Ethacrynic acid

To determine glutathione levels, cells were primed as indicated. Thereafter, the medium was removed and cells were exposed to DMEM containing 30 μM monochlorobimane (mBCl; Molecular Probes, Paisley, UK) for 30 min in the cell culture incubator. Following this incubation, the medium was removed and replaced with medium E₁. Fluorescence intensity of mBCl-loaded cells was captured using the same imaging system described above, but using a dicroic mirror of 400 nm and a 420 nm long pass emission filter. Cells were excited at 380 nm using the monochromator. Images were captured for at least four different visual fields from at least two independent experiments for each condition using the Live Acquisition software (Till Photonics, Munich, Germany). Background corrected fluorescence intensities of individual cells were determined using ImageJ v1.50 (NIH, Bethesda, USA), while “n” represents the number of cells analyzed. To check the specificity of mBCL for glutathione over other thiols, 500 μM ethacrynic acid (Santa Cruz Biotechnology, Dallas, USA), an inhibitor of glutathione S-transferase, was added 10 min prior to exposure of cells to mBCl in a paired well, and mBCl fluorescence intensities were corrected for these values.