Figure 3GC-MS-based quantification of intracellular essential amino acid levels in MeWo cells treated for 72 hr with LDHAi Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 B GC-MS-based quantification of intracellular essential amino acid levels in MeWo cells mock or si-SLC1A5-transfected for 24 hr and then treated with LDHAi for 48 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 C (left) Immunoblotting of SLC7A5 in MeWo cells transfected with non-targeting (NT) or SLC7A5-specific siRNAs for 48 hr. (right) GC-MS-based quantification of intracellular essential amino acid levels in MeWo cells mock or si-SLC7A5-transfected for 24 hr and then treated with LDHAi for 48 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 Immunoblotting of total and phosphorylated S6, ULK1, and eIF4EBP1 in melanoma cells (D) treated with LDHAi for 96 hr Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 Immunoblotting of total and phosphorylated S6, ULK1, and eIF4EBP1 in melanoma cells (E) transfected with non-targeting (NT) or LDHA-specific siRNAs for 96 hr Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 Immunoblotting of total and phosphorylated S6, ULK1, and eIF4EBP1 in melanoma cells (F) si-SLC1A5-transfected for 24 hr followed by treatment with LDHAi for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. 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 Model summarizing Gln uptake-mediated cellular build-up of EAA and the consequent mTORC1 activation. H Proliferation of melanoma cells treated with LDHAi, m-TORC1 inhibitor (Rapamycin, 100 nM), or a combination of the two. 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, and two-way Anova was used for the proliferation. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 I (top) Long-term viability of melanoma cells treated as indicated for 10 days. (bottom) 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. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 J Immunoblotting of cleaved caspase-3 and p-(S780)-Rb in melanoma cells treated as indicated for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001