fig5

Figure 5. Nef and Dox combination induced ROS mediated LRP down regulation. A549 and A549/Dox cells with Dox or Nef alone or their combination: A: intracellular ROS concentration was determined using DCF; B: intracellular GSH was assessed using OPT and the fluorescence was measured by fluorometry as explained under Materials and methods. 50 µM H2O2 was used as positive control. The results shown are mean ± SEM and *** represents P < 0.01 or ** represents P < 0.05, which are three separate experiments performed in triplicate. A549 and A549/Dox cells treated with Dox/Nef alone, NAC alone and NAC and Nef + Dox combination; C, D : the cell lysate was analysed for LRP expression, a representative image of three experiments and densitometric analysis are shown. GAPDH served as loading control and (E) the cell viability was assessed, the results shown are mean ± SEM and *** represents P < 0.01 which are 3 separate experiments performed in triplicate. Nef: Neferine; Dox: Doxorubicin; LRP: lung resistance protein; NAC: N-acetyl cysteine; OPT: ortho-pthalaldehyde; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; ROS: Reactive oxygen species; GSH: Reduced glutathione; DCF: 2’, 7’ –dichlorofluorescein