Cannabinoids and bone: endocannabinoids modulate human osteoclast function in vitro
Version of Record online: 23 MAR 2012
© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society
British Journal of Pharmacology
Special Issue: Themed Section: Cannabinoids in Biology and Medicine, Part II. Guest Editors: Itai Bab and Steve Alexander
Volume 165, Issue 8, pages 2584–2597, April 2012
How to Cite
Whyte, L., Ford, L., Ridge, S., Cameron, G., Rogers, M. and Ross, R. (2012), Cannabinoids and bone: endocannabinoids modulate human osteoclast function in vitro. British Journal of Pharmacology, 165: 2584–2597. doi: 10.1111/j.1476-5381.2011.01519.x
- Issue online: 23 MAR 2012
- Version of Record online: 23 MAR 2012
- Accepted manuscript online: 7 JUN 2011 08:47AM EST
- Received; 21 January 2011; Revised; 27 April 2011; Accepted; 20 May 2011
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'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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