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. 2023 Dec 8;5(12):2148–2168. doi: 10.1038/s42255-023-00936-2

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 6 — a. Schematic of DOX-inducible iCas9-GFP - NTCs, SLC6A14/12A4 or SLC6A14/25A15 gRNAs cell line generation. b. Left; mRNA expression levels of SLC6A14 and SLC12A4 genes in HCT116 PHGDH-depleted cells expressing iCas9-GFP; NTC or SLC6A14/SLC12A4 gRNAs following 72 hours of DOX. Right; mRNA expression levels of SLC6A14 and SLC25A15 genes in HCT116 PHGDH-depleted cells expressing iCas9-GFP; NTC or SLC6A14/SLC25A15 gRNAs following 72 hours of DOX. Values are mean±s.d. from n = 3 independent experiments. Statistical significance assessed with two-tailed one-sample t-test on natural log-transformed values. c. Cell number (Ratio to DO) over time (hours) of HCT116 PHGDH-depleted cells expressing iCas9-GFP; NTC or SLC6A14/SLC12A4 gRNAs (left) or NTC or SLC6A14/SLC25A15 gRNAs (right) following 72 hours of DOX. Values are mean±s.d. from n = 3 biological replicates. d. [¹³C₃,¹⁵N]L-Serine consumption in DOX-inducible iCas9-GFP; PHGDH-depleted HCT116 cells expressing either a combination of NTCs, SLC6A14/12A4 or SLC6A14/25A15 gRNAs and treated with ±DOX for 48 hr and FACS sorted for selecting GFPneg and GFPpos populations. Values are mean±s.d. from n = 3 biological replicates. Statistical significance was assessed with ordinary one-way analysis of variance (ANOVA) and Dunnett’s multiple comparisons test comparing the GFPpos ( + DOX 48 hr) group. e. Left; Tumor volume over time of tumors from PHGDH-depleted HCT116 cells expressing SLC6A14/25A15 gRNAs ±DOX. Values are mean±s.e.m. from n = 9 -DOX and n = 11 + DOX mice. Statistical significance was assessed with two-tailed unpaired t-test. Right; Kaplan–Meier plot showing probability of survival on mice from left. Statistical significance was assessed with Gehan–Breslow-Wilcoxon test. f. Leucine levels in tumors formed by PHGDH-depleted HCT116 cells expressing NTC, SLC6A14/SLC12A4 or SLC6A14/SLC25A15 gRNAs ±DOX induction. g. Leucine levels in circulating blood serum of mice with tumors formed by PHGDH-depleted HCT116 cells expressing NTC, SLC6A14/SLC12A4 or SLC6A14/SLC25A15 gRNAs ±DOX induction. In f, g: Values are mean±s.d. from n = 6 mice per condition. h. Levels of detected amino acids, glucose, lactic acid and ornithine in tumors formed by PHGDH-depleted HCT116 cells expressing NTC, SLC6A14/SLC12A4 or SLC6A14/SLC25A15 gRNAs ±DOX induction. i. Levels of detected amino acids, glucose, lactic acid and ornithine in circulating blood serum of mice with tumors formed by PHGDH-depleted HCT116 cells expressing NTC, SLC6A14/SLC12A4 or SLC6A14/SLC25A15 gRNAs ±DOX induction. In h, i: Values are log-scale of mean±s.d. from n = 6 mice per condition.