Fig 1: Localization of SRY and cMyb in primary osteoblast cells of male origin.Localization of SRY-GFP fusion protein in male primary osteoblasts. Color legend for merged image: blue, Hoechst 33342 nuclear stain; green, SRY-GFP fusion protein; red, antibody targeting endogenous cMyb. POB primary osteoblasts; GFP green fluorescent protein. Scale bar: 10 μm
Fig 2: 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 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: Expression of RANKL, c-Myb, and Sry in human bone tissues is dependent on gender and health state.qPCR was used to measure gene expression in human bone samples from male osteoporotic (OP, n = 12), osteoarthritic (OA, n = 13) and healthy (ctl. n = 12) patients and from female osteoporotic (OP, n = 46) and osteoarthritic (OA, n = 29) patients. Mean mRNA levels were normalized to RPLP0. Asterisk, significant differences between groups. a Comparison of RANKL gene expression in bone samples from male and female patients. b Comparison of RANKL gene expression in bone samples from OP, OA, and ctl male patients. c Comparison of RANKL gene expression in bone samples from OP and OA female patients. d Comparison of Sry gene expression in bone samples from OP, OA, and ctl male patients. e Comparison of c-Myb gene expression in bone samples from OP, OA, and ctl male patients. f Comparison of c-Myb gene expression in bone samples from OP and OA female patients. g Comparison of c-Myb gene expression in bone samples from male and female patients. h Comparison of Sry: c-Myb expression ratios in bone samples from OP, OA, and ctl male patients
Fig 5: Immunolocalization of cMyb and SRY in human bone tissue.a–c Bone tissue of a male patient with osteoporotic fracture. a cMyb staining is observed in the nuclei of lining cells, individual bone marrow cells, and osteocytes. b In inactive bone, SRY staining is observed in various cells of the bone marrow (blue arrow) and in the lining cells (black arrows), while osteocytes are negative for SRY (red arrow), similar to what was observed with cMyb. c In bone with active processes of bone remodeling, in addition to the cells in bone marrow, SRY was observed in active osteoblasts (closed arrows) and osteocytes (open arrows) just beneath the trabecular bone surface, which suggests its role in bone remodeling via RANKL. d No positive SRY staining was observed in female patients with osteoporotic fracture. e, f Bone tissue of a male patient with osteoporotic fractures showing costaining of cMyb and SRY in osteoblast-like cells (arrows) on the surface of trabecular bone (e); single-positive SRY or cMyb cells (arrowheads, e, f) are present in bone marrow (BM); and cMyb single-positive cells are also present on the surface of trabecular bone (TB) (e, f). g, h Costaining for SRY and cMyb in bone tissue of a male patient with osteoarthritis (g) and in a male donor with no musculoskeletal disease (h) show similar patterns of localization; i.e., SRY single-positive cells (arrowheads) and SRY and cMyb double-positive cells (arrows) in bone marrow (BM). Immunohistochemistry (a–d) and double immunofluorescence (e–h) of human bone tissue. N = 3 donors per group (fracture, osteoarthritis and control group). Scale bars: 50 µm
Supplier Page from Santa Cruz Biotechnology, Inc. for SRY siRNA (h)