Pretreatment with RuRed prevents mitochondrial calcium uptake and protects 32D cells from apoptosis induced by VP-16. (A) 32D and (B) C2 cells were pretreated with either 30 μM RuRed (RR), or RR plus zVAD as indicated, followed by VP-16 treatment. After 16 hours cells were loaded with CaGreen-1 (for cytoplasmic Ca2+) or Rhod-2 (for mitochondrial Ca2+) and analyzed by flow cytometry. Representative histograms, corresponding to untreated cells (filled histograms), and VP-16 treated (open histograms) are shown. (C) 32D and (D) C2 cells were preincubated with 30 μM RR alone or with 50 μM zVAD as indicated for 1 hour followed by 7.5 μg/mL VP-16 treatment. Cell death was quantified at the time points indicated by measuring PI uptake by flow cytometry. Percent of dead cells is shown as the mean ± SD of 3 independent experiments. Four groups of treatment are shown: ○, treatment with VP-16; ♦, pretreatment with 50 μM zVAD followed by VP-16 treatment; ▪, pretreatment with 30 μM RR followed by VP-16 treatment; and X, pretreatment with 30 μM RR and 50 μM zVAD followed by VP-16 treatment.
