The Effect of OPA1 on Mitochondrial Ca2+ Signaling (original) (raw)
Figure 5
Mitochondrial Ca2+ uptake in permeabilized H295R cells transfected with OPA1 or Mfn1 siRNA.
The cells were transfected with control RNA or siRNA on the day following plating (day 2) and with inverse Pericam targetted into the mitochondria (mt-inv-Pericam) on day 2 or 3. On day 5 the cells were permeabilized, superfused with a cytosol-like medium, Ψm was dissipated with 10 µM rotenon, 8 µg/ml oligomycin, 10 µM FCCP and 50 ng/ml valinomycin for 2 minutes. Then, in the presence of the drugs, [Ca2+] was raised from 0 to 5 µM. [Ca2+]m was monitored by means of confocal microscopy, applying mt-inv-Pericam, the fluorescence of which exhibits inverse correlation with [Ca2+]. Fluorescence measured at saturating [Ca2+] (Fmin) was subtracted from each fluorescence value. The data were normalized for the control period. Representative mitochondrial Ca2+ uptake curves are shown for cells transfected with Mfn1 or OPA1 siRNA (note that decreasing F/F0 values indicate increasing [Ca2+]m!) (A); effect of OPA1 siRNA as compared with that of control RNA (B) or Mfn1 siRNA (C) on the slope of initial decrease of normalized mt-inv-Pericam fluorescence (indicating the slope of initial increase in [Ca2+]m). Results represent mean + SEM, the number of observations is shown within the columns.