Effect of TFM-C on cytokine production from activated U937 macrophages. A. Lay-out of cytokine-specific antibody spots in the 16-plex cytokine Stripwell array (upper image) and visualization of cytokine-specific chemiluminescence in culture medium of LPS/PMA-treated U937 cells in the absence or presence of TFM-C (lower images). The grey-shaded cytokines in the upper images are those showing the highest production in LPS/PMA-treated U937 cells at 24 h. B. Effect of TFM-C treatment (50 μM) on the viability of macrophages (PMA-stimulated U937 cells). Apoptotic cells were measured by DAPI staining, and the percentage of damaged DNA and condensed chromatin was calculated following 6, 12 and 24 h of TFM-C treatment (upper graph). Metabolic activity of cells, measured by AlarmBlue®, was expressed as growth inhibition percentage of untreated controls for 6, 12 and 24 h of TFM-C treatment (lower graph). Bars show average of three independent experiments with corresponding error bars. C. Quantification of the kinetics of cytokine secretion and mRNA production (IL-1β, IL-6, IL-8, IL-10 IL-12 and TNF-α) in differentiated macrophages treated with LPS/PMA in the absence (open squares) or presence (solid circles) of TFM-C. All values represent the averages of three independent experiments. For each cytokine, the upper graph represents amount of secreted cytokine quantified using Quansys 16-plex Stripwells, while the lower graph represents cytokine-specific mRNA quantified by QPCR. Asterisks indicate significant differences at * P < 0.05 between TFM-C-treated and -untreated cells at each time point using Student's t-test. D. Effect of 50 μM TFM-C on IL-23p19, HERP and GAPDH mRNAs (QPCR) in differentiated macrophages, stimulated by LPS and PMA. The levels of mRNA levels are shown as 2-Ct. Asterisks indicate significant differences at * P < 0.05 compared with baseline condition LPS/PMA-only using Student's t-test.