Fig. 2.

Knocking down of LNX2 expression markedly inhibits osteoclast formation. (A) BMMs were transduced with recombinant lenti-viruses expressing a control shRNA targeting fire fly luciferase (LUC-sh) or two LNX2-targeting shRNAs (LNX2-sh1 and LNX2-sh2), respectively. After selected with 6 µg/ml puromycin for 3 days, the cells were cultured with M-CSF plus RANKL for 5 days. Total RNAs were isolated from three independent cultures of each group. Quantitative real-time RT-PCR analysis of the expression of LNX2 was performed. Data were presented as mean ± S.D. *** p < 0.001 vs LUC-sh by one way ANOVA. (B) Lenti-viruses transduced BMMs were cultured with M-CSF and RANKL for 5 days. The cells were fixed and stained for TRAP. The scale bar = 20 µm. (C) The mRNA expression of osteoclast marker genes, TRAP (encoded by Acp5) and Cathepsin K (encoded by Ctsk), was measured by quantitative real-time PCR using TaqMan assay primers from Life Technologies., Data were presented as mean ± S.D, n = 3, ** p <0.01 vs LUC-sh by Student’s t-test. (D) The protein expression of Cathepsin K was detected by western blots. Tubulin served as a loading control. All experiments were repeated two to three times.