Extracellular and intracellular lactate measurements

For extracellular lactate determination, cells were seeded at 5 × 10⁴ cells/well in 24-well cell culture plates and incubated overnight for attachment prior to treatment with 2.5 μM PFK158 or 50 μM YN1 for 72 h. Media supernatants were harvested and analyzed using a Lactate Plus Meter (Nova Biomedical) and Lactate Plus Meter Test Strips (Nova Biomedical). Graphs were generated in GraphPad Prism 8, and significance was determined by a two-way ANOVA followed by Tukey’s multiple comparisons test. For intracellular lactate determination, cells were seeded in triplicate at 2 × 10⁴ cells/well in a 96-well cell culture plate and incubated overnight prior to treatment with 2.5 μM PFK158 for 72 h. Cell lysates were prepared with 0.6 N HCL followed by 1 M Tris Base. Lactate concentration was determined using the Lactate-Glo Assay (Promega) by luminescence with gain adjustment normalized to a 5000 RLU maximum using a Synergy HT Microplate Reader. Using R, RLUs were converted to the log scale and correlated to LDHA gene expression using SCLC cell line transcriptional data [37].