Figure 7. PKC is involved in IFNα and LPS-mediated mA3 induction.

(A and B) Immortalized wild-type BMDM on C57BL/6 background were stimulated with 1000 units/ml of endotoxin-free recombinant IFNα for 4 hours, 1μg/ml LPS for 6 hours, or vehicle. Cells were washed and medium containing 6μM of the PKC inhibitor Rottlerin was added with or without IFNα or LPS. Cells were cultured for additional 4 or 6 hours and then used for RNA extraction, cDNA synthesis and examination of mA3 mRNA levels by qPCR. (C ) Immortalized wild-type BMDM on C57BL/6 background were pre-treated with different concentrations (0.5, 6, 10, 20, 50 μM) of Rottlerin as indicated on the figure or vehicle for 1 hour followed by stimulation with 1000 units/ml of endotoxin-free recombinant IFNα for 4 hours, 1μg/ml LPS for 6 hours, or vehicle. Cells were used for RNA extraction, cDNA synthesis and qPCR examination of mA3 mRNA levels. (D and E) Immortalized wild-type BMDM on C57BL/6 background were pre-treated with Rottlerin (6μM) or vehicle for 1 hour followed by stimulation with 1000 units/ml of endotoxin-free recombinant IFNα for 4 hours, 1μg/ml LPS for 6 hours, or vehicle. Cells were used for RNA extraction, cDNA synthesis and qPCR examination of STAT1, STAT2, and mA3 mRNA levels. (F) Immortalized IRF3−/− BMDM on C57BL/6 background were pre-treated with Rottlerin (6μM) or vehicle for 1 hour followed by stimulation with 1000 units/ml of endotoxin-free recombinant IFNα for 4 hours, 1μg/ml LPS for 6 hours, or vehicle. Cells were used for RNA extraction, cDNA synthesis and qPCR examination of mA3 mRNA levels. Data were normalized to GAPDH and presented as fold change relative to vehicle treated cells. Error bars are standard error, * is significance with p value less than 0.05 and ** is significance with p value less than 0.01. Experiments were repeated at least three different times with similar results.