Figure 5.

Characterization of CHL cells expressing COX-2 after DEN treatment. CHL cells were stably transfected with empty pPyCAGIP-GFP vector (CHL-V) or pPyCAGIP-hCOX-2-GFP encoding human COX-2 (CHL-C). For in vitro PB/DEN protocol, CHL-C and CHL-V cells were incubated with 1.5 mmol/L PB for 16 hours following by 15 mmol/L DEN for 24 to 48 hours. A: hCOX-2-GFP and COX-1 expression in whole cell extracts were detected by Western blot and normalized with β-actin. B: qPCR analysis of COX-2 mRNA expression. COX-2 mRNA amounts were calculated as RQ and normalized to the expression of 36b4 mRNA. Values represent fold change relative to CHL-V. C: PGE₂ production determined by enzyme immunoassay in the culture medium of CHL-V and CHL-C cells. D: Growth rate of CHL-V and CHL-C cells. E: Cell viability was measured after PB/DEN treatment at 24 and 48 hours as indicated in Materials and Methods using the MTT assay. F: Oxidative damage of DNA was analyzed in cultured cells under PB/DEN treatment by measuring the levels of 8-OH-dG by immunoassay. Intracellular content of ROS (G) and glutathione (GSH) (H) were measured by using the fluorescent probes H₂DCFDA and monochlorobimane, respectively. Data are reported as means ± SD of four independent experiments. *P < 0.05 and **P < 0.001 vs the CHL-V control cells treated under identical conditions. ***P < 0.05 vs CHL-C cells without DFU treatment.