Figure 4. The effects of F. nucleatum LPS on IL-6 production, and of pioglitazone on F. nucleatum LPS-induced IL-6 production in adipocytes. Mouse 3T3-L1 adipocytes were differentiated in 24-well tissue culture plates as described in MATERIALS & METHODS. After confirming maturation of adipocity by staining the cells with oil red O, we stimulated the cells with indicated concentrations of F. nucleatum LPS. In some cultures, the cells were co-incubated with 10 µM of pioglitazone. Twenty-four hrs later, the cell-culture supernatants were harvested, and the IL-6 concentration was measured by ELISA. All experiments were done in triplicate, and the statistical differences were calculated by Student’s t test. In pioglitazone-treated cells, the IL-6 production was compared with that of untreated cells when stimulated with the identical concentration of F. nucleatum LPS. Mean IL-6 concentration ± standard deviation in each culture condition was calculated (1094.2 ± 68.6 pg/mL for LPS-unstimulated cells, 1308.3 ± 33.6 pg/mL for 1 ng/mL of LPS-stimulated cells, 1283.8 ± 6.4 pg/mL for 10 ng/mL of LPS-stimulated cells, 1552.9 ± 42.7 pg/mL for 100 ng/mL of LPS-stimulated cells, 365.1 ± 5.0 pg/mL for LPS-unstimulated cells co-incubated with pioglitazone, 223.7 ± 11.8 pg/mL for 1 ng/mL of LPS-stimulated cells co-incubated with pioglitazone, 236.0 ± 5.9 pg/mL for 10 ng/mL of LPS-stimulated cells co-incubated with pioglitazone, and 383.8 ± 5.5 pg/mL for 100 ng/mL of LPS-stimulated cells co-incubated with pioglitazone, N = 3 for all groups). **p < 0.0001, compared with the cells stimulated with the same concentration of LPS in the absence of pioglitazone.