(A) Expression of MTR in the HCT116, 8988T, and HepG2 cell lines as reported in the Cancer Cell Line Encyclopedia[63]. (B) Cell growth curves in the media containing indicated folate sources (mean ± SD, n = 2). (C) Cell growth curves in media containing indicated folate and methionine concentrations (mean ± SD, n = 3). (D) Individual tumor volumes for HCT116 xenografts in female CD-1 nude mice (n = 10 mice). (E) Terminal tumor mass of HCT116 xenografts in female CD-1 nude mice (mean ± SEM, n = 10 mice). P values were determined by a two-sided paired Student’s t-test comparing ΔMTR-1 to wild-type, and ΔMTR-2 to CRISPR control (control-1). (F) Growth of subcutaneous HCT116 xenografts in male CD-1 nude mice on a standard folate (4ppm) or low folate diet (mean ± SEM, n = 10 mice). (G) Western blot analysis of MTR and eGFP in HCT116 wild-type (WT), CRISPR control-1 or ΔMTR-1 which was also engineered to express a vector containing either eGFP or MTR cDNA. Loading control (COXIV) was analyzed on a separate gel from parallel experiments. Results are representative of 2 independent biological replicates with similar results.