Figure 2. Disulfiram suppressed osteoclast-related genes expression and down-regulated the expression of NFATc1. (A) qPCR was used to measure relative expression levels, normalized to that of β-actin, of RANKL-induced OC-related genes at different times of treatment with 50 mM ethanol (n = 3). (B) qPCR was used to measure relative expression levels, normalized to that of β-actin, BMMs were treated with indicated conditions indicated below figure 2B for 3 days (n = 3). (C) Ethanol increased the RANKL-induced NFATc1 protein expression but did not affect c-Fos and c-Jun. Total cellular proteins were extracted from BMM-derived OCs co-treated with RANKL and 50 mM ethanol for 0, 1, and 3 days. (D) Relative expression of c-Fos, c-Jun, and NFATc1 was determined by densitometric analysis of each band and expressed as a ratio to that of β-actin using ImageJ. (E) Ethanol stimulated NFATc1 transcriptional activity. RAW 264.7 cells stably expressing the NFATc1-TA-Luc luciferase reporter were pretreated with 50 mM ethanol for 1 h and then stimulated for 6 h with RANKL, after which luciferase activity was measured. Results are expressed as fold-changes compared to the levels in unstimulated controls (n = 3). (F) Disulfiram inhibited the expression of NFATc1 in a dose-dependent manner. (G) Relative expression of NFATc1 was determined by densitometric analysis of each band and expressed as a ratio to that of β-actin using ImageJ software. Bar graphs are presented as the mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001.