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. Author manuscript; available in PMC: 2010 Nov 13.
Published in final edited form as: Mol Cell. 2009 Nov 13;36(3):500–511. doi: 10.1016/j.molcel.2009.10.021

Figure 6. Ca2+ Blockers and CsA Prevent Apoptosis, Rescue ATP Levels and Abolish Abnormal ROS Production in GM1-Storing Cells.

Figure 6

(A) Flow cytometry analysis of Annexin V β-gal+/+, GM1-loaded β-gal+/+ and β-gal−/− MEFs. In some instances, cells were preincubated with BAPTA, CsA and Z-VAD-fmk. Values are expressed as mean ± standard deviation of three independent experiments. Groups were compared by the Student t-test for unpaired samples. P< 0.05 (*)

(B) ATP levels in β-gal+/+, GM1-loaded β-gal+/+ and β-gal−/− MEFs were measured using a luciferase-based assay. Where indicated, cells were pre-incubated with BAPTA and CsA prior to ATP analysis. Staurosporine was used as positive controls. Values are expressed as mean ± standard deviation of three independent experiments. Groups were compared by the Student t-test for unpaired samples. P< 0.05 (*)

(C) Reactive oxygen species (ROS) were measured in β-gal+/+, GM1-loaded β-gal+/+ and β-gal−/− MEFs with the fluorescent probe 2′,7′-dichlorofluorescein (H2DCFDA). The progressive increase in ROS levels was monitored by the fluorescence emitted after reaction with intracellular ROS. Addition of CsA and BAPTA normalized ROS levels in GM1-accumulating cells. Data are expressed as mean ± SD of 4 distinct experiments; groups were compared by the Student t-test for unpaired samples. Asterisks denote mean values ±s.d significantly different from control or from untreated GM1-loaded β-gal+/+ and β-gal−/− (*P<0.05, **P<0.01).

(D) Caspase activation was assessed by immunofluorescence analysis using a fluorescent pan caspace inhibitor, caspACE FITC VAD-fmk. Staurosporine was used as positive control.