Fig 2: 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 3: Mild, Transient Perturbations in Mitochondrial Function Are Associated with Increased Survival in Sepsis(A and B) CRIF1 and Cre recombinase protein expression (A) and Crif1 mRNA expression (B) in Crif1lox/lox or Crif1lox/− mice 7 days after injection of AAV8-TBG-expressing Cre recombinase (Ad.Cre) or GFP as a control (Ad.GFP).(C) Survival of Crif1lox/lox or Crif1lox/− mice, previously injected with AAV8-TBG-CRE, after infection with E. coli.(D) Bacterial loads in Crif1lox/− mouse blood 24 h after infection.(E) Survival of infected mice treated with phenformin at the time of infection.(F) Bacterial loads in blood 24 h after infection.(G) Enzymatic activity of the ETC complex I in liver at 12 h. ETC activity is expressed as % of PBS-treated control, normalized for CS activity.(H) HPLC-MS analysis of FAO metabolites in liver 8 h after infection and phenformin treatment. Each square represents one mouse.Data in (A) is from a single experiment. (B) represents mean ± SD of four mice per group assayed in triplicate. (C) represents pooled data from three independent experiments. (D) represents data from a single experiment. Bars indicate the mean. (E) represents pooled data from four independent experiments. (F) represents pooled data from two independent experiments. Bars indicate the mean. (G) represents mean ± SD of five mice per group from a single experiment. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. See also Figure S6.

Fig 4: SLC6A7 protein is upregulated and shows ectopic expression in CamiCre+ hippocampus(A) Western blotting results from hippocampal lysates.(B) Quantification results of A. Values were normalized against α-tubulin expression level and are shown as fold increases against Cre- controls. Mean ± SD is shown. ∗∗p < 0.01. N = 4 (biological replicates). Statistical tests were performed using two-tailed, unpaired t-tests.(C) Immunofluorescent images of SLC6A7 or Synapsin1/2 staining in the hippocampal region of CamiCre mice. Arrows point to mossy fibers projecting from granule cells in the dentate gyrus. Scale bars: 250 μm.

Fig 5: Establishment of transgenic mice. (A) Principle of tissue-specific inducible transgenic mice system. (B) Cre was detected in lung tissue by IHC staining (scale bar, 100 µm). (C) Cre was detected by IHC staining in the peripheral lung tissue and then SPC was detected by IHC staining after antibody stripping. The solid arrows indicate that Cre and SPC were co-localized in the same cell. (D) SPC was detected by IHC staining and then mEGFP was detected by IHC staining after antibody stripping. The solid arrows indicate that SPC and mEGFP were co-localized in the same cell, while the hollow arrows indicate that SPC and mEGFP were not co-localized in the same cell (scale bars, 50 µm). IHC, immunohistochemistry; mEGFP, cell membrane-localized enhanced green fluorescence protein; SPC, surfactant protein C; ACTB, β-actin.