Figure 7

Mitochondrial Ca²⁺ uptake in HeLa cells is promoted by E73. (A) Representative confocal images of permeabilized HeLa cells loaded with Rhod2 after transiently transfection with VDAC expression constructs. Mitochondrial Rhod2 fluorescence is minimal at the basal state (left). Upon addition of Ca²⁺, the Rhod2 fluorescence rapidly concentrates in mitochondria (right). (B-D) Mitochondrial Ca²⁺ uptake assays of HeLa cells transiently transfected with VDAC constructs; (B) mitochondrial Ca²⁺ uptake was observed in empty vector transfected control cells (empty, ∆F/F₀ = 2.64 ± 0.82, n = 18), which was significantly enhanced by overexpression of VDAC2 (∆F/F₀ = 3.99 ± 0.80, n = 22) but not VDAC3 (∆F/F₀ = 2.47 ± 0.61, n = 22) and significantly inhibited by ruthenium red (∆F/F₀ = 1.06 ± 0.23, n = 12). (C) While wild-type VDAC2 enhanced mitochondrial Ca²⁺ uptake (∆F/F₀ = 1.83 ± 0.52 for empty cells vs. ∆F/F₀ = 4.18 ± 1.4 for VDAC2, n = 22 and 24, respectively), VDAC2^E73Q failed to induce this effect (∆F/F₀ = 2.09 ± 1.15, n = 13). (D) Conversely, wild-type VDAC3 did not induce a significant increase in mitochondrial Ca²⁺ uptake (∆F/F₀ = 2.63 ± 0.70 for empty cells vs. ∆F/F₀ = 2.59 ± 0.81 for VDAC3, n = 15 and 18, respectively); however, VDAC3^Q73E significantly increased mitochondrial Ca²⁺ uptake to ∆F/F₀ = 4.85 ± 1.99 (n = 18).