Figure 6

Transfection of siRNA to reduce IRAG expression reduced cell spreading. (a) Phase photograph of osteoclasts (green) after transfection with fluorescently tagged siRNA (red). Approximately 70% of cells are labeled, indicating the transfection efficiency. (b) Cells with inhibited IRAG production had mean cell diameter reduced by ∼30%; the difference is significant (P<0.05); n=10. (c) Phase photomicrographs of osteoclast cultures after transfection with IRAG-specific siRNA (1, left) or control siRNA (2, right). No increase in floating or apparently apoptotic cells were observed in IRAG suppression, although cell spreading was reduced. Photographs in (a) and (c) are of 220 μm² fields. (d) Photomicrographs of control and IRAG-specific siRNA-transfected cells, as in (a) (red), with additional labeling using phalloidin (green) to show podosomes and Hoechst (blue) to label nuclei, as in Figure 4. The attachment sites of osteoclasts are randomly ordered (arrowheads) when IRAG is deficient (top frames), whereas cells with scrambled siRNA retain normal attachment rings (arrows).¹ Fields are 50 μm².