Fig. 5. Calcium-activated chloride channel Ano1 promotes RANKL signaling via its activity-dependent interaction with RANK.

a Microarray assays were performed in osteoclasts originated from Ano1^fl/fl (n = 3) and Ctsk-Cre;Ano1^fl/fl (n = 3) mice. The relative mRNA expression is depicted according to the color, red indicates upregulation and blue indicates downregulation. Shown are the mRNAs that changed more than 2 folds. b Pathway enrichment analysis (Gene Analytics) for genes expressed differentially between Ano1^fl/fl and Ctsk-Cre;Ano1^fl/fl osteoclasts. c Western blot analysis of NFATc1 protein level in Ano1^fl/fl and Ctsk-Cre;Ano1^fl/fl osteoclasts after treatment with or without RANKL mice (top). The quantification of NFATc1 protein level in osteoclasts (below). d Western blot analysis of NFATc1 protein level in WT and Ano1 TG osteoclasts after treatment with or without RANKL (top). The quantification of NFATc1 protein level in osteoclasts (below). e Immunofluorescence of Ano1 (green) and RANK (red) in osteoclasts treated with or without RANKL by confocal microscopy. Representative images are shown. Scale bar, 10 μm. f Coimmunoprecipitation of Ano1 and RANK in osteoclasts after treatment with or without RANKL. g Coimmunoprecipitation of Ano1 and RANK in RANKL-induced osteoclasts after treatment with Ano1 inhibitor Benzbromarone (10 μM). h Coimmunoprecipitation of Ano1 and RANK in RANKL-induced osteoclasts after overexpression of Ano1 or Ano1 with E702Q and E705Q mutants. i Coimmunoprecipitation of RANK and TRAF6 in RANKL-induced osteoclasts transfected with Ano1 siRNA or its negative control. j Western blot analysis of the phosphorylation levels of Syk, Btk, and Plcγ2 in osteoclasts (top). The quantification of the phosphorylation level of Syk, Btk, and Plcγ2 in osteoclasts (below). k Resting [Ca2+]_i in osteoclasts from Ano1^fl/fl mice and Ctsk-Cre;Ano1^fl/fl mice, n = 40 (Ano1^fl/fl) and n = 55 (Ctsk-Cre;Ano1^fl/fl) cells pooled from three independent experiments. l Western blot analysis of p-CaMKIV, p-Creb, and c-Fos protein levels in osteoclasts (top). The quantification of p-CaMKIV, p-Creb, and c-Fos protein levels in osteoclasts (below). All data are the mean ± s.e.m. from three independent experiments. Two-tailed unpaired Student’s t-test was used for statistical evaluations of two group comparisons. Statistical analysis with more than two groups was performed with two-way analysis of variance (ANOVA) with Šídák post-hoc test to determine group differences. Source data are provided as a Source Data file.