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. 2016 Jul 19;5:214–221. doi: 10.1016/j.bonr.2016.07.002

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© 2016 Published by Elsevier Inc.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Fig. 1 — Swap-70^−/− BMOCs are impaired in podosome dynamics but not in assembly of podosomes.

(A) Immunofluorescence staining of podosomes (vinculin, red; F-actin, green; nuclei, blue) from wild-type (left) and Swap-70^−/− (right) BMOCs at day 4, day 5 and day 6 of in vitro culture. (B) Quantification of BMOCs containing podosome clusters (grey), rings (green) or belts (yellow) as assessed in (A) at day 4, day 5 and day 6. For quantification five fields of view per sample (day 4: 3 slides, day 5: 3 slides, day 6: 4 slides per genotype) were used as assessed in (A). All values are presented as mean ± standard deviation. (C) Size of F-actin rings in wild-type BMOCs and Swap-70^−/− BMOCs (d4 to d6, five fields of view of 11 slides in total) as assessed in (A). (D–F) Immunofluorescence staining of podosome-associated proteins (D) vinculin (vinculin, red; F-actin, green; nuclei, blue), (E) talin (talin, red; F-actin, green; nuclei, blue), (F) paxillin (paxillin, red; F-actin, green; nuclei, blue) from Swap-70^+/+ and Swap-70^−/− BMOCs at day 4 or day 5 of in vitro culture. The two sets of images arranged in columns in Fig. 1D–F display the different patterns of podosome organization in Swap-70^+/+ osteoclasts (columns 1–4, from left to right), i.e. ring (columns 1 and 2) and cluster (columns 3 and 4) formation, and cluster formation (columns 5 and 6) in Swap-70^−/− osteoclasts. The small pictures in columns 2, 4 and 6 of each figure represent detail enlargements of the actin cores surrounded by vinculin (D), talin (E) und paxillin (F), respectively. (G) F-actin ring reformation assay was performed as described by Kim et al. (2006). F-actin was visualized with Phallodin-Alexa Fluor 488. Left: cells before podosome disassembly (ctrl), middle: cells after treatment with serum-free medium (disassembled) and right: cells after RANKL-induced re-assembly of F-actin rings and podosome clusters (re-assembled). All experiments were performed in 2–3 replicates. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)