GoKawiilCell culture, drug treatment and fractionation
143B cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco, 11965-126) supplemented with 10% Nu-serum IV (Corning, 355504), 10 mM HEPES (Corning, 25-060-CI), 1× GlutaMAX (Gibco, 35050061), 1× antibiotic–antimycotic solution (Gibco, 15240062), 2.5 µg ml−1 plasmocin prophylactic (InvivoGen, ant-mpp), 1 mM methyl pyruvate (Sigma-Aldrich, 371173) and 400 µM uridine (Sigma-Aldrich, U3003). Mouse ES cells were cultured in 2i medium consisting of 0.5× Neurobasal medium (Gibco, 21103049), 0.5× DMEM/F12 (Gibco, 11320033), 0.5× N-2 supplement (Gibco, 17502048), 0.5× B-27 supplement (Gibco, 17504-044), 0.05% bovine albumin fraction V (Gibco, 15260037), 1× antibiotic–antimycotic solution (Gibco, 15240062), 2.5 µg ml−1 plasmocin prophylactic (InvivoGen, ant-mpp), 1 μM PD0325901 (Reprocell, 04-0006-02), 3 μM CHIR99021 (Reprocell, 04-0004-02), 2 mM glutamine (Gibco, 25030081), 150 μM 1-thioglycerol (Sigma-Aldrich, M6145), 1,000 U ml–1 leukaemia inhibitory factor (Sigma-Aldrich, ESG1106), 1 mM methyl pyruvate (Sigma-Aldrich, 371173) and 400 µM uridine (Sigma-Aldrich, U3003). DMSO, ethanol, piericidin A (Cayman, 15379), 3-NPA (Sigma-Aldrich, 164603), atpenin A5 (Cayman, 11898), antimycin A (Sigma-Aldrich, A8674), myxothiazol (Sigma-Aldrich, T5580), sodium azide (NaN 3 ) (Sigma-Aldrich, S8032), l-ascorbic acid (Sigma-Aldrich, A92902), TMPD (Sigma-Aldrich, T7394), AOA (Sigma-Aldrich, C13408), DMKG (Sigma-Aldrich, 349631), 4-CBA (Sigma-Aldrich, 135585) and MDH2 inhibitor (Ambinter, Amb5965675) were used to treat cells in different experiments. 143B cells were treated with various drugs for 24 h, whereas the mouse ES cells were treated for 16 h unless mentioned otherwise. Cells were incubated at 37 °C, 5% CO 2 and 95% humidity. For anoxia experiments, cells were incubated for 24 h in a H35 HEPA Hypoxystation (Don Whitley Scientific) at 0% O 2 , 5% CO 2 , 95% N 2 and 37 °C under humidified conditions.
For 13C 5 -l-Gln tracing, the cell culture medium was switched to a labelling medium containing DMEM without glutamine (Gibco, 11960044), 2 mM 13C 5 -l-glutamine (Cambridge Isotope, CLM-1822-H-0.01), 10 mM HEPES (Corning, 25-060-CI), 1× antibiotic–antimycotic solution (Gibco, 15240062), 2.5 µg ml−1 plasmocin prophylactic (InvivoGen, ant-mpp), 1 mM methyl pyruvate (Sigma-Aldrich, 371173), 400 µM uridine (Sigma-Aldrich, U3003) and 10% dialysed FBS (PEAK serum, PS-FB2).
Mitochondrial and cytosolic fractions were prepared with a Cell Fractionation kit-standard (Abcam, ab109719). Nuclei were isolated with a Nuclear Extraction kit (Abcam, ab113474).
Cell lines
WT 143B cells and 143B ΔCYTB cells transduced with EV or expressing LbNOX-mito, LbNOX-cyto or AOX have been previously described26.
To generate the 143B ΔCYTB NT and MDH2 KO cell lines, a NT sgRNA and a sgRNA targeting MDH2 (sgMDH2), respectively, were separately cloned into pSpCas9(BB)-2A-GFP (PX458) plasmids (Addgene, plasmid no. 48138; a gift from F. Zhang), according to the provider’s instructions. Oligonucleotide sequences were as follows: sgMDH2: 5′-CGATATCATAGAGGGTCAGG-3′ (targeting the negative strand of exon 2); NT sgRNA: 5′-GTAGCGAACGTGTCCGGCGT-3′. 143B ΔCYTB cells were independently transfected with either construct using jetPRIME transfection reagent (Polyplus). Forty-eight hours after transfection, the GFP+ cells were single-cell sorted into 96-well plates using a BD FACSAria cell sorter. The sorted cells were grown in culture for 2–3 weeks, and the resultant clonal cell lines were expanded. KO of MDH2 was confirmed by Simple Western analysis, and our MDH2 KO clone was identified.
For lentiviral expression, full-length coding sequences of human MDH2, rat L2hgdh, rat L2hgdh-cyto (rat L2hgdh without the mitochondrial targeting sequence), LbNOX-mito (Addgene, plasmid no. 74448; V. Mootha laboratory) and LbNOX-cyto (Addgene, plasmid no. 75285; V. Mootha laboratory) were subcloned into the pLV-EF1A-IRES-mRFP1 vector (VectorBuilder, vector ID VB160708-1059xrd) using BamHI (5′) and SalI (3′) restriction sites. Human MDH2, rat L2hgdh and rat L2hgdh-cyto sequences are provided in the Supplementary Methods. L2hgdh-cyto was cloned using the GenScript service. These constructs or EV control, along with pMD2.G and psPAX2 lentiviral packaging vectors, were then transfected into 293T cells (American Type Culture Collection, CRL-3216) using jetOPTIMUS (Polyplus) to generate lentivirus. Cells were transduced with lentivirus, and RFP+ cells were sorted using a BD FACSAria cell sorter. The cells were periodically sorted to maintain high RFP expression. L2HGDH, L2HGDH-cyto and MDH2 overexpression were confirmed by Simple Western analysis. All cell lines tested negative for Mycoplasma contamination.
Simple Western analysis
Cells and tissues were lysed in NP40 cell lysis buffer (ThermoFisher Scientific, FNN0021) supplemented with 1× Halt protease inhibitor cocktail (ThermoFisher Scientific, 78430). Protein concentrations were measured using a Pierce BCA Protein Assay kit (ThermoFisher Scientific, 23225). Simple Western analysis was performed using the Wes or Jess platform (Bio-Techne) according to the manufacturer’s instructions. For p-eIF2α and eIF2α, RePlex Module (Bio-Techne, RP-001) was used. Protein abundance was quantified using Compass software. Primary antibodies used were anti-L2HGDH (Abcam, ab230230; 1:250; 1:1,000), anti-cofilin (Cell Signaling Technology (CST), 5175; 1:2,500), anti-vinculin (CST, 13901; 1:50,000), anti-MDH2 (Abcam, ab181873; 1:5,000), anti-TOM20 (CST, 42406; 1:50), anti-eIF2α (CST, 9722; 1:200) and anti-p-eIF2α (Ser51) (CST, 9721; 1:20).
... continue reading