Figure 4: Effects of WCUP on LPS-induced production of inflammatory cytokines and MAPK/NF-κB/STAT3 activation in murine macrophage J774A.1 cells.

(A) J774A.1 cells were pretreated with 250, 500, and 1000 μg/mL WCUP for 1 h and then stimulated with 200 ng/mL LPS for 24 h. Culture supernatants were collected and measured for NO production. The data are representative of independent experiments performed in triplicate and expressed as means ± SD. Statistical significance was evaluated with Student t-test. ^#p < 0.001 vs. untreated control, *p < 0.05 and **p < 0.01 vs. WCUP-untreated control cells. (B) J774A.1 cells pretreated with the indicated concentrations of WCUP for 1 h were stimulated with 200 ng/mL LPS for 24 h. mRNA and protein levels of iNOS were examined by RT-PCR and Western blotting, respectively. Relative band intensities were calculated after normalization to GAPDH and tubulin using ImageJ software. The full size gels and blots were shown in the Supplementary Fig. S4 and band of interest is indicated with an arrow. (C) The levels of IL-6, TNF-α, and IL-1β in culture supernatants collected as described in (A) were measured by ELISA. The data are representative of independent experiments performed in triplicate and expressed as means ± SD. Statistical significance was evaluated with Student t-test. ^#p < 0.001 vs. untreated control, *p < 0.05, **p < 0.01, and ***p < 0.001 vs. WCUP-untreated control cells. (D) J774A.1 cells were pretreated with 500 and 1000 μg/mL WCUP for 12 h and then stimulated with 200 ng/mL LPS for 30 min. After extracting proteins, the levels of p38, ERK, JNK, IκBα, STAT3, and their phosphorylated forms were detected by Western blotting. The band intensities relative to untreated control cells were calculated using ImageJ software after normalization to tubulin. The full size gels and blots were shown in the Supplementary Fig. S7 and band of interest is indicated with an arrow.