TNF-α treatment prevents loss of H3H9me3 at intronic L1Md and HSC gene repression in vitro. (A–C) NFKB1 expression in HSCs 1 mo after TBI. (B) mRNA expression measured by qRT-PCR. Ct values were normalized to mean of RPL32 and HPRT. Results are expressed as fold change from the mean value of the NIR condition. Means ± SEM. Each dot represents a pool of three (NIR) or four (IR) mice from three independent experiments. t test. (C) Representative images and quantification of NFKB1 protein mean immunofluorescence (IF) intensity. Bars, 5 µM. Each dot represents a cell. Results are expressed as fold change from the mean value of the NIR condition from two independent experiments and represented as means ± SEM. t test. (D) Experimental design analyzing the effects of IR and TNF-α in vitro in WT and Nfkb1^−/− mice. (E) Representative images and quantification of NFKB1 staining. Bars, 5 µM. Each dot represents a cell. Results are represented as mean ± SEM of NFKB1 IF intensity. One-way ANOVA with Tukey’s multiple comparison test. (F) Gene expression evaluated by qRT-PCR in WT mice. Means ± SEM from two independent experiments. One-way ANOVA with Tukey’s multiple comparison test. (G) H3K9me3 enrichment at intronic L1Md evaluated by ChIP-qPCR. Results are expressed as in legend to Fig. 5 D. Means ± SEM from two to three independent experiments. One-way ANOVA with Bonferroni’s multiple comparison test. (H and I) mRNA expression measured by qRT-PCR in HSCs sorted from Nfkb1^−/− mice (KO) vs. WT without IR (H) or after IR and with or without prior TNF-α treatment in vitro (I). Ct values were normalized to the mean of RPL32 and HPRT. (J) H3K9me3 enrichment at intronic L1Md evaluated by ChIP-qPCR. For each experiment (H–J), a pool of 14–16 mice was used and divided in two to four culture replicates. Each dot represents one replicate of two to five independent experiments. Means ± SEM, t test (H); one-way ANOVA with Tukey’s multiple comparison test (I and J). *, P < 0.05; **, P < 0.01; ***, P < 0.001.