Cell treatments 35.

For the dichloroacetate (DCA) treatments, 0.3 g of sodium dichloroacetate (MW 150.92) was dissolved in 5 ml UltraMEM and filter-sterilised, giving a stock solution of approximately 400 mM DCA.This meant that adding 500 µl stock to 10 ml UltraMEM gave a treatment concentration of approximately 20 mM and adding 250 μl stock (with an additional 250 µl UltraMEM) gave a final treatment concentration of approximately 10 mM DCA. Further dilutions of the stock were performed in order to achieve lower DCA concentrations whilst still adding a total of 500 µl to each 10 ml culture. Control cells were incubated in a total of 10.5 ml UltraMEM.

For the acetic acid treatments, 115 µl acetic acid (99.5 % solution; 17.4 M) was added to 4885 µl UltraMEM and filter-sterilised to give a final stock concentration of approximately 400 mM. This meant that adding 500 µl stock to 10 ml UltraMEM gave a treatment concentration of 20 mM and adding 250 μl stock (with an additional 250 µl UltraMEM) gave a treatment concentration of 10 mM.

For the sodium acetate (MW = 82.034) treatments, 0.16406 g was dissolved in 5 ml UltraMEM and filter-sterilised to give a final stock concentration of approximately 400 mM. This meant that adding 500 µl stock to 10 ml UltraMEM gave a treatment concentration of 20 mM and adding 250 μl stock (with an additional 250 µl UltraMEM) gave a treatment concentration of 10 mM.

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For the Reactivating p53 and Inducing Tumour Apoptosis (RITA) treatments, RITA (CAY10006426- Cambridge Bioscience; MW = 292.37) was obtained as a 10 mg lyophilised sample. DMSO (1369.86 µl) was added to this sample, giving a stock concentration of 25 mM. The stock (10 μl) was then added to 90 µl DMSO, to give a final stock concentration of 2.5 mM. This meant that adding 10 µl of final stock on 10 ml UltraMem gave a treatment concentration of 25 µM, 4 µl of final stock gave a treatment concentration of 10 µM, and 2 µl of final stock gave a treatment concentration of 5 µM. This stock was then serially diluted 10- fold, meaning that adding 2 µl of the first serial dilution gave a treatment concentration of 0.5 μM and 2 µl of the second serial dilution gave a treatment concentration of 0.05 µM.

For the GSK2837808A lactate dehydrogenase inhibitor (Tocris, Abingdon, UK; MW = 649.62) treatment, 10 mg of inhibitor was reconstituted in 30.5754 ml DMSO, which was then divided into 1 ml aliquots and frozen at -20 °C in order to give a 10 x stock. One of these aliquots was diluted 10-fold with DMSO to give 1 x stocks which were divided into 200 µl aliquots and frozen. When using 10 ml culture volumes, 10 µl of the 1 x stock was added to the medium to give a final inhibitor concentration of 50 nM. All other final concentrations were produced by further dilution of the 1 x stock with DMSO, prior to adding 10 µl of these dilutions to the cultures. For 96 well plate cultures containing 200 µl medium per well, the 1 x stock was diluted 10-fold with DMSO and 2 µl of this was added to the wells to give a final inhibitor concentration of 50 nM.

For the FX11 lactate dehydrogenase inhibitor (Merck Millipore, Darmstadt, Germany; MW = 350) treatment, 10 mg FXII LDHA compound was dissolved in 285.71 µl DMSO, giving a stock concentration of 1 mM. This meant that adding 10 µl stock to 10 ml UltraMEM gave a final treatment concentration of 100 µM in T75 cm2 _{flasks. For all other inhibitor}

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concentrations, the stock was diluted such that the same volume (10 µl) could be added to cultures to give lower final inhibitor concentrations. For 96 well plate cultures containing 200 µl of medium per well, the stock was diluted 10-fold with DMSO and 2 µl of this was added to each well to give a 100 µM final inhibitor concentration.

For the pyruvate treatments, it is worth noting initially that the basal concentration of pyruvate in UltraMEM is 1 mM. As such, the UltraMEM ‘control’ for these experiments actually represented 1 mM pyruvate and the mass calculations for all other pyruvate concentrations were performed taking this initial 1 mM concentration into account. As such, 0.54648 g of sodium pyruvate (Sigma-Aldrich Ltd, Pool, UK; MW = 110.4) was dissolved in 50 ml UltraMEM and filter-sterilised. Cells were cultured directly in this solution without any further dilution, such that the final pyruvate concentration was 100 mM. Other final pyruvate concentrations were obtained simply by adjusting the weight of dry powder dissolved in the UltraMEM in which the cells were cultured.

For the lactic acid treatments, 0.54048 g of lactic acid (Sigma-Aldrich Ltd, Poole, UK; MW = 90.08) was dissolved in 10 ml UltraMEM and filter-sterilised to give a final stock solution of 600 mM. This meant that adding 200 µl of stock solution to 10 ml UltraMEM gave a final treatment concentration of 12 mM.

For the N-acetyl cysteine (NAC) treatments, 0.163195 g NAC (Sigma-Aldrich Ltd, Poole, UK; MW = 163.195) was dissolved in 10 ml distilled water and filter-sterilised to give a final stock concentration of 100 mM. This meant that adding 100 µl of stock solution to 10 ml UltraMEM gave a final treatment concentration of 1 mM.

For the ammonium chloride treatments, 0.5349 g of ammonium chloride (Sigma-Aldrich Ltd, Poole, UK; MW = 53.49) was dissolved in 10 ml UltraMEM and

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filter-sterilised to give a stock solution of 1 M. This meant that adding 100 µl of stock to 10 ml UltraMEM gave a final treatment concentration of 10 mM.

For the chloroquine diphosphate treatments, 0.25793 g of chloroquine diphosphate (Sigma-Aldrich Ltd, Poole, UK; MW = 515.86) was dissolved in 100 ml UltraMEM to give a final stock concentration of 5 mM. This meant that adding 100 µl of stock solution to 10 ml UltraMEM gave a final treatment concentration of 50 µM.

For the batimastat treatments, 10 mg batimastat (Biotechne, Abingdon, UK; MW = 78.13) was dissolved in 12.7992 ml DMSO to give a stock solution of 10 mM. This meant that adding 5 µl to 10 ml UltraMEM gave a final treatment concentration of 5 µM.