Figure 2.
Figure 2. Tks5 as a potential mediator of fusion-competent protrusion formation. (A) Venn diagram summarizing the results of screening for molecules required for osteoclast fusion downstream of PI 3-kinase. Microarray analysis of RAW264.7 cells stimulated with 10 ng/ml RANKL for 72 h or not treated (NT) revealed 7,088 genes whose expression was up-regulated by RANKL, and database analysis yielded 1,027 proteins that contain a PH or PX domain; 114 proteins were present in both datasets. See also Tables S1 and S2. (B) Domain organization of Tks5. SH3, Src homology 3 domain. (C and D) RAW264.7 macrophages cultured in the presence of 10 ng/ml RANKL for the indicated times were subjected either to quantitative RT-PCR analysis of the amount of Sh3pxd2a mRNA (normalized by that of Gapdh mRNA; C) or to immunoblot analysis with antibodies to Tks5, to NFATc1, or to γ-tubulin (loading control; D). Quantitative data are means ± SD from three independent experiments. (E and F) Mouse bone marrow–derived macrophages cultured in the presence of 10 ng/ml M-CSF with or without 10 ng/ml RANKL for the indicated times were subjected either to quantitative RT-PCR analysis of the amount of Sh3pxd2a mRNA (normalized by that of Actb mRNA; E) or to immunoblot analysis with the indicated antibodies (F). Quantitative data are means ± SD from three independent experiments. (G and H) Live imaging of RAW264.7 macrophages expressing RFP-tagged mouse Tks5. Images were obtained after stimulation with 10 ng/ml RANKL for 48 h. ZsGreen1 was separately expressed as an internal control, and differential interference contrast (DIC) images were also obtained. Arrows indicate the outer edge of an expanding podosome (G) or transient expansion of a podosome-related protrusion (H). Arrowheads in H represent a fusing plasma membrane manifesting RFP-Tks5 accumulation at the plasma membrane. Pixel intensities on the traversing dashed lines were measured using LAS AF or FluoView software and shown in boxed areas (arbitrary units). Bars, 25 µm. See also Videos 5 and 6.

Tks5 as a potential mediator of fusion-competent protrusion formation. (A) Venn diagram summarizing the results of screening for molecules required for osteoclast fusion downstream of PI 3-kinase. Microarray analysis of RAW264.7 cells stimulated with 10 ng/ml RANKL for 72 h or not treated (NT) revealed 7,088 genes whose expression was up-regulated by RANKL, and database analysis yielded 1,027 proteins that contain a PH or PX domain; 114 proteins were present in both datasets. See also Tables S1 and S2. (B) Domain organization of Tks5. SH3, Src homology 3 domain. (C and D) RAW264.7 macrophages cultured in the presence of 10 ng/ml RANKL for the indicated times were subjected either to quantitative RT-PCR analysis of the amount of Sh3pxd2a mRNA (normalized by that of Gapdh mRNA; C) or to immunoblot analysis with antibodies to Tks5, to NFATc1, or to γ-tubulin (loading control; D). Quantitative data are means ± SD from three independent experiments. (E and F) Mouse bone marrow–derived macrophages cultured in the presence of 10 ng/ml M-CSF with or without 10 ng/ml RANKL for the indicated times were subjected either to quantitative RT-PCR analysis of the amount of Sh3pxd2a mRNA (normalized by that of Actb mRNA; E) or to immunoblot analysis with the indicated antibodies (F). Quantitative data are means ± SD from three independent experiments. (G and H) Live imaging of RAW264.7 macrophages expressing RFP-tagged mouse Tks5. Images were obtained after stimulation with 10 ng/ml RANKL for 48 h. ZsGreen1 was separately expressed as an internal control, and differential interference contrast (DIC) images were also obtained. Arrows indicate the outer edge of an expanding podosome (G) or transient expansion of a podosome-related protrusion (H). Arrowheads in H represent a fusing plasma membrane manifesting RFP-Tks5 accumulation at the plasma membrane. Pixel intensities on the traversing dashed lines were measured using LAS AF or FluoView software and shown in boxed areas (arbitrary units). Bars, 25 µm. See also Videos 5 and 6.

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