Fig. 5.

Role of ROS on the induction of Hsp70 and MnSOD by heat shock. a Confocal micrographs of V79 fibroblasts treated with single dose heat shock of 43°C for 40 min in the presence and absence of NAC. Cells were grown on coverslips and first incubated with NAC for 2 h. After that, the cell-containing petridishes were given three washes with fresh medium and were subjected to heat shock at 43°C for 40 min. Thereafter, the cells were incubated with the ester form of the H₂DCF dye for 15 min at 37°C in Hanks buffer. Cells were visualized under confocal microscope for determining extent of ROS production at the excitation wavelength of 506 nm. Vctrl- untreated cells; V + HS- V79 fibroblasts treated with single dose heat shock of 43°C for 40 min; V + NAC + HS- V79 fibroblasts pretreated with 5 mM NAC and subsequently treated with single dose heat shock of 43°C for 40 min; Vctrl—First picture from left is visualized by dye fluorescence and second picture from the left is differential interference contrast image of the same field. V + HS- Third picture from left is visualized by dye fluorescence and fourth picture from the left is differential interference contrast image of the same field. V + NAC + HS- fifth picture from left is visualized by dye fluorescence and sixth picture from the left is differential interference contrast image of the same field. b Western blot showing p38MAPK phosphorylation and Hsp70 and MnSOD protein levels of V79 fibroblasts treated with single dose heat shock of 43°C for 40 min in the presence and absence of NAC. Vctrl—untreated cells; V + HS- V79 fibroblasts treated with single dose heat shock of 43°C for 40 min; V + NAC + HS- V79 fibroblasts pretreated with 5 mM NAC subsequently treated with single dose heat shock of 43°C for 40 min; The numbers above the panels indicate band densities with respect to control bands normalized to 1. Details of the experimental procedures are described in the “Materials and methods” section