10.1371/journal.pone.0028081.g003 Figure 3  Inhibition of PKC or NF-κB induces apoptosis in MDMs.
(A) MDMs were pre-incubated in RPMI medium containing inhibitors (Ro-31-8220: 5 µM, Gö-6976: 5 µM, BAPTA/AM: 2.5 µM) for 30 minutes prior to the addition of M-CSF. As a control, untreated cells were incubated with dimethyl sulfoxide (DMSO). Cell lysates were resolved by SDS-PAGE and immunoblotted with antibody recognizing the active cleaved form of caspase-3. The blots were reblotted with β-actin that served as a loading control. The ratio of active caspase-3 bands (17 kD and 19 kD) to β-actin control was determined by densitometry analysis (bottom panel). Data represents the mean ± S.E.M from two independent donors. (B) Apoptosis of the treated MDMs was also measured by Annexin V-FITC and propidium iodine (PI) staining and analyzed by flow cytometry. The percentage of surviving cells (Annexin V/PI negative) for the cells treated with vehicle/M-CSF was arbitrarily set as 100. Data shown represent the mean ± S.E.M from three independent experiments. *The p-values of inhibitors/M-CSF compared to vehicle/M-CSF were ≤0.05.