Fig 1.

Cot induces Ca²⁺ oscillation/calcineurin-independent osteoclastogenesis. (A) (Left) TRAP staining of wild-type (WT) and IP₃R2/3KO (2/3KO) BMMs infected with retroviruses encoding either an active form of Cot plus GFP (trCot) or GFP alone (−) in the absence or presence of FK506. Bar, 50 μm. (Right) Percentage of TRAP-positive multinuclear osteoclasts (MNCs). Values are means ± standard deviations (SD); n = 4 for each group. (B) Bone resorption assay. Arrows show bone resorption pits. Bars, 200 μm. (C) NFATc1 transcriptional activity based on luciferase reporter assays in RAW264.7 cells following overexpression of an active form of Cot (trCot), a kinase-deficient mutant of Cot (KMCot) or empty vector (vec.) in the absence or presence of FK506. Values are means ± SD; n = 3 for each group. **, P < 0.01. (D) The expression levels of NFATc1 protein in BMMs infected with retroviruses encoding either an active form of Cot plus GFP (tr) or GFP alone (−) in the presence or absence of FK506. β-Tubulin signal served as an internal control. (E) Expression and localization of NFATc1 (red) in WT and 2/3KO osteoclasts infected with retroviruses encoding HA-tagged trCot plus GFP (HA-trCot, dark blue) or GFP alone (−) in the absence or presence of FK506. Nuclei are stained with DAPI (light blue). Bar, 50 μm.