair bulb (lower panels); DAPI is depicted in blue (scale bar = 20 μm); yellow arrows point toward DCT⁺/Nestin⁺/GFP⁺ cells (upper, middle panels) and DCT⁺/Nestin^neg/GFP⁺ cells (middle, lower panels); yellow arrowheads point toward DCT^neg/Nestin^neg/GFP⁺ cells (upper panels) and DCT⁺/Nestin^neg/GFP^neg cells (middle panels (Sox9KO), lower panels).

(E) Quantification of the mean cell number of DCT⁺/GFP⁺ cells in the bulge/ORS and bulb in Sox9KO mice and Ctrl mice 7 dpi; n = 4 mice per group; 5–12 HF per mice; data are presented as mean ± SEM.

(F) Quantification of the mean cell number of DCT⁺/Nestin⁺/GFP⁺ cells in the bulge in Sox9KO mice and Ctrl mice 7 dpi; n = 4 mice per group; 5–12 HF per mice; data are presented as mean ± SEM.

(G) Quantification of the mean cell number of DCT⁺ cells per HF in Sox9KO mice and Ctrl mice 7 dpi; n = 4 mice per group; 12–15 HF per mice; data are represented as mean ± SEM. (B, E, F, G) students two-sided t-test was applied to determine statistical significance (∗: p < 0.05, ∗∗: p < 0.01).