Fig 1. MitoNEET plays a role for oxidative stress induced apoptosis in cardiac HL-1 cells.

HL-1 cells were transfected with silencing RNA (siRNA) directed against mitoNEET and non-specific-siRNA as control and subjected to 3h hypoxia followed by 1h of reoxygenation (H/R). (A) Densitometric analysis of Western Blots revealed aggravated activation of caspase-3 in mitoNEET-knockdown (mitoN-KD) cells after H/R compared to hypoxic controls (n = 11). (B) MitoNEET-KD showed no effect on lactate dehydrogenase (LDH) release after H/R measured in culture supernatants (n = 10). LDH was quantified as U/L by a routine clinical analyzer and is expressed as % of H/R control. (C,D) Overexpression of mitoNEET in HL-1 cells caused a significant decrease in apoptosis in mitoNEET overexpressing cells compared to H/R control cells. Expression of mitoNEET (n = 5) and cleaved caspase (n = 7) is shown in representative Western Blots and was densitometrically measured as % of H/R control. (E-G) H/R-induced apoptosis in control- and mitoNEET-KD cells was reduced by two different antioxidants, superoxide scavenger Tiron (10 mM, n = 5) and esterified glutathione compound GSH-MEE (glutathione reduced ethyl ester, 2 mM, n = 6) as demonstrated by representative Western Blots (F-G). Data were analyzed densitometrically, normalized to housekeeping gene expression and are expressed as % of H/R control.