RelA/p65 promotes osteoclast differentiation by blocking a RANKL-induced apoptotic JNK pathway in mice
J. Clin. Invest. Sergio Vaira, et al. 118:2088 doi:10.1172/JCI33392 [Go to this article.]

Figure 5
Knockdown of Bid rescues Rela^–/– osteoclastogenesis. (A) Rela^+/+ and Rela^–/– BMMs were stimulated with RANKL at the indicated times, and total cell lysates were examined by immunoblot for expression of Bid. β-actin was used as loading control. (B) Rela^–/– BMMs were transduced with retrovirus expressing 2 different siRNAs for Bid (siBid1 and siBid2) or luciferase (siLuc). Following puromycin selection, levels of Bid were examined by immunoblot, demonstrating reduction of Bid by the specific siRNAs. β-actin was used as loading control. (C) Rela^+/+ and Rela^–/– BMMs transduced with siLuc or siBid1 or -2 were cultured with RANKL for 36 hours and DNA fragmentation was measured. The data are representative of 3 independent experiments. *P < 0.05 versus Rela^+/+ siLuc. (D) The same BMMs in C were cultured in osteoclastogenic conditions for 6 days, then stained for TRAP, showing rescue of differentiation with knockdown of Bid. Scale bar: 200 μm. (E) BMMs cultured on cortical bone slices were stained with horseradish peroxidase-conjugated wheat germ agglutinin to demonstrate resorption. Scale bar: 200 μm. (F) Pits in E were quantified, showing that reduction in apoptosis correlates with differentiation and bone resorption. Black bars indicate Rela^+/+; gray bars indicate Rela^–/–; *P < 0.05 versus Rela^+/+ siLuc.