Supplementary Figure 7: Depletion of Slc1a3^CreER+ cells in tail skin.

(a) Lineage tracing of surviving Slc1a3^CreER+ cells after DTA-mediated cell killing. Whole-mount immunostaining of the tail epidermis in Slc1a3^CreERRosa-DTA;GFP/Rosa-tdTomato triple-transgenic mice shows increased apoptosis, as indicated by caspase3⁺ cells, and the loss of tdTomato⁺ cells and GFP upon TM induction. (b) Expression of tdTomato and GFP in tail whole-mounts. Small numbers of tdTomato⁺ cells survived in some scales and showed moderate expansion of clones by 2 weeks. (c,d) Two days of BrdU administration marked proliferating cells. The average number of BrdU⁺ cells per tdTomato⁺ cells is shown; data from two mice are shown as individual bars. (e) One week of BrdU administration marks proliferating cells in the BL of the epidermis. Following 5 weeks of chase, infrequently dividing cells were detected as BrdU LRCs and were located preferentially within interscales. The non-LRC (scale) area is marked in the Slc1a3^CreER/Rosa-tdTomato mice. (f) Experimental scheme of BrdU and TM treatment in Slc1a3^CreER/Rosa-DTA mice. (g,h) Whole-mount (g) or section staining (h) of Slc1a3^CreER/Rosa-DTA mice with BrdU pulse-chase, showing that LRCs became more proliferative (Ki67⁺; arrowheads) by 1 week after the loss of Slc1a3⁺ cells. Controls were either single-transgenic Slc1a3^CreER or Rosa-DTA mice. (i) Quantification of Ki67⁺ LRCs in scale and interscale regions at 1-week post-induction shows activation of proliferation in all areas (n = 3 mice). Statistical analyses were performed using a two-tailed Student’s t-test. Error bars show s.e.m.; ∗: P < 0.01; ∗: P < 0.05. (Interscale, P = 0.02; scale, P = 0.0006.) Significantly increased LRC proliferation was observed in both scales and interscales indicating that LRCs are activated to migrate and proliferate upon the loss of Slc1a3^CreER+ cells. Ki67, cell proliferation. The dashed lines represents the boundaries of the tail epidermis structures (circle is scale, while the remaining marked area is interscale). Asterisks indicate HFs. Scale bars, 100 μm (a,b,c,e,g,h). Experiments are repeated twice with 2 mice for all representative images (a,b,c,e,g,h).