Fluoro carbonyl cyanide phenylhydrazone (fccp)

Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured using the extracellular flux analyser XFe96 from Agilent Seahorse Bioscience as previously optimized for PCa cells [10 (link), 12 (link), 19 (link)]. Cells were plated at 10 000 cells/well confluency in a 96‐well Seahorse microplate in phenol red‐free RPMI medium supplemented with 5% CSS. After 48 h, cells were treated with androgens and/or IDH1 inhibitors. After another 48 h, media was changed for Seahorse XF RPMI Medium supplemented with 10 mm glucose, 2 mm glutamine, 1 mm sodium pyruvate, and 100 U·mL⁻¹ penicillin–100 μg·mL⁻¹ streptomycin. The microplate was then transferred to a CO₂‐free incubator at 37 °C for 1 h. After equilibration, the microplate was inserted into the XFe96 instrument. Basal respiration as well as respiration during a mitochondrial stress test were measured by three consecutive measurements of OCR and ECAR before and after injection of oligomycin (Sigma), FCCP (Santa Cruz), and antimycin A (Sigma) + rotenone (Sigma). For normalization, cell counting was performed using CyQUANT (Thermo Fisher Scientific) assay as described previously [25 ].

The following drugs were used: 6‐Diazo‐5‐oxo‐l‐norleucine (Don, D2141, Sigma‐Aldrich); Sodium dichloroacetate (DCA, 347795, Sigma‐Aldrich); NCT‐502 and PHGDH inactive (19716 and 19717, Cayman); Doxycycline (Dox, D9891, Sigma‐Aldrich), Oligomycin (sc‐203342, Santa Cruz Biotechnology); FCCP (sc‐203578, Santa Cruz Biotechnology); Antimycin (sc‐202467, Santa Cruz Biotechnology); Rotenone (sc‐203242, Santa Cruz Biotechnology); Cyt.C (C2037, Sigma‐Aldrich) CB‐839 (10‐4556, Focus Biomolecules); Adenosine diphosphate (ADP, A2754, Sigma‐Aldrich).