Figure 2. PHB1 is required to maintain mitochondrial homeostasis and membrane permeability.

• A, B
  GFP‐expressing HeLa cells with stable Phb1 knockdown were fixed and subjected to immunofluorescence (IF) analysis to detect mitochondria (MitoTracker, red; A) or ΔΨm (TMRM, red; B). Images shown are representative of at least three independent experiments. White boxed regions in the panels in (A) are enlarged. Scale bar: 10 μm.
• C
  Flow cytometric analysis of ΔΨm (TMRM). HeLa cells with stable Phb1 knockdown were treated with CsA (2 μM) for 30 min or FCCP (4 μM) for 5 min.
• D
  The mitochondrial respiratory capacity of J774A.1 cells with or without Phb1 knockdown was measured with a Seahorse XFe96 analyzer. Data are from at least three independent experiments (mean ± SEM). n.s., no significance. **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student's t‐test).
• E
  HeLa cells with stable PHB1 ablation were incubated with H₂O₂ (2 μM) and CsA (2 μM) for 30 min. Flow cytometric analysis of intracellular ROS levels (DHE).
• F, G
  Confocal microscopy analyses of mitochondrial Ca²⁺ in time‐series mode. HeLa cells stably expressing 4mt‐RCaMPh with or without Phb1 knockdown were treated with CsA (2 μM) for 30 min. During the acquisition of fluorescence images (F), cells were treated with histamine (200 μM). Traces of mitochondrial Ca²⁺ (4mt‐RCaMPh) dynamics are shown in (G) (mean ± SEM). n = 16, 16, 15, 16 (from the top to the bottom). Scale bar: 10 μm. *P < 0.05 relative to Scramble group, #P < 0.05 relative to shPHB1 group (Student's t‐test).

Source data are available online for this figure.