Fig 1: Depletion of pyrimidine de novo synthesis enzymes activates autophagy.A, schematic representation of the pyrimidine synthesis pathways. B, a LentiCRISPR-mediated bulk knockout was performed with GFP-LC3-RFP-expressing HEK293T cells using two independent sgRNAs, each for CAD and DHODH. Infected cells were selected using puromycin. The ratio of the autophagy-activated population to the total population was measured using flow cytometry after at least 1 week of culture after infection. C, immunoblotting of LentiCRISPR-mediated bulk knockout HEK293T GFP-LC3-RFP cells. 5-FU, 5-fluorouracil; CAD, carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase; DHODH, dihydroorotate dehydrogenase; HEK, human embryonic kidney; LC3, microtubule-associated protein 1 light chain 3; sgRNA, single guide RNA; TYMS, thymidylate synthase.