Figure 2A Schematic representation of the regulation of glucose levels and glycolysis by LDHA activity. Suppressed NAD+ regeneration consequent to LDHA inhibition compromises the activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), an enzyme required for the conversion of glyceraldehyde 3-phosphate to 1,3-biphosphoglycerate. This in turn triggers a build-up of the glycolytic intermediates in the first few steps of glycolysis; increase in cellular levels of unused glucose; and a suppression in glucose uptake. B, C Glucose (B) and glutamine (Gln, C) levels in the medium after treatment of melanoma cell lines with LDHAi for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. D Medium Gln levels after transfection of melanoma cells with non-targeting (NT) or LDHA-specific siRNA for 96 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. E GC-MS-based quantification of intracellular Gln levels in cells Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. F qRT-PCR analysis of expression of the indicated SLC family transporters in MeWo (left) and A375 (right) melanoma cells after treatment with LDHAi for 48 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. G Immunoblotting of SLC1A5 in melanoma cells treated with LDHAi for 96 hr. H Immunoblotting of LDHA and SLC1A5 in melanoma cell lines transfected with NT-siRNA or indicated si-LDHA for 96 hr. I Immunoblotting of SLC1A5 and LDHA in MeWo cells stably transduced with empty vector (EV) or four different LDHA-­specific shRNAs. J Immunoblotting of SLC1A5 in MeWo cells mock (NT-siRNA + DMSO) or si-SLC1A5-treated for 24 hr and then treated with LDHAi for 48 hr. K Medium Gln levels (left), intracellular Gln levels (right) of MeWo cells Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. L Proliferation of melanoma cell lines for the indicated timepoints following treatment with LDHA-i, si-SLC1A5 or both. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. two-way Anova was used for the proliferation. N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. M (left) Long-term viability of melanoma cells treated as indicated for 8 days. (right) corresponding quantification showing change in viability. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. N Immunoblotting of SLC1A5 and cleaved caspase-3 in melanoma cells subjected to mock (NT-siRNA + DMSO) or si-SLC1A5 treatment for 24 hr followed by exposure to LDHAi for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.