Fig. 3.

Digitoxin and MonoD modulate intracellular levels of reactive species. A: Cells were treated with 50 nM Digitoxin and MonoD and assayed for nitric oxide, hydrogen peroxide, and superoxide production using DAF, DCF and DHE fluorescent probes. Cr(VI) (100 μM) was used as a positive control. Fluorescence intensity was recorded using a multi-mode fluorescence plate reader. Plots show relative fluorescence intensity over no drug treated control at the peak response time of 15 min. B: Cells treated with Digitoxin and MonoD were lysed and lysates separated on a SDS–PAGE and probed for SOD. C: Cells were pre-treated with 300 μM aminoguanidine for 1 h prior to treatment with 50 nM Digitoxin and MonoD. Superoxide production was assayed using DHE (Fig. 2D) and fluorescence intensity was measured at the peak response time of 15 min post-drug treatment. D: Cells were pre-treated with 300 μM aminoguanidine for 1 h prior to treatment with 50 nM Digitoxin and MonoD. Following this, cells were treated with 50 nM Digitoxin and MonoD for 1 h and lysed and assayed for autophagy regulatory proteins such as Beclin-1, NF-κB, and p38 MAPK levels by immunoblot analysis. Blots were re-probed with GAPDH antibody to confirm equal loading of the samples. E: Cells pre-treated for 1 h with 300 μM aminoguanidine prior to 1 h treatment with 50 nM Digitoxin and MonoD were analyzed for autophagosome formation by Cyto-ID^®. Rapamycin (2 nM) was used as a positive control. Following treatment with test drugs, cells were incubated with Cyto-ID^® for 30 min at 37°C and intracellular Cyto-ID^® fluorescence imaged using EVOS^® FL Cell Imaging System at 40× resolution. F: Autophagic vacuoles were quantified using ImageJ after performing background subtraction and plotted as Corrected Total Cell Fluorescence. (*P < 0.05 for each drug treated data point as compared to non-treated control; #P < 0.05 versus Digitoxin- and MonoD-treated datasets).