Figure 2.

miR-25 Inhibits Mitochondrial Ca²⁺ Uptake without Causing Morphological Rearrangement or Changes in the Electrochemical Gradient

(A) Cytosolic Ca²⁺ concentration peaks: Ctrl miR: 1.72 ± 0.25 μM; miR-25: 1.726 ± 0.08 μM. n = 12 independent experiments.

(B) Reticular Ca²⁺ concentration levels: Ctrl miR: 328.2 ± 19.68 μM; miR-25: 319.4 ± 13.18 μM. n = 12 independent experiments.

(C) TMRM fluorescence measurements: miR-25-expressing HeLa cells show no difference in TMRM loading (−2.25 ± 1.18% compared to control cells). a.u., arbitrary units. n = 32 independent experiments.

(D) Fluorescence images of mtDsRed- and erGFP-labeled mitochondria and ER, respectively, in control- and miR-25-expressing HeLa cells. Mitochondrial volume and number were deduced by calculating object size (Ctrl miR: 191.49 ± 54.64 μm³; miR-25: 221.16 ± 74.4 μm³) and number (Ctrl miR: 115.56 ± 49 μm³; miR-25: 151.67 ± 63.2 μm³). ER/mitochondria colocalization was estimated by the average volume of overlapping areas (Ctrl miR: 267.89 ± 123.93 μm³; miR-25: 230.7 ± 103.26 μm³). n = 10 independent experiments.

(E and F) [Ca²⁺]_m in permeabilized cells stimulated with 4 μM (mitochondrial Ca²⁺ uptake rate: Ctrl miR: 11.44 ± 0.49 μM/s; miR-25: 6.01 ± 0.31 μM/s; E) or 1 μM (mitochondrial Ca²⁺ uptake rate: Ctrl miR: 0.61 ± 0.04 μM/s; miR-25: 0.32 ± 0.01 μM/s; F) EGTA-buffered fixed [Ca²⁺]. n = 14 independent experiments.

^∗p < 0.05; error bars correspond to mean ± SEM. See also Figure S2.