(A) Genetic correction of Fanca deficiency abolishes Nlrp12 induction under persistent DNA damage. LSK cells from Fanca^–/– mice were transduced with lentiviral vector expressing Venus or Venus/FANCA. Two thousand sorted Venus⁺ cells were transplanted into lethally irradiated BoyJ recipients followed by a single dose of MMC injection at 2 weeks posttransplant. Venus⁺ SLAM cells were then sorted for qPCR analysis for Nlrp12 expression at the indicated time points. The kinetics of NLRP12 expression is shown. Samples were normalized to the level of GAPDH mRNA (n = 9 per group). (B) Knockdown of Eya2 increases Nlrp12 expression in WT HSCs. LSK cells from WT mice were transduced with lentiviral vector expressing scramble shRNA or shRNA targeting Eya2. Two thousand sorted GFP⁺ cells were transplanted into lethally irradiated BoyJ recipients followed by single dose of MMC injection. GFP⁺ SLAM cells were then sorted for qPCR analysis for Nlrp12 at the indicated time points post–MMC treatment. Results are shown as means ± SD of 3 independent experiments (n = 9 per group). CTL, untreated control. (C) Eya2 knockdown induces persistent DNA damage in WT HSCs. WBMCs from the mice described in B were isolated for flow cytometry analysis for γ-H2AX in donor-derived SLAM cells at the indicated time points after MMC injection. (D and E) Persistent DNA damage in HSCs from mice deficient in Brca2, DNAPKcs, and Parp1 genes. Quantification of γ-H2AX levels (D) and Nlrp12 expression (E) in Brca2^–/–, DNAPKcs^3A/3A, and Parp1^–/– HSCs following MMC treatment is shown. (F) Serum TNF-α levels in WT and Fanca^–/– mice subjected to DNA damage. WT and Fanca^–/– mice were i.p. injected with a single dose of MMC (0.75 mg/kg). The serum of BM was subjected to ELISA 16 hours later for TNF-α levels (n = 5). (G) NF-κB activation in HSCs from WT and Fanca^–/– mice subjected to DNA damage. WBMCs from mice described in F were subjected to flow cytometry analysis for phosphorylated p65 (p-p65) in SLAM cells. (H and I) Suppression of inflammatory signaling failed to prevent DNA damage–induced Nlrp12 upregulation in Fanca^–/– HSCs. Fanca^–/– mice or their WT littermates were injected with a single dose of MMC injection. Anti-TNF antibody (H) or NF-κB inhibitor BAY11-7082 (I) was administered 30 minutes before and after MMC injection. Sixteen hours after MMC treatment, SLAM cells were sorted for qPCR analysis for Nlrp12 expression. Samples were normalized to the level of GAPDH mRNA. Results are shown as means ± SD of 3 independent experiments (n = 6–8 per group). **P < 0.01; ***P < 0.001. Paired or unpaired 2-tailed Student’s t test was used for 2-group comparison and 1-way ANOVA for comparison of more than 2 groups.