Figure 2. Exogenous pyruvate relieves NAD⁺ depletion and glycolysis inhibition.

(A) The average extracellular acidification rates (ECAR) were analyzed with Seahorse analyzer in 143B and 143B206 cells with 1 mM pyruvate or not. Error bars indicate SD from 4 replicates. (B) 143B206 and 143B cells were cultured with indicated conditions for 24 h. Cellular ATP levels were measured and normalized by protein content. Error bars indicate SD from triplicates. (C) Phosphorylated ACC (Ser79) and total ACC in whole lysates of 143B and 143B206 cells were analyzed with immunoblotting. α-tubulin was used as loading control. Representative result from triplicates is shown. (D) NAD⁺ concentrations in the lysates of 143B and 143B206 cells were measured and used to calculate the total amount of NAD⁺ in both cell lines (per 10⁶ cells). (E and F) NAD⁺/NADH ratio in 143B and 143B206 cells was calculated and presented. Error bars indicate SD of triplicates. (G–I) 143B206 cells were cultured in media with 1 mM of pyruvate and 50 μg/mL uridine. The knockdown efficiency of LDHA with siRNA was analyzed with immunoblotting and shown in (G) The effects of LDHA knockdown on ECAR were shown in (H) The effects of LDHA knockdown on 143B206 proliferation were shown in (I) Error bars indicate SD of triplicates. **p < 0.01.