Fig. 4

Elevation of 12(S)-HETE in LPS-stimulated CYP1A1/RAW cells is CYP1A1 hydroxylase-dependent rather than 12 lipoxygenase-dependent. a, b CYP1A1/RAW and NC/RAW were stimulated with vehicle or LPS (10 μg/ml) for 2 h for qRT-PCR or 12 h for western blotting. a 12-LOX mRNA levels were detected using qRT-PCR. 12-LOX protein levels were assessed by western blotting. b, c CYP1A1/RAW and NC/RAW were pre-treated with the selective 12-LOX inhibitor ML355 (10 μM) for 2 h and then stimulated with LPS for 12 h. b Supernatants were collected for CYP1A1 AHH activity measurement using a standard AHH activity assay protocol. c Alternatively, TNF-α, IL-6 and 12(S)-HETE levels were detected in supernatants using ELISA. d Schematic of CYP1A1 cDNA nucleotide sequence containing two mutant positions that impair CYP1A1 AHH activity. e, h CY1A1/RAW, NC/RAW and CYP1A1 mutant/RAW were treated with LPS for 12 h. e CYP1A1 protein levels were measured from cell lysate by western blotting. CYP1A1 AHH activity was measured in supernatants using a standard AHH activity assay. f, g CY1A1/RAW, NC/RAW and CYP1A1 mutant/RAW were treated with LPS for 2 h. f Treated cells were lysed and subjected to western blotting analysis. g The nuclear-extract proteins of treated cells were incubated with AP-1-binding site probe and binding activity measured by EMSA. h Supernatants were collected for analysis of TNF-α, IL-6, 12(S)-HETE and 14, 15-EET levels using ELISA. Data shown as mean ± SEM of three independent experiments. Results were compared by one-way ANOVA. *p < 0.05. NS, no statistical difference