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. 2014 Jun 16;111(29):10580–10585. doi: 10.1073/pnas.1401591111

Fig. 4.

Fig. 4.

Altering c-subunit expression or packing regulates IMM conductance and sensitivity to excitotoxic and ROS-mediated cell death. (A) Immunoblot using the indicated antibodies. (B and C) Images and group data of propidium iodide (PI) staining of cultured hippocampal neurons at 24 h after 30-min exposure to 100 µM glutamate or 20 µM H2O2 with or without 0.5 µM CsA (n ≥ 3 independent cultures, 56–65 micrographs of each condition; G1, ATP5G1; G3, ATP5G3). (D) Schematic of the WT and M4 c-subunit rings. Increased channel conductance of M4 mutant suggests decreased packing and a larger central pore due to the Gly-Val mutation. (E) Representative patch clamp recordings of liposomes reconstituted with WT or M4 c-subunit. (F) Peak single channel conductance for liposome recordings with the indicated protein (WT, n = 38; M1, n = 7; M2, n = 17; M3, n = 7 M4, n = 22; ***P < 0.0001 for WT vs. M1–M4). (G) Normalized NPO before and after addition of 1 mM ATP to patches with the indicated protein (WT, n = 21; M1, n = 3; M2, n = 6; M3, n = 5; M4, n = 8; ***P = 0.0009 comparing WT to M4 ATP response, NPO, number of channels multiplied times the probability of opening of each channel). (H) Immunoblot using the indicated antibodies. The uppermost bands labeled with anti–c-subunit antibody represent the myc/FLAG tagged overexpressed protein. The lower bands have lost one of the tags (blot is representative of >5 blots). (I and J) Percent pyknotic nuclei (I) and cell survival (MTT assay) (J), measured in HEK 293T cells expressing the indicated constructs (n = 2 experiments with three coverslips, two fields per coverslip per condition (**P < 0.01, ***P < 0.0001). Measurements were made 24 h after switching to galactose-containing, glucose-free medium. (K and L) Images and group data of PI staining of hippocampal neurons at 18 h after 30-min exposure to 100 µM glutamate or 20 µM H2O2 with vehicle or 0.5 µM CsA (n = 3 independent cultures, 38–53 micrographs of each condition. (Scale bars: B, C, K, and L, 20 µm.) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.