Figure 1.
Effect of OPA1 and Mfn1 siRNA on the expression of OPA1.
The cells were transfected with OPA1 siRNA, Mfn1 siRNA or a non-silencing RNA on the day following plating (day 2). The samples were lysed on day 5, run on SDS-PAGE, transferred onto nitrocellulose membrane, incubated with anti-OPA1 mouse monoclonal antibody then with anti-mouse immunoglobulin-horseradish-peroxidase conjugate. Protein disulphide isomerase was used as loading control.
Figure 2.
Effect of OPA1 or Mfn1 silencing on Ca2+ signaling in intact H295R cells.
The cells were transfected with control RNA or siRNA and 4mt-D2-cpV on the day following plating (day 2)and once again with control RNA or siRNA on day 3. On day 5, after preloading with Fura-2 AM, the cells were stimulated with 25 mM K+. Changes in cytosolic [Ca2+] ([Ca2+]c) were monitored by measuring Fura-2 excitation ratio while [Ca2+]m was indicated by the FRET ratio of 4mt-D2-cpV. Both ratios are normalized to those obtained in the control period. Representative cytosolic Ca2+ signals (A) and mitochondrial Ca2+ uptake curves (B) are shown for cells transfected with control RNA, Mfn1 or OPA1 siRNA. C: peak [Ca2+]c, D: peak [Ca2+]m E: [Ca2+]m response normalized to peak [Ca2+]c (Δ[Ca2+]m/[Ca2+]c) indicating mitochondrial responsiveness; F: the slope of [Ca2+]m rise related to peak [Ca2+]c (cells not displaying a mitochondrial Ca2+ response were omitted from this statistics). Data are shown for control (C), Mfn1 siRNA (M) or OPA1 siRNA-transfected (O) groups. Results represent mean + SEM, the number of observations is shown within the columns.
Figure 3.
Effect of OPA1 or Mfn1 knockdown on Ca2+ signaling in intact HeLa cells.
The cells were transfected with control RNA or siRNA on the day following plating (day 2) and with 4mt-D1-cpV on day 3. On day 5, after preloading with Fura-FF AM, the cells were stimulated with 1, 5 or 50 µM histamine. Changes in [Ca2+]c were monitored by measuring Fura-FF excitation ratio while [Ca2+]m was indicated by the FRET ratio of 4mt-D1-pcV. Both ratios are normalized to the values obtained in the control period. [Ca2+]m for the pooled data, as indicated by the FRET signal of 4mt-D2-cpV, is shown in function of [Ca2+]c. Figure 4 contains statistics for the cells displaying normalized Fura-FF ratios greater than 1.25 (i.e. for the data shown right to the broken vertical line).
Figure 4.
Mitochondrial Ca2+ signaling in intact, OPA1 or Mfn1 siRNA-treated HeLa cells displaying large [Ca2+]c peak.
(displaying normalized Fura-FF ratios greater than 1.25), shown in Figure 3 right to the broken vertical line. Representative curves are shown for [Ca2+]c measured with Fura-FF (A) and for [Ca2+]m measured with 4mt-D1-cpV (B) after stimulation with 5 µM histamine in cells transfected with control RNA, Mfn1 siRNA or OPA1 siRNA. Statistics for peak [Ca2+]c (C), peak [Ca2+]m (D), Δ[Ca2+]m/[Ca2+]c indicating mitochondrial responsiveness (E) and the slope of [Ca2+]m rise related to peak [Ca2+]c (F) are shown for control (C), Mfn1 siRNA (M) or OPA1 siRNA-transfected (O) groups. Results represent mean + SEM, the number of observations is shown within the columns.
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.
Figure 6.
Effect of OPA1 or Mfn1 silencing on mitochondrial Ca2+ uptake in permeabilized HeLa cells.
For the transfection protocol and measurement of fluorescence see legend of Figure 5. On day 5, after the dissipation of Ψm (see legend of Figure 5), [Ca2+] of the superfusion medium was raised from 0 to 2 or 5 µM. A: mitochondrial response to 5 µM Ca2+. Representative curves are shown for cells transfected with Mfn or OPA1 siRNA (note that decreasing F/F0 values indicating increasing [Ca2+]m!). B: Western blot shows that 2-min exposure to the depolarizing medium did not change the pattern of immunoreactive OPA1. P: permeabilisation, D: dissipation of Ψm, C: control. The slope of initial increase in [Ca2+]m in permeabilized cells superfused with 2 µM (C) or 5 µM Ca2+ (D and E) is shown in cells transfected with control RNA (D), Mfn1 siRNA (C and E) and OPA1 siRNA (C, D, E). C: control RNA, M: Mfn1 siRNA, O: OPA1 siRNA. Results represent mean + SEM, the number of observations is shown within the columns.