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. 2012 May 14;197(4):553–568. doi: 10.1083/jcb.201111116

Figure 6.

Figure 6.

Circumferential podosomes/invadopodia mediate invasive and fusion activities. (A) B16F0 murine melanoma cells transfected with control (Ctr) or Tks5 siRNAs were cultured in the presence of 10 ng/ml RANKL alone or together with 5 ng/ml TGF-β and 5 ng/ml TNF-α for 48 h. (left) The cells were then stained with rhodamine-phalloidin to visualize F-actin, with antibodies to Tks5, and with DAPI to visualize nuclei. Arrowheads indicate circumferential invadopodia. Pixel intensities on the traversing dashed line were measured using LAS AF software and shown in a boxed area (arbitrary units). Bars, 25 µm. (right) The percentage of cells with circumferential invadopodia among >100 cells scored was also determined. Data are means ± SD from three independent experiments. *, P < 0.05; **, P < 0.001 (Student’s t test). See also Fig. S3 B for immunoblot. (B) B16F0 cells cultured in the presence of 10 ng/ml RANKL alone or together with 5 ng/ml TGF-β and 5 ng/ml TNF-α for 48 h were subjected to immunoprecipitation with antibodies to Tks5, and the resulting precipitates as well as the original cell lysates (Input) were subjected to immunoblot analysis with the indicated antibodies. The minus sign indicates no stimulus added to the culture. (C) B16F0 cells transfected with control (Ctr) or Tks5 siRNAs were cultured in the presence of 10 ng/ml RANKL alone or together with 5 ng/ml TGF-β and 5 ng/ml TNF-α for 48 h. The cells were then replated in Matrigel chambers and assayed for invasive activity in the same conditions. Cells that had invaded through the Matrigel at 24 h after plating were stained with crystal violet, and those in three different sampling areas were counted. Data are means ± SD from three independent experiments. **, P < 0.001 (Student’s t test). (D, left) Experimental protocol for co-culture of B16F0 melanoma cells and osteoclasts derived either from parental RAW264.7 macrophages or from those expressing hTks5-WT, hTks5-ΔPX, hTks5-Y(#2,#3)F, or hTks5-Y(#2,#3)E. See also the Materials and methods section for details. (right) The cultures were then stained with rhodamine-phalloidin (red), antibodies to GFP, and DAPI (blue). Bars, 50 µm. (E) Quantification of the number of osteoclasts harboring at least one GFP-positive nucleus after co-culture with B16F0 cells on an 11 × 22–mm coverslip as in D. Data are means ± SD from more than three independent experiments. *, P < 0.05; **, P < 0.01 (Student’s t test). IP, immunoprecipitation.