Figure 7
NO2-FAs inhibit LPS/IFNγ-mediated inflammatory markers iNOS in RAW264.7 cells. Raw cells were treated with 10 μM NCE-1, -2, -8, -10, OA-NO2, DMF or 100 nM CDDO-Im for 6 and 16 hr. Expression of iNOS mRNA was determined at 6 hr (A, left) and 16 hr (A, right). Raw cells were treated with 2.5, 5 and 10 μM NCE-1, -2, -8, -10, OA-NO2, DMF or 10 and 100 nM CDDO-Im for 16 hr and expression of iNOS mRNA was determined (B). Raw cells were treated with 5 μM NCE-1, -2, -8, -10, OA-NO2, DMF or 100 nM CDDO-Im for 20 hr. Representative Western blot of iNOS, HO1, actin and gapdh protein expression levels following treatment with electrophilic compounds for 20 hr (C). Uncropped membranes with superimposed protein ladder (Bio-Rad, cat# 161–0374) are presented in Supplementary Figure 2. Densitometric analysis was performed using housekeeping protein gapdh (D). Within an individual experiment, there was a minimum of an n = 2/treatment which was normalized to the media alone treatment (cell media was replaced only). These technical replicates were averaged for each treatment. The values in the bar graph represent mean ± SEM for at least three independent experiments (n = 3–12). #p < 0.001 and !p < 0.01 indicates statistically significant over media alone treatment without LPS/IFNγ at each time point; Compared to the vehicle (DMSO) treatment group at each time point, statistical significance is indicated by: (a) p < 0.05; (b) p < 0.01 and (c) p < 0.001.
