JCS031534 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. 3D reconstruction shows localization of PKCα-EGFP to the basolateral membrane of bone-marrow-derived osteoclasts. Osteoclast expressing PKCα-EGFP was stimulated with BzATP (150 µM) for 60 seconds to induce translocation, then fixed. Cell was imaged on the confocal microscope using a pin-hole setting of 72 µm and focal plane of 0.45 µm. 102 images were assembled for this high-resolution reconstruction. Panel at top shows image in x-y orientation, with PKCα localized at basolateral membrane of the osteoclast, and only a small septum evident in the apical membrane domain (arrows). Lower panel shows the same cell viewed in the z-x plane, with location of substratum indicated by the dashed line. Width of field is 65 µm. See supplementary material Movie 2 for animation of the reconstructed view of this cell.

• Supplemental Figure S2 -

  Fig. S2. Functional characterization of osteoclast-like cells derived from RAW 264.7 cells and osteoclasts generated from murine bone-marrow cells. (A-C) RAW 264.7 cells were seeded onto the indicated substrates and maintained for 2 (A) or 6 (B,C) days in medium supplemented with RANKL (100 ng/ml). (D-F) Osteoclasts derived from mouse bone-marrow cells were plated on the indicated substrates in the presence of RANKL (100 ng/ml) and M-CSF (50 ng/ml) for 1-2 days. (A,D) Large multinucleated cells stained positively for tartrate-resistant acid phosphatase (TRAP) activity. Image in A is phase-contrast, and D is bright field. (B,E) Resorptive capacity of osteoclast-like cells was confirmed using calcium phosphate-coated disks, where multinucleated cells were adjacent to areas of resorption, as indicated by arrows. (C,F) Cells were plated on dentine for 2 days, then removed and the dentine slices were viewed using scanning electron microscopy. Scale bar in panel C is 50 µm, and 100 µm in all other cases.

• Movie 1 -

  Movie 1. BzATP induces transient translocation of PKCα from the cytosol to the basolateral membrane. RAW-264.7-derived osteoclast-like cell expressing PKCα-EGFP was stimulated with BzATP (150 µM) at the time indicated at upper left, causing redistribution of the fluorescence from cytosol to the periphery of the cell. Recovery was apparent in the continued presence of agonist. Image intervals are 12 seconds and frames are shown at 5 frames/second. Width of field is 70 µm.

• Movie 2 -

  Movie 2. 3D reconstruction shows localization of PKCα to the basolateral membrane of bone-marrow-derived osteoclast. Osteoclast expressing PKCα-EGFP was stimulated with BzATP (150 µM) 60 seconds before fixation, then imaged with high resolution on the confocal microscope. Images were obtained with pin-hole setting of 72 µm and focal plan of 0.45 µm, with 102 images combined for this reconstruction. Still frames of this cell are shown in supplementary material Fig. S1. Cell is rotated 90° to illustrate the localization of the PKCα-EGFP primarily to the basolateral membrane. Width of the field is 65 µm.