Figure 7. Nitric oxide is produced by inducible nitric oxide synthase (iNOS); iNOS expression is dependent on ERK 1/2 and JNK activation.

(A) K562 cells were treated as indicated for varying time periods and the whole cell lysates were subjected to western blot analysis with indicated antibodies. (B) After transfection of K562 cells with indicated siRNAs followed by indicated treatments for 24 h, cells were stained with DAF-FM for measurement of NO. Representative of two similar histograms. (C) K562 cells were transfected with control siRNA or indicated NOS siRNAs for 48 h and then treated with combination of BSO (100 µM) and HCH (10 µM) for 24 h. Whole cell lysates were subjected to western blot for the expression of indicated proteins to confirm knockdown of representative NOS isoforms. (D) K562 cells were subjected to annexin V/PI binding assay by flow cytometry after 36 h. Representative of two similar histograms. (E) K562 cells were transfected with JNK1 or ERK2 siRNA. Cells were then treated with combination of BSO (100 µM) and HCH (10 µM) for 18 h. Cell lysates were then immunoblotted with anti-iNOS antibody. (F) iNOS siRNA transfected K562 cells were treated with combination of BSO (100 µM) and HCH (10µM) for 18 h and subjected to immunoblot analysis with indicated antibodies. (G) K562 cells were pre-incubated with GME for 1 h before treatment with combination of BSO and HCH for 18 h and cell lysates were then immunoblotted with anti-iNOS antibody. (H) GSH measurement was done in iNOS transfected K562 cells after combined treatment with BSO and HCH for 18 h. Data represent mean ± SD of three experiments. *** p<0.001 compared to vehicle control.