Fig 2: TNF regulates IL-23A expression by phosphorylating N-WASP and degradation of G9a/GLP complex. a qRT-PCR analysis of IL-23A mRNA expression in con and ko primary keratinocytes treated for 4 h with TNFa (n: 3/3, mean ± SD, two-tailed unpaired t-test). b FACS analysis of surface TNF-RI in primary keratinocytes (n: 3/3, two-tailed unpaired t-test). c Primary keratinocytes were treated for indicated times with TNFa and then tested by immunoblot for expression of indicated proteins (n: 4/4). d Primary keratinocytes were treated for indicated time with TNF, fractionated, and analyzed by immunoblot for indicated proteins (C: cytosolic fraction, S300: soluble nuclear fraction extracted by 300 mM NaCl, P300: pellet after 300 mM NaCl extraction, SE: short exposure, LE: long exposure; n: 3). e ChIP of primary keratinocytes for H3K9me2 with qRT-PCR for indicated regions of IL-23A promoter after treatment with TNFa for indicated times (n: 5/5, mean ± SD, two-tailed unpaired t-test). f qRT-PCR analysis of IL-23A mRNA expression in con and ko primary keratinocytes pretreated with or without IOX-1 for 24 h and then stimulated with or without TNFa for 4 h as indicated. All normalized to GAPDH (n: 4/4, mean ± SD, two-tailed unpaired t-test; *p = 0.05; **p = 0.01; ***p = 0.001)

Fig 3: N-WASP represses IL-23p19 expression in keratinocytes by regulating H3K9 dimethylation. a, b Immunoblot analysis of H3K9me2 level in con and ko primary keratinocytes treated with BIX01294 (a) or IOX-1 (b) for 24 h (n: 3/3, mean ± SD, one-way ANOVA, with Tukey’s multiple comparisons). c Immunoblot for H3K9me2 in epidermis of 6-day-old mice (n: 3/3, mean ± SD, two-tailed unpaired t-test). d H3K9me2 IF of back skin of 6-day-old mice (n: 3/3). Scale bar: 100 µm. e qRT-PCR analysis of IL-23A mRNA expression in sorted interfollicular epidermal keratinocytes from con and G9a knockout. All normalized to GAPDH (n: 4/4, mean ± SD, two-tailed unpaired t-test). f ChIP of primary keratinocytes for H3K9me2 with qPCR for indicated regions of IL-23 promoter (n: 7/7, mean ± SD, two-tailed unpaired t-test). g, h Primary N-WASP fl/fl cells lentivirally transduced with GFP or Cre-GFP were analyzed by western blot for indicated genes (g) and by qRT-PCR for IL-23A expression (h; n: 4/4, mean ± SD, two-tailed unpaired t-test; *p =0.05; **p = 0.01; ***p = 0.001)

Fig 4: IL-23A expression is increased in N-WASP-null keratinocytes. a qRT-PCR analysis of indicated genes in FACS purified primary keratinocytes from 6-week-old mice (n: 4/4/4, mean ± SD, two-tailed unpaired t-test, 2 independent sorting). b qRT-PCR analysis of indicated cytokine mRNA expression in primary keratinocytes. All normalized to GAPDH (n: 3/3, mean ± SD, two-tailed unpaired t-test). c Primary keratinocytes were differentiated with 2 mM CaCl2 (High Ca2+) for 48 h and then analyzed by qRT-PCR for IL-23A expression, compared to untreated cells (Low Ca2+; n: 3/3, mean ± SD, two-tailed unpaired t-test). d qRT-PCR analysis of IL-23A mRNA expression in primary keratinocytes in response to 24 h treatment with indicated inhibitors. All normalized to GAPDH (n: 3/3, mean ± SD, two-tailed unpaired t-test). e Representative immunofluorescence of phalloidin in con and ko back skin. Scale bar: 100 µm. f Quantification of FACS mean fluorescence intensity of phalloidin in con and ko primary keratinocyte (n: 3/3, mean ± SD, two-tailed unpaired t-test). g qRT-PCR analysis of IL-23A mRNA expression in primary keratinocytes treated with indicated inhibitors for 24 h. All normalized to GAPDH. The right panel shows the same data, but normalized to the respective DMSO-treated samples, thus showing the relative effect of the treatment as fold change (n: 3/3, mean ± SD, two-tailed unpaired t-test; *p = 0.05; **p = 0.01; ***p = 0.001; ****p = 0.0001)

Fig 5: Keratinocyte-derived IL-23p19 is important for skin inflammation in N-WASP knockout. a Strategy of loxP insertion in mouse IL-23A gene. b, c CD45, K6, Ki67 immunofluorescence of back skin section from 6-week-old con, ko, and dko (b), quantification of mean fluorescence intensity of CD45, and quantification of Ki67-positive cells in interfollicular epidermis (IFE) (n: 5/5/5, mean ± SD, one-way ANOVA, with Tukey’s multiple comparisons). Scale bar: 100 µm. d qRT-PCR analysis of indicated genes from back skin RNA 6–8-week-old of con, ko, and dko mice. All normalized to GAPDH (n > = 6/6/6, mean ± SD, one-way ANOVA, with Tukey’s multiple comparisons; *p = 0.05; **p = 0.01; ***p = 0.001; ****p = 0.0001)