Suppression of RANKL-mediated induction of NFATc1 expression and Ca2+ signaling by NDGA. (a) Semiquantitative RT-PCR to detect NFATc1 mRNA in RANKL-stimulated RAW-D cells. (b) Expression of NFATc1 protein in RAW-D cells. Total cell lysates were prepared and subjected to western blot analysis with anti-NFATc1 and actin Abs. (c) RANKL-induced nuclear translocation of NFATc1 was suppressed by NDGA treatment. Mature osteoclasts from mBMMs were incubated with RANKL (50 ng/ml) in the absence or presence of NDGA (5 μM) for 30 min. The cells were fixed and stained for NFATc1 (red) and nuclei (blue). Original magnification × 40. (d) RANKL-induced Ca2+ oscillation was completely inhibited by NDGA treatment. RAW-D cells were cultured with RANKL (20 ng/ml) and TNF-α (1 ng/ml) for 24 h in the absence (upper panel) or presence (lower panel) of NDGA (5 μM). [Ca2+]i change in single cells were detected by loading Ca2+ indicators as described in Materials and Methods. Each color indicates corresponding single cells in the same field.