S 1 mitoplates

To address the function of the mitochondria, S-1 MitoPlates (Biolog) Mitochondrial Function Assays were performed following the manufacturer's protocol. The assay covers 14 metabolites in central metabolic pathways, namely, glucose, glucose-1 phosphate, glucose-6 phosphate, pyruvate, and lactate in the glycolysis pathway; citrate, 2-oxoglutarate, succinate, fumarate, malate in the TCA cycle; and amino acids glutamate, glutamine, serine, and ornithine. Specifically, assay mix (60 min at 37°C) was added to the plates to dissolve the substrates. We collected, counted, resuspended PDAC cells in provided buffer and plated them at 5 × 104 cells/well after treatment. Readings at 590 nm were taken every 5 min for 4 h at 37°C. Experiments were performed in triplicate with three biological replicates for the siAPEX1 and control PDAC cells under the normoxia condition. Raw data was analyzed using GraphPad Prism 8, and statistical significance was determined using the two-way ANOVA, and P-values < 0.05 were considered statistically significant.

S-1 Mitoplates (Biolog, Hayward, CA) were used to investigate mitochondrial function of TCA cycle substrates as previously published [16 (link)]. Assays were performed as per manufacturer's protocol. Briefly, plates were activated by adding the Assay Mix to the wells to dissolve the substrates for 60 min at 37 °C. Cas9 control cells in low glucose media (0.5 mM) were used as control and Ref-1^C65A E19 clone was treated with DMSO or APX2009 (5 μM, 24 h) in low glucose media. Following treatment, cells were collected, counted, resuspended in provided buffer and plated at 5x10⁴ cells/well. This resuspension was added to the plate, which was immediately read at 590 nm kinetically at 5 min intervals for 4h at 37 °C. Data was analyzed using Graphpad Prism 8, and statistical significance was determined using the 2-way ANOVA and p-values <0.05 were considered statistically significant.