Figure 3.

Tks5 plays an essential role in osteoclast fusion by generating circumferential podosomes. (A–D) RAW264.7 macrophages were transfected on a consecutive 2 d with control (Ctr) or one of two Tks5 siRNAs and were then stimulated with 10 ng/ml RANKL for 72 h. The cells were then subjected to immunoblot analysis (A), to quantitative RT-PCR analysis (B), or to semiquantitative RT-PCR analysis (C) as indicated. Data in B are means ± SD from three independent experiments. (D) The cells were also stained with rhodamine-phalloidin to visualize F-actin, with antibodies to Tks5, and with DAPI to visualize nuclei. Arrows indicate cells still expressing Tks5 that generate circumferential podosomes. (bottom) The percentage of cells with circumferential podosomes among >100 cells scored was also determined. Data are means ± SD from three independent experiments. **, P < 0.005 (Student’s t test). Bars, 50 µm. See also Fig. S1 A and Video 7. (E and F) RAW264.7 macrophages that express FLAG-tagged hTks5-WT or hTks5-ΔPX under the control of a Dox-responsive promoter were transfected with control or Tks5 siRNAs as described for A–D. They were then cultured in the absence or presence of 10 ng/ml RANKL or 1 µg/ml Dox for 72 h. TRAP-positive multinucleated osteoclasts of the indicated diameters were then identified and counted. Bars, 200 µm. Quantitative data are means ± SD from three independent experiments. *, P < 0.05 (Student’s t test). See also Fig. S1 (B–D). (G and H) RAW264.7 macrophages harboring Dox-sensitive FLAG–hTks5-WT (G) or FLAG–hTks5-ΔPX (H) constructs were cultured in the presence of RANKL with or without Dox for 72 h and were then stained with rhodamine-phalloidin (red), antibodies to FLAG, and DAPI (blue). Bars, 25 µm. (I) Binding proteins of hTks5-WT or hTks5-ΔPX were identified as described in the Materials and methods. A portion of the binding proteins was subjected to SDS-PAGE and silver staining. Green and red arrowheads indicate hTks5-WT and hTks5-ΔPX, respectively. Among the binding proteins identified, cytoskeletal proteins that associated with hTks5-WT or with hTks5-ΔPX are listed in the green and red circles, respectively. Accession numbers were obtained from the NCBI Protein database. (J) The binding proteins eluted as in I as well as the original cell lysates (Input) were also subjected to immunoblot analysis with the indicated antibodies. See also Fig. S1 E and Fig. S2 A. IP, immunoprecipitation; LC-MS/MS, liquid chromatography with tandem mass spectrometry; RAW, RAW264.7.