Figure 2. HK2 detachment from MAMs prompts Ca²⁺ influx and depolarization in mitochondria following IP3R opening.

• A–F
  Effect of cl‐HK2pep on cellular Ca²⁺ dynamics and IP₃ levels. ER Ca²⁺ levels are measured by the FRET‐based, D4ER fluorescent probe expressed in the lumen of ER (A); IP₃ levels are assessed with the GFP‐PHD probe; histamine (100 μM) is used as a positive control for IP₃ generation; data are reported as mean of fluorescent signals ± SEM (n = 3 independent experiments; B); changes in mitochondrial Ca²⁺ levels are recorded (C; scale bar: 20 μm) and quantified using the GCAMP6f sensor (in D, as mean of 475/410 nm ratio; signal ± SEM of at least five independent experiments and more than 20 cells analyzed), in (E) as percentage of cells with increased Ca²⁺ in mitochondria, with a threshold 475/410 ratio for positivity > 3; baseline mean ratio = 1.84 ± 0.54) or with mitochondria‐targeted aequorin (F, where data are reported as mean of [Ca²⁺] ± SEM of 3 independent experimental days).
• G–J
  Effect of cl‐HK2pep treatment on mitochondrial membrane potential assessed with the TMRM probe. Kinetic experiments (G, single cell analysis, n = 216 cells analyzed in at least 10 independent experiments; H, representative field; scale bar: 20 μm) are quantified (I and J; TMRM fluorescence is normalized to initial value and expressed in percentage for each time point, with a depolarization threshold placed at 40% of initial value).

Data information: Experiments throughout the figure are carried out on HeLa cells; cl‐SCRpep, negative control of cl‐HK2pep (2 μM each). Where indicated, cells are kept in Ca²⁺‐free medium plus 500 μM EGTA with or without 10 μM BAPTA‐AM; Xe‐C is Xestospongin C, which selectively inhibits IP3R at the 5 μM concentration used here 46.