Fig 2: c-Myb binds to the site -691/-674 bp upstream of the RANKL transcription start, and SRY binds to the site -1606/-1600 bp upstream of the RANKL transcription start.a Electrophoretic mobility shift assays using human bone osteosarcoma cell nuclear extracts and biotinylated oligonucleotide probes containing site -691/-674 bp upstream of the RANKL transcription start. Lane 1, negative control without nuclear extract; lane 2, nuclear extract added; lane 3, competitive unlabeled probes added; lane 4, anti-c-Myb antibodies added; lane 5, mutated competitive unlabeled probes added. b Electrophoretic mobility shift assays using human bone osteosarcoma cell nuclear extracts and biotinylated oligonucleotide probes containing site -1606/-1600 bp upstream of the RANKL transcription start. Lane 1, negative control without nuclear extract; lane 2, Sry-FLAG transfected nuclear extract added; lane 3, nontransfected nuclear extract added; lane 4, competitive unlabeled probes added; lane 5, mutated competitive unlabeled probes added; lane 6, anti-FLAG antibodies added. c Western blotting confirmation of Sry-FLAG transfection in the nuclear extract (NE) used for the electrophoretic mobility shift assays in b

Fig 3: c-Myb and SRY affect the expression of the RANKL gene in human primary osteoblasts.a, b Female (a) and male (b) human primary osteoblasts were transfected with empty expression vector (ctl) and Sry-FLAG or c-Myb-HA. RANKL gene expression was measured at 48 h after treatment using q-PCR. c, d Male human primary osteoblasts were nucleofected with negative control siRNA and siRNAs against c-Myb (c) or Sry (d). RANKL gene expression was measured at 72 h after treatment using q-PCR. All of the data are presented as the mean relative RANKL expression after normalization ± SEM. Asterisks indicate significant differences between two samples. Data are representative of at least three independent experiments

Fig 4: c-Myb increases and SRY decreases the luciferase activity of the RANKL promoter region.a Different lengths of the RANKL proximal promoter region (F1, F2, F3, F4, F5) were cloned into the luciferase reporter vector pGL3-basic. Mutations were induced in the predicted SRY binding site (-731 AA > CC) and the predicted c-Myb binding site (-680 TT > GG) in the F4 vector using site-directed mutagenesis. b HOS (human bone osteosarcoma) cells were transfected with pGL3-F1/-F2/-F3/-F4/-F4-SRY/-F4-c-Myb and pRL normalization vector. Luciferase activities were measured 24 h after transfection. c Luciferase assays of HOS cells cotransfected with the pGl3-F4 RANKL promoter region and pcDNA3 (empty) or c-Myb-HA and cotransfected with the mutated pGl3-F4 RANKL promoter regions and c-Myb-HA. d Luciferase assays of HOS cells cotransfected with the pGl3-F4 RANKL promoter region and pcDNA3 (empty) or Sry-FLAG and cotransfected with the mutated pGl3-F4 RANKL promoter regions and Sry-FLAG. e Luciferase assays of HOS cells cotransfected with the pGl3-F5 RANKL promoter region and pcDNA3 (empty) or Sry-FLAG. f Luciferase assays of human primary osteoblasts nucleofected with pGL3-F5 RANKL promoter region and pcDNA3 (empty) or Sry-FLAG or c-Myb-HA. g Western blotting of human bone osteosarcoma cells after transfection with Sry-FLAG or c-Myb-HA expression vector and of empty human bone osteosarcoma cells. All of the data are presented as the mean luciferase activities after normalization ± SEM. Asterisks indicate significant differences between luciferase activities

Fig 5: Colocalization of SRY and cMyb with T-lymphocyte and B-lymphocyte markers in human bone tissue.a Bone tissue of a male patient with osteoporotic fracture showing costaining of SRY with the T-lymphocyte marker CD3 (arrow) in bone marrow (BM) cells. Double-positive cells (arrows) and single-positive cells for SRY or CD3 (arrowhead) are observed within bone marrow cells. b Bone tissue of a male patient with osteoporotic fracture showing costaining of cMyb with the B-lymphocyte marker CD19. Double-positive cells (arrow) and single-positive cells for cMyb or CD19 (arrowhead) are observed within bone marrow (BM) cells. c Bone tissue of a male patient with osteoporotic fracture showing costaining of cMyb with the T-lymphocyte marker CD3 (arrow) in bone marrow (BM) cells. Double-positive cells (arrows) are observed within bone marrow cells. Single-positive cells for cMyb (arrowhead) are observed within bone marrow cells, on the trabecular bone (TB) surface, and in a few osteocytes. d Bone tissue of female patients with osteoporotic fracture showing negative staining for SRY and similar patterns of cMyb staining as in the male osteoporotic fracture tissue. Scale bars: 50 µm