Extended Data Fig. 5: Establishment of an in vitro keratinocyte competition assay. | Nature Aging

Extended Data Fig. 5: Establishment of an in vitro keratinocyte competition assay.

From: Distinct types of stem cell divisions determine organ regeneration and aging in hair follicles

Extended Data Fig. 5

a, b, IF images of COL17A1 (a) or aPKCλ (b) in HaCaT cells at 72 hr after transfection of EmGFP-tagged sh SCRAMBLE (SCR) or sh COL17A1 or sh aPKCλ expressing vectors. COL17A1 or aPKCλ expression were decreased in EGFP tagged HaCaT cells (dashed lines), respectively. c, Western blot hybridization analysis of aPKCλ in EmGFP-tagged sh SCR or sh aPKCλ expressing stable HaCaT cell lines. d, Quantification of cell proliferation rates in EGFP tagged sh SCR or sh COL17A1 or sh aPKCλ stable HaCaT cell lines (n = 4 wells each lines). Cell proliferation was not significantly different in any of these cell lines. e, Western blot hybridization analysis of COL17A1 and EmGFP in the doxycycline (Dox) inducible EmGFP tagged sh SCR or sh COL17A1 expressing stable HaCaT cell lines with or without Dox. Dox treatments induce EmGFP and COL17A1 knockdown. f, g, h, IF images of COL17A1, EmGFP and KRT10 after 3D-coculture with sh SCR or sh COL17A1 stable HaCaT cell lines and wild-type HaCaT cells at ratios of 10:0 (f) and 1:10 (g, h) with DOX from -3 days (h) or 1 day (f, g) after plating. Arrows, EmGFP+ basal cells; White dashed line, basement membrane. i, Quantification of EmGFP+ cell contribution in the basal cell layer (sh SCR, Dox 1d, n = 15; sh COL17A1, Dox 1d, n = 15; sh SCR, Dox -3d, n = 10; sh COL17A1, Dox -3d, n = 11 images). COL17A1 KD cells were significantly eliminated by wild-type cells and did not depend on their ability to attach to the base of the dish. Error bars, means ± SEM; Kruskal-Wallis test with Dunn’s post hoc test (d); two tailed Student’s t-test (i).

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