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Figure S1 High throughput quantification of VNR fluorescence using a plate reader as a measure of osteoclast formation. Human osteoclasts derived from highly enriched M-CSF-dependent mononuclear cells were cultured in the presence of M-CSF, M-CSF and RANKL or M-CSF, RANKL and 10 ng·mL−1 TGFβ for 7 days. Cells were fixed and stained for the vitronectin receptor and immunofluorescence intensity measured as an indication of osteoclast number (A). Results are expressed as a percentage of M-CSF and RANKL treated cells – mean ± SEM; n = 4–8 experiments – 5 replicates each. ***P < 0.001; Student&apos;s t-test. (B) Comparison of osteoclast number determined by counting versus plate reader VNR fluorescence. (C) Representative images of osteoclasts formed in the presence of M-CSF alone, M-CSF and RANKL or M-CSF, RANKL and TGFβ are shown with vitronectin receptor in (green) and nuclei (blue).

Figure S2 Anandamide, 2-AG and CP 55 940 do not have a toxic effect on osteoclast precursors. M-CSF-dependent human PBMCs were cultured in the presence of RANKL and treated with anandamide (A), 2-AG (B) or CP 55 940 (C) for 7 days, after which an AlamarBlue (Invitrogen) assay was performed to assess the number of viable cells. Results are expressed as mean ± SEM (mean of 4–7 experiments with 5 replicates per experiment).

Figure S3 Cannabinoid receptor antagonist AM251 increases human osteoclast formation. Human osteoclasts derived from highly enriched M-CSF-dependent mononuclear cells were cultured in the presence of vh (control), 10 ng·mL−1 TGFβ (positive control) or cannabinoid receptor antagonists SR141716A (141), SR144528 (144), AM251 and AM630 for 7 days and then fixed and stained for VNR. Immunofluorescence intensity was measured and expressed relative to control cultures as an indication of osteoclast number. Mean ± SEM; n = 4 experiments – 5 replicates each. **P < 0.01, ***P < 0.001 compared with control – one way ANOVA with Bonferroni post-test.

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