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. 2021 Jan 14;10(1):109. doi: 10.3390/antiox10010109

Figure 2.

Figure 2

Different carbon sources have distinct effects on NO and cytokine production in LPS-treated BV2 cells. (A) BV2 cells were grown overnight in low-glucose medium and then transferred to medium containing only the indicated carbon source and either untreated or treated for 4 h with 10 µM IAA or rotenone. Cells extracts were prepared and assayed for ATP, the levels of which were normalized to the protein content of the extracts. The results are the average of a minimum of three independent experiments. * p < 0.05, *** p < 0.001 as compared to cells not treated with IAA or rotenone. ns, not significant. (B) BV2 cells were grown overnight in low-glucose medium and then transferred to medium containing the indicated carbon source(s) and then treated with 25 ng/mL LPS for 24 h. The cell culture supernatants were assayed for NO, IL6 and TNFα production. The results are the average of a minimum of three independent experiments. * p < 0.05, *** p < 0.001 as compared to medium containing glucose, pyruvate and glutamine. ns, not significant. (C) BV2 cells were grown overnight in low-glucose medium and then transferred to medium containing the indicated carbon source(s) and then left untreated or treated with 25 ng/mL LPS for 24 h. Cell extracts were prepared and equal amounts of protein analyzed by SDS-PAGE and Western blotting for iNOS and actin as a loading control. (D) BV2 cells were grown overnight in low-glucose medium and then transferred to high-glucose medium before treatment with 25 ng/mL LPS for 24 h in the absence or presence of 10 µM of the iNOS inhibitors SEITU or 1400W. As a complement to the inhibitors, cells were transfected overnight with iNOS siRNA. The next day, the cells were plated for experiments and grown overnight in low-glucose medium and then transferred to high-glucose medium before treatment with 25 ng/mL LPS for 24 h. In all cases, the cell culture supernatants were assayed for NO, IL6 and TNFα production. The results are the average of a minimum of three independent experiments. *** p < 0.001 as compared to LPS alone.