Supplementary Figure 1: H2B-GFP tet-off system to count cell division frequency in skin cells in vivo.

(a) Models of cellular lineage hierarchy in the epidermis. (b) Schematic of microscopic inspection of skin sections or whole-mounts. (c) Adult H2B-GFP tet-off mice subjected to doxy chases. After 1 week of doxy administration, many epidermal cells still have high levels of GFP signal. A chase period of 2 weeks can detect a fraction of bright H2B-GFP clustered cells, indicative of a more infrequent division history. After 3 weeks of doxy chase, rare H2B-GFP bright cells are present (arrowhead). (d) Mice were subjected to 2 weeks of doxy and 2 days of EdU to verify inverse correlation between proliferation and H2B-GFP retention. Data from two mice are shown as individual bars. (e) Unchased control for the tail epidermis whole-mount. Both interscale (K10⁺) and scale (K10⁻) show similar level of the original H2B-GFP signal. (f) Whole-mount immunostaining of the tail skin after 6 weeks of doxy chase and 2 days of BrdU, showing basal cells on the scale are more proliferative. The dashed line surrounds non-LRC area (e,f) or represents epidermal-dermal junctions (c). Asterisks indicate HFs. Scale bars, 100 μm (c,e,f). (g) Number of hair follicles within each structure. Data from two mice are shown as individual bars. Experiments are repeated twice with 2 mice for all representative images (c,e,f). (h) Number of blood vessel branch points within each structure detected in back skin. Data from two mice are shown as individual bars.