Generation and Characterization of ATF3 Knockout Cells

HT1080 cells expressing ATF3 and empty vector were generated previously [26 (link)]. Retinal pigment epithelial (RPE) cells immortalized with hTERT were obtained from Dr. Todd Waldman [27 (link)]. To generate ATF3-knockout cells, a target sequence (5′-AAAATGATGCTTCAACACCC-3′) spanning the ATF3 start codon was inserted into pSpCas9(BB)-2A-puro, and used to transfect RPE and U2OS cells as described [28 (link)]. ATF3-knockout HCT116 cells were generated using rAAV-mediated homologous recombination as described previously [14 ]. These cells were cultured in media supplemented with 10% fetal bovine serum. We used DMEM for HT1080, RPE, DU145, and U2OS cells, RPMI 1640 for PC3 cells, and McCoy’s 5A for HCT116 cells. Erastin, RSL3, ferrostatin-1, BSO, Z-VAD-FMK, and FIN56 were purchased from Cayman Chemical. N-acetylcysteine (NAC) was purchased from Thermo Fisher Scientific. Other reagents were obtained from Sigma-Aldrich.