Erk and IEX-1 are necessary for DNA-PK activation in human and mouse HSPCs. (A-B) Ser2056-pDNA-PK foci number 30 minutes after IR of CD34⁺ human progenitor cells cultured without TPO or in complete medium (Comp) in the presence or absence of 10 µM U0126 (A) or 1 µM NF-κB I or 10 µM Nemo inhibitory peptide (B). Each dot represents an individual cell. Data are means + SEM from cells counted in 3 independent experiments. (C) Number of pSer2056-pDNA-PK 30 minutes after IR of CD34⁺ human cells infected with lentiviruses encoding control GFP-shRNA or IEX-1-shRNA and cultured and analyzed as in panel A (left). Data are means + SEM foci per cell counted from 3 independent experiments. IEX-1 mRNA expression in the shRNA-infected cells analyzed on the left (right). (D) Number of Thr2609-pDNA-PK and (E) pErk/DNA-PKc interaction using PLA in UT7-Mpl infected with control (ΔU3) or with IEX-1-ΔFTF vectors and treated with TPO and IR as in panel A. Representative experiments. (F) Representative model illustrating the cooperation between NF-κB and Erk activation upon TPO and IR, leading to Iex-1 induction and Iex-1 activation, respectively. The complex formed between pErk/Iex-1/DNA-PKc is required for DNA-PK phosphorylation and DNA-repair promotion.