Figure 3

Effect of SA exposure on GSH level in N9 cells and involvement of MRP1 in depleting cellular GSH.

(a) N9 cells were exposed to SA as well as LPS for 24 h and cellular GSH level measured. SA exposure increased cellular GSH level to 2.5 μmol/mg protein significantly higher than control where as insignificant alteration observed following LPS exposure. (b) N9 cells were exposed to SA as well as LPS in presence of BSO for 24 h, one SA treated group was supplemented with exogenous GSH ethyl ester (15 mM). Viability checked following 24 h incubation. BSO did not affect viability of control as well as LPS treated N9 cells but severely compromised the viability of SA exposed group (≈7%). Supplementation of exogenous GSH increased N9 viability to 66%. (c) N9 cells were exposed to SA as well as LPS and cultured 4 h in presence or absence of BSO. Following 4 h incubation with BSO, level of cellular GSH decreased almost 50% in all the groups compared to BSO unexposed group. (d) N9 cells were exposed 4 h to SA in presence of BSO with or without MRP1 inhibitor, Cyclosporin-A (CsA) and MK571. Following 4 h of incubation, cellular GSH depleted in SA treated cells (0.7 μmol/mg protein) significantly more than the control (1.2 μmol/mg protein) cells whereas addition of CsA and MK571 inhibited GSH (0.94 and 1.3 μmol/mg protein respectively) depletion. N9 cells were seeded in 6 well culture plate and treated 4 h as in figure d western blot analysis for the expression of MRP1, (e) representative western blot and (f) densitometric graph of MRP1 expression. (g) Similarly, N9 cells were seeded on cover slips and treated 4 h as in figure D followed by immunostaining to check the MRP1 expression level. Data shown in bar graphs represents Mean ± SE (Standard Error) of two-three independent experiments. ‘p’ denotes level of significance in comparison to control, *p < 0.05, **p < 0.01, ***p < 0.001, ns: non significant.