Fig. 5. Mg²⁺-dependent MCU inactivation in live cells.

(See also Fig. S6).

(A) Permeabilized HEK293T cells were pulsed with 20 μM Ca²⁺ in the presence of MgCl₂ as indicated. Representative traces show changes in extramitochondrial Ca²⁺ concentrations ([Ca²⁺]_out); (n=3 independent experiments).

(B) Representative traces show ΔΨ_m (i.e. JC-1 ratio) in permeabilized HEK293T cells in response to Ca²⁺ and CCCP. The [Ca²⁺]_out and ΔΨ_m traces were acquired simultaneously; (n=3 independent experiments).

(C) Overlay of kinetic changes in [Ca²⁺]_out during 300 s in the presence of different MgCl₂ concentrations.

(D) The rate of mitochondrial Ca²⁺ uptake was calculated as 1/τ. Mean ± SE. *P < 0.05, ***P < 0.001; (n=3 independent experiments).

(E) Quantification of ΔΨ_m before the addition of CCCP. Mean ± SE. n.s., not significant; (n=3 independent experiments).

(F) and (G) Quantification of [Ca²⁺]_m after CCCP addition. Mean ± SE. *P < 0.05, ***P < 0.001; (n=3 independent experiments).