A) Western blot of 3 clonal ACLY deficient (KO) cell lines (PC7, PC8, and PC9) generated from Aclyf/f MEFs. B) Proliferation curve of Aclyf/f and ACLY-KO MEFs over 5 days, mean +/− SEM of triplicate wells, statistical significance compared to Aclyf/f. C) Western blot verification of ACLY knockout by CRISPR-Cas9 in LN229 glioblastoma cells. D) Proliferation curve of LN229 and two ACLY-knockout clonal cell lines over 5 days, mean +/− SEM of triplicate wells, statistical significance compared to LN229. E) Western blot of nuclear and cytoplasmic fractions of Aclyf/f, PC9, and reconstituted ACLY-WT and ACLY-H760A PC9 cells. FASN and LMNA are cytoplasmic and nuclear markers, respectively. F) Proliferation curve of Aclyf/f MEF and PC9 lines compared to PC9 reconstituted with ACLY-WT or ACLY-H760A over 5 days, mean +/− SEM of triplicate wells, statistical significance compared to PC9. G) Western blot of ACLY and ACSS2 protein levels in Aclyf/f MEFs over 144 hours following administration of Cre recombinase. H) Western blot of ACLY and ACSS2 protein levels in Aclyf/f MEFs over 144 hours with pharmacological inhibition of ACLY (50 μM BMS-303141). For all panels: **, p<0.01; ***, p<0.001; ****, p<0.0001. See also Figure S1.