Requirement of Gimap5 for the association of Mcl-1 with HSC70, Mcl-1 stability, mitochondria integrity, and the survival of BM-derived B cell and hematopoietic progenitors. (A) Total cell lysates from IL-7 BM culture–derived wild-type or Gimap5-deficient B cell progenitors were subjected to Western blot analysis with anti-Gimap5 or anti-Actin. (B) Cell lysates from wild-type or Gimap5-deficient IL-7 BM culture–derived B cell progenitors were immunoprecipitated with anti–Mcl-1 or anti–Bcl-xL, followed by Western blot analysis with anti-HSC70. (C and D) The cytosol and mitochondrial fractions were extracted from IL-7 BM culture–derived wild-type or Gimap5-deficient B cell progenitors (C) or IL-3 + IL-6 + SCF BM culture–derived wild-type or Gimap5-deficient CD34⁺ hematopoietic progenitors (D). These fractions were subjected to Western blot analysis with the indicated antibodies. Tom20 was used as a marker of mitochondrial fraction. *, nonspecific bands. (E and F) IL-7 BM culture–derived wild-type or Gimap5-deficient B cell progenitors (E) or purified IL-3 + IL-6 + SCF BM culture–derived wild-type or Gimap5-deficient CD34⁺ hematopoietic progenitors (F) were cultured without cytokines for the indicated times. The total cell lysates were subjected to Western blot analysis with the indicated antibodies. Data shown are representative of five independent experiments. (G) IL-3 + IL-6 + SCF BM culture–derived wild-type and Gimap5-deficient CD34⁺ hematopoietic progenitors were cultured in the presence or absence of IL-3 + IL-6 + SCF for 16 h, followed by incubation with Δϕ_m indicator JC-1. The green (FITC) and red (PE) fluorescence were measured by flow cytometry. The percentages of cells with decreased mitochondrial Δψ_m in total alive cells are shown. Data shown are representative of two independent experiments. (H and I) IL-7 BM culture–derived wild-type or Gimap5-deficient B cell progenitors (H) or IL-3 + IL-6 + SCF BM culture–derived wild-type and Gimap5-deficient hematopoietic progenitors (I) were cultured in the presence of BrdU for 1 h, followed by BrdU and 7-AAD or DAPI staining. The percentages of BrdU⁺ cells in S/G₂/M phase are shown. Data shown are representative of three (H) or two (I) independent experiments. (J and K) IL-7 BM culture–derived wild-type or Gimap5-deficient B cell progenitors (J) or IL-3 + IL-6 + SCF BM culture–derived wild-type and Gimap5-deficient hematopoietic progenitors (K) were cultured in the absence of cytokines for the indicated times. The cell apoptosis was examined by Annexin V and 7-AAD staining. The percentages indicate cells in total (J) or Lin⁻c-Kit⁺Sca⁺ population (K). Data shown are representative of three independent experiments.