Figure 4
Over-expression of Trx1 increases cell survival and ameliorates asbestos-induced ROS generation in LP9 cells. (A) LP9 cells were exposed to two doses of crocidolite asbestos (15 × 106 μm2/cm2 and 75 × 106 μm2/cm2) for 24 h and incubated with NBT for 45 min at 37°C. The absorbance of the solubilized formazan formed after incubation with NBT was then read at 630 nm to determine ROS levels after asbestos exposure. (B) Over-expression of Trx1 in LP9 cells using the pCMV-SPORT6 plasmid was confirmed by qRT-PCR 48 and 72 h after transfection. (C) Trx1 transfected cells were exposed to crocidolite asbestos for 2 h and incubated with NBT for 45 min to determine ROS levels. Solubilized formazan was measured spetrophotometrically at 630 nm on a plate reader. (D) Analysis of the oxidation state of Trx1 after asbestos exposure of Trx1 over-expressing LP9 cells was determined by redox Western blot analysis and densitometry of the blot was performed (n = 2 per group). (E) LP9 cells transfected with the Trx1 over-expressing plasmid, pCMV-SPORT6 were exposed to crocidolite asbestos for the times indicated. Cells were then trypsinized and counted to estimate cell survival (*p < 0.05 compared to control; †p < 0.05 compared to Trx1 OE). Cell survival and NTB graphs are the average results of 3 experiments.