Journal: The Journal of Experimental Medicine

Article Title: Strawberry notch homologue 2 regulates osteoclast fusion by enhancing the expression of DC-STAMP

doi: 10.1084/jem.20130512

Figure Lengend Snippet: Sbno2 plays a key role in fine tuning DC-STAMP expression. (A) MDMs from wild-type mice were treated with 100 ng/ml RANKL for the indicated times. Sbno2 mRNA levels were measured by qPCR. (B) qPCR analysis of Jdp2, TRAP, DC-STAMP, and Sbno2 in wild-type and Jdp2 −/− MDMs stimulated with 50 ng/ml RANKL. (C) MDMs were transfected with control siRNA (cont) or c-Fos–specific siRNA (siFos) and stimulated with 50 ng/ml RANKL. c-Fos and Sbno2 levels were measured by qPCR. (D) MDMs were treated with 3 µg/ml FK506 (FK506) or DMSO (cont) and stimulated with 50 ng/ml RANKL. NFATc1 and Sbno2 levels were measured by qPCR. (E) RANK and c-fms mRNA levels in unstimulated MDMs from wild-type and Sbno2 −/− mice were measured by qPCR. (F) MDMs from wild-type mice were treated with 150 ng/ml RANKL for the indicated times. Nuclear extracts were harvested and DNA-binding activity of NFATc1 and NF-κB p65 were measured using a TransAM Transcription Factor Assay kit. (G) MDMs from wild-type and Sbno2 −/− mice were treated with 100 ng/ml RANKL for 24 h and Ca 2+ imaging was performed. Three representative traces of change in the fura-2 fluorescence ratio in cells are shown. (H) qPCR analysis of DC-STAMP level in wild-type and Sbno2 −/− MDMs stimulated with 50 ng/ml RANKL for the indicated number of hours. (I) Chromatin immunoprecipitation analyses with indicated antibodies of lysates from wild-type and Sbno2 −/− MDMs stimulated with or without 100 ng/ml RANKL for 84 h. DNA fragments of the DC-STAMP promoter region were detected by PCR. (J–L) Effect of exogenous DC-STAMP expression on the osteoclastogenesis of Sbno2 −/− MDMs stimulated with 50 ng/ml RANKL for 4 d. Nuclei numbers (J) in TRAP + cells were analyzed ( n = 4). DC-STAMP expression levels were measured by qPCR (K) and representative TRAP staining is shown (L). (M) Immunoprecipitation of wild-type MDM lysates. After anti-Tal1 and control IgG immunoprecipitation, immunoprecipitates were analyzed by Western blotting with anti-Sbno2 and anti-Tal1 antibodies. Sbno2 −/− MDMs were also used as Input. (N) Association between Sbno2 and Tal1 was analyzed by coimmunoprecipitation. HEK293T cells were transfected with the following constructs: Sbno2 1–500 Flag (aa residues 1–500), Sbno2 501–1000 Flag (aa residues 501–1,000), Sbno2 1001–1349 (aa residues 1,001–1,349), and Tal1 Myc. (O–R) Effect of Tal1 knockdown on the osteoclastogenesis of Sbno2 −/− MDMs stimulated with 50 ng/ml RANKL for 4 d. Nuclei numbers (O) in TRAP + cells were analyzed ( n = 4). DC-STAMP expression levels were measured by qPCR (P) and Western blotting (Q). Representative TRAP staining is shown (R). (S) Luciferase assays examining the effects of Sbno2 on the transcriptional activity of DC-STAMP. Results are representative of three (A–E and H–S) or two (F and G) independent experiments. Error bars, SE. *, P < 0.05, n = 3 unless indicated.

Article Snippet: For immunoprecipitation, precleared cell lysates were incubated with protein A–Sepharose (GE Healthcare) containing 3 μg anti-Tal1 antibodies (H-60; Santa Cruz Biotechnology, Inc.) or rabbit polyclonal IgG (Y-11; Santa Cruz Biotechnology, Inc.) for 1 h at 4°C.

Techniques: Expressing, Transfection, Control, Binding Assay, Activity Assay, Transcription Factor Assay, Imaging, Fluorescence, Chromatin Immunoprecipitation, Staining, Immunoprecipitation, Western Blot, Construct, Knockdown, Luciferase