NF-κB is required for TPO-induced Iex-1 expression and DSB repair in HSPCs. (A) EMSA for NF-κB DNA-binding activity in WT and Mpl^+/− LSK cells cultured in complete medium (Comp) or without TPO for 45 minutes and then irradiated (2 Gy) or not. Analysis was performed 30 minutes after IR. Representative experiment out of 4 similar performed. (B) Supershift analysis after addition of anti-p65RelA antibody or no antibody (−) in WT LSK cells treated as in (B) 30 minutes post-IR in the presence of TPO. (C) Iex-1 mRNA expression 30 min after IR in LSK cells precultured in medium without TPO or in complete medium containing either the Nemo peptide or a control peptide (10 µM). Data are means + SEM from 4 independent experiments performed with cells from 3 to 5 mice. (D) Comet tail moment of WT LSK cultured as in panel C and 2 hours after IR. Representative pictures of comets labeled with SYTOXGreen and means + SEM of the tail moment values measured in at least 60 cells are shown. The scale bars on the pictures represent a 107 µm × 10 objective. One representative experiment out of 3 experiments is shown. (E) γH2AX staining in LSK cells cultured like in panel D in the presence or absence or of the NF-κB inhibitor (NF-κB I, 1 µM) 24 hours after IR. Data are means + SEM of 3 independent experiments with at least 100 cells counted per condition.