Unless otherwise stated, human ND1-mutant (3796A>G) cybrid, human MELAS (A3243G-Leucine tRNA) cybrid, human LHON ND6 (G14459A) cybrids, and control cybrid cells were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) containing 10% fetal bovine serum, 100 U/mL penicillin and streptomycin, 1 mM sodium pyruvate, and 4 mM l-glutamine. Mouse Rieske knockout cells were cultured in the same media, but also supplemented with 5 μg/mL uridine. Depending on the experiment, media also contained either 10 mM galactose or 25 mM glucose. For cell survival experiments: 100,000 cells were seeded directly in galactose-media with or without compounds or in glucose media containing varying dosages of tunicamycin. Cells were trypsinized and counted 48 or 72 h later unless otherwise stated. Drug treatments included: 25 μM glimepiride unless otherwise stated (Santa Cruz Biotechnology Inc.), 25 μm glyburide (Sigma-Aldrich), 25 μM repaglinide (Sigma-Aldrich), 25 μM SB203580 (Santa Cruz Biotechnology Inc.), 2 μM SB202190 (Sigma-Aldrich), 20 μM SP600125 (Sigma-Aldrich), 1 mM sodium 4-phenylbutyrate, 500 μM Tauroursodeoxycholic acid sodium salt (Fisher Scientific), 100 μM d-(+)-glucosamine (Sigma-Aldrich), 25 μM B I09 (Tocris), 20 μM 4 μ8C (7-hydroxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde, Sigma), tunicamycin (Sigma-Aldrich) at varying dosages. Drug treatments were typically performed for either 48 or 72 h in galactose media unless otherwise noted. CRISPR constructs: The lentiCRISPR v2 plasmid (addgene #52961) was used to clone all CRISPR constructs. Guide sequences for Kir6.2 were as follows. Kir6.2#1, 5′-CACCGAAGTGACTATTGGCTTTGGG-3′; Kir6.2#2, 5′-CACCGGAGTGGATGCTGGTGACAC-3′.
Soustek M.S., Balsa E., Barrow J.J., Jedrychowski M., Vogel R., Jan S.m.e.i.t.i.n.k., Gygi S.P, & Puigserver P. (2018). Inhibition of the ER stress IRE1α inflammatory pathway protects against cell death in mitochondrial complex I mutant cells. Cell Death & Disease, 9(6), 658.
Presence or absence of compounds (glimepiride, glyburide, repaglinide, SB203580, SB202190, SP600125, sodium 4-phenylbutyrate, Tauroursodeoxycholic acid sodium salt, D-(+)-glucosamine, B I09, 4 μ8C, tunicamycin)
Dosage of tunicamycin
dependent variables
Cell survival/count measured 48 or 72 hours after treatment
control variables
Cell type (human ND1-mutant, human MELAS, human LHON ND6 cybrids, control cybrids, mouse Rieske knockout cells)
Culture media (DMEM with 10% fetal bovine serum, 100 U/mL penicillin and streptomycin, 1 mM sodium pyruvate, 4 mM L-glutamine, with or without 5 μg/mL uridine, and 10 mM galactose or 25 mM glucose)
Cell seeding density (100,000 cells)
controls
Positive control: Cells cultured in glucose media
Negative control: Cells cultured in galactose media without any compounds
Annotations
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