Fig. 5

Loss of IRF2 induces stem cell potential whereas loss of YY1 or SNAI2 reduces it. a Assessment of CRISPR–Cas9 mediated knockdown of YY1 (left panel) or SNAI2 (right panel) on colony formation and migration (as in Fig. 1) in HSCP-HK compared to control cells, with protein levels monitored by Western blot analysis utilizing β-actin protein level as a loading control. b Comparison of SNAI2 KD and YY1 KD to control cells in their functional ability to generate an epidermis in human dermo-epidermal 3D model. # indicates Epidermis layer. Epidermis thickness (μm) was quantified for each condition and differences assessed using One-way ANOVA with Holm–Šídák multiple comparisons test. ^****p < 0.0001. Scale bar = 167 μm. Means ± S.D. of n = 4 biological replicates. c Assessment of colony formation and migration in LSCP-HK cells with CRISPR–Cas9 mediated knockdown of IRF2 compared to control cells, with protein levels monitored by Western blot analysis utilizing β-actin protein level as a loading control. d Epidermis formation in human dermo-epidermal 3D model comparing IRF2 KD cells to controls. # indicates epidermis layer. Epidermis thickness (μm) was quantified for each condition (means ± S.D. of n = 3 biological replicates for CT and n = 1 for IRF2 KD). Scale bar = 167 μm. e Comparison of ability to clonally expand aHPEK-IRF2-KD versus control cells, where individual keratinocytes either fail to form a colony or form a colony belonging to three broad morphological types termed holo-, mero-, and paraclones (pictured, scale bar = 1.1 mm). Total colonies of each morphological type were counted and plotted as means ± S.D. of n = 4 biological replicates. *p < 0.05, ***p < 0.001, ****p < 0.0001 (Ordinary One-way ANOVA with Holm–Šídák multiple comparisons test)