Extracellular acidification rate (ECAR) measurements

Glycolysis in iPSCs was assessed using the Glycolysis Stress Test (Agilent) and Seahorse XFe24 Analyzer (Agilent). Approximately 48 h prior to the experiment, dissociated iPSCs were seeded onto cell culture microplates (Agilent Seahorse XF24) in culture medium (StemMACS™ iPS‐Brew, Miltenyi Biotec) supplemented with 1 μM of ROCK inhibitor (Y‐27632, Miltenyi Biotec, Germany). On the next day, fresh culture medium without ROCK inhibitor was added and cells cultured for an additional 24 h. On the day of the experiment, growth media was replaced with Seahorse XF base medium (Agilent), supplemented with 2 mM of glutamine. Cells were then equilibrated for 60 min in a CO₂‐free incubator at 37°C before being placed into the Seahorse XFe24 Analyzer. Following three baseline measurements, glucose (10 mM of final concentration), oligomycin (1 μM final concentration) and 2‐Deoxy‐D‐glucose (2‐DG, 50 mM of final concentration) were sequentially added. Changes in the extracellular acidification rate (ECAR) were recorded and used to assess glycolytic function. After the assay, cells were collected lysed and sonicated in RIPA buffer. Protein concentrations were then determined via Bradford assay (Sigma) and raw ECAR values normalized to the respective protein content.