Vialight plus assay

Cellular ATP content was assessed using the ViaLight Plus assay (Lonza, Walkersville, MD, USA). Cells were lysed with 10 μL of the assay cell lysis reagent for 10 min, followed by 25 μL of ATP-monitoring reagent (AMR) containing firefly luciferase, luciferin, pyrophosphate, BSA and magnesium ions. After 2 min incubation in the dark, the cellular ATP content was determined by top reading luminescence measurements using LMax luminometer. Luminescence (rate of photon emission) is directly proportional to concentration of ATP substrates). The ATP assay was performed in three independent experiments (n = 3), with triplicate wells within each experiment.

MIA PaCa-2 cells were seeded at 10,000 cells/well into clear-bottomed, white-walled 96-well plates (Corning) and allowed to adhere overnight. The following day, wells were treated with PFK15, inhibitor cocktail (4 μM CCCP, 10 μM oligomycin, 200 μM bromopyruvate and 2 mM iodoacetate) or vehicle (DMSO). Global ATP was quantified using a Vialight plus assay (Lonza, Slough, UK), following the manufacturer’s instructions, using a Biotek HT plate reader.