Silencing of Krox20 in preosteoclasts increases cFms signaling. (A) Wild type splenocytes were cultured for 3 days with 100 ng/mL M-CSF, and Krox20 expression was silenced using shKrox20-2 lentiviruses. cFms mRNA levels were determined by RT-qPCR and corrected for the corresponding expression of rpL10A. Bars represent mean ± SEM of expression levels relative to cells transduced with a nonspecific (NS) shRNA lentivirus (n = 5; *P < .05 vs NS). (B) Western blot analysis of cFms was performed on preosteoclasts obtained as in panel A. Tubulin was used as loading control. (C) Preosteoclasts obtained as in panel A were serum-starved for 2 hours and treated with M-CSF for 5 and 30 minutes as indicated, followed by Western blot analysis of phospho- and total Akt and ERK. A vertical line has been inserted to indicate a repositioned gel lane.