Fig. 2.

Arsenic trioxide-mediated ROS generation and UPR signaling activation are inter-dependent. ROS generation was assessed using fluorescent DCFH-DA dye. (A-I) Raw 264.7 cells were exposed to ATO (0.2 and 2 μM) treatment for 1.5 and 3 h alone as well as in the presence of NAC (10 mM for 24 h) and PBA (1 mM for 24 h). Fluorescence intensity was measured at excitation wavelength at 485 nm and emission wavelength at 528 nm by micro-plate reader and changes were calculated as percent of control. (A-II) Representative microphotographs showing ATO-induced ROS generation in Raw 264.7 cells. Images were snapped using upright fluorescence microscope. The positive control (Post. control) group consisted of Raw 264.7 cells pretreated (1 h) with 500 μM of H₂O₂. (B) Cells were pre-incubated with NAC (10 mM for 2 h and 10 mM for 24 h) followed by ATO (2 μM for 3 h) treatment. UPR regulating proteins p-PERK, GRP78, GRP94, p-eIF2α and CHOP were assessed by western blot analysis. (C-I) Raw 264.7 cells were pretreated with PBA and co-treated with thapsigargin at concentrations 1 mM and 2.5 μM for 24 h and 3 h respectively. Following various pretreatments/co-treatments, cells were incubated with ATO (2 μM for 3 h) and levels of proteins including p-PERK, GRP78, p-eIF2α and CHOP were examined by western blot analysis. (C-II) Relative densitometry analysis of band intensity expressed in fold change. β-Actin was used as an endogenous control. Saline-treated cells served as control in all experiments. Data are expressed as Mean ± SE of at least three independent samples. Statistical significance was determined using Student’s t test. #,*P > 0.05, ##,**P > 0.01 and ###,***P > 0.001 show significance levels. * indicates significant level when compared to saline-treated control cells. # indicates significant level when compared to ATO-treated cells.