FIGURE 6.

C/EBPα silencing does not affect actin ring formation but attenuates bone resorption by OCs. A, MBM cells were first treated with M-CSF (10 ng/ml) and RANKL (10 ng/ml) for 2 days before infection with a lentivirus encoding scramble shRNA control (Scr-sh) or C/EBPα shRNA construct 1 or 5 (C/EBPα-sh 1 or 5) for 1 day (d). The treatments were then continued for 2 more days. Gene expression was examined by Western blotting using β-actin as loading control. B, the same set of assays as in A was repeated, and some cells were stained with Alexa Fluor 488-phalloidin (Phalloidin Staining) for actin ring analysis in three independent experiments. The other cells were submitted to AO staining (AO Staining) for analysis of extracellular acidification in three independent experiments. Scale bars = 200 μm. C, the same set of experiments as in A was carried out with cells seeded on bone slices, but after infection, the cells were cultured for 5 more days before SEM analysis of the bone resorption pits. D, quantification for the bone resorption assays shown in C from three independent experiments. The numbers in parentheses show concentration in nanograms per milliliter. Error bars show mean ± S.D. *, p < 0.05.