Emetine dihydrochloride

Mitochondrial translation products were labelled using ³⁵S-methionine as previously described (24 ). Fibroblasts were washed twice with methionine/cysteine free DMEM (Life Technologies) supplemented with 1 mM L-glutamax, 96 μg/ml cysteine (Sigma), 1 mM pyruvate and 5% (v/v) dialyzed FBS, and incubated in the same medium for 10 min at 37°C. A total of 100 μg/ml emetine dihydrochloride (Sigma) was then added to inhibit cytosolic translation, before pulse-labelling with 100 μCi [³⁵S]-methionine for 45–60 min. Cells were chased for 10 min at 37°C in regular DMEM with 10% FBS, washed three times with PBS and collected. Labelled cells were lysed in PBS, 0.1% n-dodecyl-D-maltoside (DDM), 1% SDS, 50 units Benzonase (Millipore), 1X protease inhibitor cocktail (Roche). Protein concentration was measured by DC protein assay kit (Biorad) and 20 μg of protein were separated by 12% SDS-PAGE. Gel were fixed and vacuum dried, and exposed to X-ray film for 1 week.

To label newly synthesised mtDNA-encoded proteins, growth media was replaced with methionine/cysteine-free DMEM supplemented with 2 mM Glutamax, 110 mg/ml sodium pyruvate and 48 μg/ml cysteine. Cells were incubated for 2 × 10 min in this medium before replacement with fresh methionine/cysteine-free DMEM medium containing 10% dialysed FCS and emetine dihydrochloride (Sigma-Aldrich, 100 μg/ml) to inhibit cytosolic translation. Cells were incubated for 20 min before addition of 120 μCi/ml of [³⁵S]- methionine (Perkin Elmer). Labelling was performed for 30 min and cells were washed twice with PBS and cells were pelleted. Cells were lysed and aliquots of samples (30 μg) were separated on 10–20% Tris-Glycine SDS-PAGE gels. Gels were dried and products were visualized and quantified with a PhosphorImager system with ImageQuant software.

Mitochondrial translation products were labelled using ³⁵S-methionine³⁷ . Fibroblasts were washed twice with methionine/cysteine-free DMEM (Life Technologies) supplemented with 1 mM L-glutamax, 96 μg/ml cysteine (Sigma), 1 mM pyruvate and 5% (v/v) dialyzed FBS, and incubated in the same medium for 10 min at 37 °C. Then, 100 μg/ml emetine dihydrochloride (Sigma) was added to inhibit cytosolic translation, before pulse labelling with 100 μCi [³⁵S]-methionine for 45–60 min. Cells were chased for 10 min at 37 °C in regular DMEM with 10% FBS, washed three times with PBS and harvested. Labelled cells were lysed in PBS, 0.1% n-dodecyl-D-maltoside (DDM), 1% SDS, 50 U Benzonase (Millipore), 1x protease inhibitor cocktail (Roche). Protein concentration was measured by DC protein assay kit (Biorad) and 20 µg of protein were separated by 12% SDS-PAGE. The gels were then stained with the Coomassie staining solution (50% Methanol (Fisher Scientific), 10% Acetic Acid (Sigma), 0.1% Coomassie Brilliant Blue R250 (Biorad)) to confirm equal loading. Gels were then dried and exposed to phosphor screens (GE Healthcare). The signal was detected by using Typhoon^TM Phosphoimager (GE Healthcare).

In vivo³⁵S-metabolic labelling studies were performed as described previously with the following modifications. Cells, cultured to 60–70% confluency in T25 mm flasks, washed with phosphate-buffered saline (PBS; Sigma) and washed by incubating twice for 10 min at 37°C/5% CO₂ in methionine/cysteine-free DMEM (Sigma, Poole, UK), with the media replaced between each incubation. Cells were then incubated for 15 min at 37°C/5% CO₂ in methionine/cysteine-free DMEM supplemented with 5% (v/v) dialyzed FBS, 0.1 mg/ml emetine dihydrochloride (Sigma). Following addition of 200 mCi/ml ³⁵S-methionine/cysteine (³⁵S EasyTag EXPRESS; Perkin Elmer), cells were incubated for 15 min at 37°C/5% CO₂, then washed twice with ice-cold DMEM supplemented with 7.5 mg/ml methionine. Cell pellets were prepared after washing once with ice-cold PBS. Radio-labelled proteins were then analysed using SDS–PAGE as described previously.

In some experiments, cells were treated with the following drugs: olaparib (S1060; Selleck Chemicals), emetine dihydrochloride (E0100000; Sigma-Aldrich) - EME, adarotene (HY-14808; MedChemExpress) - ADA, CD437 (C5865; Sigma-Aldrich) - CD, hydroxyurea (H8627; Sigma-Aldrich) - HU, cycloheximide (C4859; Sigma-Aldrich) - CHX, and PARG inhibitor (PDD 0017273; Tocris) - —PARGi.

Mitochondrial translation products in cultured cells were labelled as described previously (16 (link)). HeLa cells, transfected with non-targeting siRNAs or siRNAs targeting MPV17L2, or mock transfected, were washed twice with methionine/cysteine-free DMEM (Sigma) supplemented with 2 mM l-glutamine, 96 μg/ml cysteine and 5% (v/v) dialyzed FBS followed by 10 min incubation in this media at 37°C. Cytosolic translation was subsequently inhibited by incubating the cells for 20 min with 100 μg/ml emetine dihydrochloride (Sigma). 100 μCi [³⁵S]-methionine was added and labelling was performed for 1 h at 37°C, after which cells were washed three times with PBS (Life Technologies) before lysis in 1× PBS, 0.1% n-dodecyl-β-d-maltoside (DDM), 1% SDS, 50 units benzonase (Novagen), 1:50 (v/v) Roche protease inhibitor cocktail. Twenty micrograms lots of protein were resolved via SDS-PAGE (Novex) and the radiolabelled proteins were detected by Phosphorimager of the dried gels (Typhoon Molecular Imager FX, GE Healthcare).

Mitochondrial protein synthesis in cultured cells was performed essentially as described previously (Chomyn, 1996). Fibroblasts were labelled for 1 h in methionine/cysteine‐free DMEM (Sigma) with 200 μCi/ml of a [35S]‐methionine/cysteine mixture (Perkin Elmer) and 100 μg/ml emetine dihydrochloride (Sigma) followed by either 4 or 8 h chase in standard DMEM with additional 7.5 μg/ml cold methionine. Aliquots (50 μg) of total cell protein were separated by 15% SDS–PAGE. Signals were detected using the Typhoon FLA9500 Phosphorimager and ImageQuant software (GE Healthcare).