Fig 2: NAC1 promotes the expression of LDHA in tumor cells. (A) Heatmap of normalized RNA-seq reads (Z score), top upregulated glycolysis-associated genes in the TCGA SKCM database. (B, C) qRT-PCR analysis of mRNA of LDHA, LDHB, LDHC, and HK2 in WT and NAC1 KO B16-OVA cells (B) or WT and NAC1 KO A2058 cells (C). The results are presented relative to the level of GAPDH. n=5 per condition from two independent experiments. *p=0.05, **p=0.01, ***p=0.001. (D) LDHA expression in whole-cell extracts by Western blots. ß-actin was used as a loading control. One representative experiment out of three is shown. (E) Correlations of NACC1 with LDHA gene expression (Z-score) were determined in a dataset including 470 melanoma tumors and 368 melanoma-metastatic tumors (‘R2: Tumor Skin Cutaneous Melanoma-TCGA-470-rsem-tcgars’). Pearson’s correlation was calculated. (F) Correlations of CD8+ T cell infiltration with NACC1 expression in melanoma (SKCM, n=470) and metastasis melanoma (SKCM-metastasis, n=368), (G) Correlations of CD8+ T cell infiltration level with LDHA expression in melanoma (SKCM, n=470) and metastasis melanoma (SKCM-metastasis, n=368), were estimated using TIMER algorithm. Pearson’s correlation was calculated. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LDHA, lactate dehydrogenase A; NAC1, nucleus accumbens-associated protein-1; SKCM, skin cutaneous melanoma; TCGA, The Cancer Genome Atlas; TIMER, Tumor Immune Estimation Resource.

Fig 3: Effects of LDHA expression and lactic acid level on cytokine production, apoptosis, and exhaustion of CD8+ T cells. Mouse or human CD8+ T cells were incubated with CM from mouse WT or NAC1 KO B16-OVA cells, or human WT or NAC1 KO A2058 tumor cells for 24 hours. In parallel, some T cells were incubated with CM from NAC1 KO tumor cells overexpressing LDHA (NAC1 KO LDHA OE) or an empty control (NAC1 KO Mock), or CM supplemented with LA (2–5 mM for mouse T cells; 5–10 mM for human T cells). (A) The production of cytokines TNF-α or IFN-γ and expression of PD-1 and TIM-3 of mouse CD8+ T cells with CM from mouse WT or NAC1 KO B16-OVA cells, or CM supplemented with LA by flow cytometry. One-way ANOVA with multiple comparisons correction. *p≤0.05, **p≤0.01. (B) Apoptosis of mouse CD8+ T cells with CM from mouse WT or NAC1 KO B16-OVA cells, or CM supplemented with LA. (C) The production of cytokines and expression of PD-1 of mouse CD8+ T cells with CM from mouse WT or NAC1 KO B16-OVA cells, or CM from NAC1 KO tumor cells overexpressing LDHA or an empty control by flow cytometry. One-way ANOVA with multiple comparisons correction. **p≤0.01. (D) Apoptosis of mouse CD8+ T cells with CM from mouse WT or NAC1 KO B16-OVA cells, or CM from NAC1 KO tumor cells overexpressing LDHA or an empty control by Annexin V staining. (E, F) Apoptosis of human CD8+ T cells with CM from human WT or NAC1 KO A2058 cells, or CM supplemented with LA or CM from human NAC1 KO tumor cells overexpressing LDHA or an empty control by Annexin V staining. (G, H) The production of IFN-γ of human CD8+ T cells with CM from human WT or NAC1 KO A2058 cells, CM supplemented with LA, or CM from human NAC1 KO tumor cells overexpressing LDHA or an empty control. (I, J) The production of IL-2 of human CD8+ T cells with CM from human WT or NAC1 KO A2058 cells, CM supplemented with LA, or CM from human NAC1 KO tumor cells overexpressing LDHA or an empty control. (K, L) Expression of PD-1 of human CD8+ T cells with CM supplemented with LA, or CM from human NAC1 KO tumor cells overexpressing LDHA or an empty control. (M) ECAR of mouse tumor cells measured by the Seahorse Metabolic Analyzer. Data are represented as mean±SEM; n=6 per condition from two independent experiments. (N) Basal glycolysis rates (left panel) and maximum glycolysis rate (right panel) of mouse tumor cells. One-way ANOVA with multiple comparisons correction. ***p≤0.001. (O) ECAR of human tumor cells measured by the Seahorse Metabolic Analyzer. Data are represented as mean±SEM; n=6 per condition from two independent experiments. (P) Basal glycolysis rates (left panel) and maximum glycolysis rate (right panel) of human tumor cells. One-way ANOVA with multiple comparisons correction. ***p≤0.001. ANOVA, analysis of variance; CM, conditional medium; ECAR, extracellular acidification rate; LDHA, lactate dehydrogenase A; NAC1, nucleus accumbens-associated protein-1; OVA, ovalbumin.

Fig 4: Metformin increased glucose uptake and glycolysis in HepG2/DDP cells. (A) Metformin increased glucose uptake. Indicated cells were treated with or without metformin (1mM) for 24 hours and glucose uptake assay were conducted with Glucose Uptake-Glo. Cell Titer-Glo was also carried out to measure the relative viability, which was used to normalize the data in glucose uptake assay. Data from 3 independent biological samples of 3 replicates were statistically analyzed by student’s t-test (*** P<0.0001). (B) Metformin increased the expression of glucose transporter Glut1 and Glut4. HepG2/DDP cells were treated with or without metformin (1mM) for 24 hours and total cell lysates were separated by SDS-PAGE. Glut1 and Glut4 protein levels were detected by Western blot using specific antibodies to Glut1 and Glut4. Tubulin was used as internal control. Representative images of were shown. Quantification of N= 2 biological repeats were shown in bar graph. (C) Metformin increased intracellular glucose concentration in HepG2/DDP cells. HepG2/DDP cells were treated with or without metformin (1mM) for 24 hours, washed extensively and intracellular glucose concentration was measured by using Glucose-Glo kit. Data from 2 independent biological samples of 3 replicates were plotted and statistically analyzed by student’s t-test (** P<0.001). (D) Metformin increased the protein levels of glycolytic enzymes HK2 and LDHA. Experiment was conducted as in (B) except HK2 and LDHA antibodies were used. Representative images of were shown. Quantification of N= 3 biological repeats were shown in bar graph. (E) Metformin increased intracellular lactate production. Indicated cells were treated with or without metformin (1mM) for 24 hours, washed extensively then intracellular lactate concentration was measured by using lactate-Glo kit. Data from 2 independent biological samples of 3 replicates were plotted and statistically analyzed by student’s t-test (** P<0.001, *** P<0.0001). (F) Metformin increased intracellular NAD/NADH production. HepG2/DDP cells were treated with or without metformin (1mM) for 24 hours and lactate concentration was measured by using NAD/NADH -Glo kit. Data from 2 independent biological samples of 3 replicates plotted and statistically analyzed by student’s t-test (* P<0.05, **P<0.001). (G) Metformin increased intracellular NADP/NADPH production. HepG2/DDP cells were treated with or without metformin (1mM) for 24 hours and lactate concentration was measured by using NADP/NADPH -Glo kit. Data from 2 independent biological samples of 3 replicates were plotted and statistically analyzed by student’s t-test (*** P<0.0001).