Fig 1: EcsitPrx1 mice show defects in skeletogenesis, fracture healing, and the bone marrow niche.a Immunostaining of ECSIT in 2-month-old wild-type femurs. tb: trabecular bone, cb: cortical bone. b Immunofluorescence showing the localization of ECSIT in P21 mouse femurs. Arrows indicate co-localization of ECSIT and NDUFS3. c. Immunoblot analysis showing mitochondrial fractionation of ECSIT in human BMSCs. d, e X-radiography (d, left) and microCT showing P21 whole body and hindlimbs (d, middle) and quantification of femoral bone mass (e, n = 5). Alizarin red/alcian blue staining of P1 neonates (d, right). Arrows indicate spontaneous fractures. Tra. BV/TV: Trabecular bone volume/total volume. f H&E staining of E16.5 and P0 femurs (left, middle). Immunofluorescence showing osteocalcin (OCN) expression in the femurs (right). Arrows indicate femoral trabecular bone. g Skeletal progenitors isolated by FACS from P0 Ecsit;Prx1Rosa26mTmG (cKO) and Prx1;Rosa26mTmG (WT) limbs using GFP+CD45-Ter119-Tie2- markers. Osteogenic gene expression (n = 4) and alkaline phosphatase activity (ALP, n = 3) were assessed for osteogenic differentiation. h Flow cytometry analysis showing frequency of the indicated bone marrow cell populations at P10 (right, HSCs (Ecsitfl/fl, n = 7; EcsitPrx1, n = 5); other subpopulations, n = 4-5) and total number of the isolated cells (left, Ecsitfl/fl, n = 7; EcsitPrx1, n = 6). Supplemental Fig. 2 demonstrates the gating strategy for this analysis. i GFP-expressing PRX1-lineage cells residing in the diaphyseal periosteum of P1 PRX1-cre;Rosa26mTmG mice. m: muscle, bm: bone marrow. j P21 femoral cortical bones were stained for Von Kossa (top), H&E (bottom) and immunofluorescence for OCN (middle). k H&E staining of EcsitPrx1 femurs at the age of P0, P10, and P21. l, m Flow cytometry analysis showing frequency of SSCs, pre-BCSP, and BCSP in skeletal cells (l, n = 5). RT-PCR shows expression of Ecsit in pre-BCSP (m, n = 3). Supplemental Fig. 3 demonstrates the gating strategy for this analysis. A two-tailed unpaired Student’s t-test for comparing two groups (e, g, h, l, m; error bars, data represent mean ± SD). Data are representative of three independent experiments. Scale bars = a, 100 µm; b, 50 µm; d (left, middle), 2 mm; d (right), 1 mm; f, 60 µm; i, 75 µm; j, 40 µm; k, 500 µm.