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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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Fig. 2 — a, Schematic of serine synthesis pathway. b, Immunoblots of PHGDH, PSAT1, PSPH and ACTIN (loading control) in control (NTC) or PHGDH-depleted HCT116 cells (left). Blots are representative of three independent experiments. Densitometric quantification of protein (right). Values are mean ± s.d. from n = 3 independent experiments. Statistical significance assessed by two-tailed one-sample t-test on natural log-transformed values. c, Growth curves of HCT116 NTC (control) or PHGDH-depleted cells in medium ± SG (left). SG, serine and glycine. Growth curves of HCT116 PHGDH-depleted cells in medium ± serine and/or glycine ± formic acid (FA) (right). Values are mean ± s.e.m. from n = 3 biological replicates and relative to t = 0 (D0). d, Schematic of [U-¹³C₆]d-glucose administration to monitor serine synthesis. e, Incorporation of glucose-derived, labeled carbons into 3-phosphoglycerate (3-PG) and serine in HCT116 NTC (control) or PHGDH-depleted cells following 15 min and 180 min incubation in medium supplemented with [U-¹³C₆]d-glucose. Values are mean ± s.d. from n = 3 biological replicates. f, Schematic of [¹³C₃,¹⁵N]l-serine administration to monitor serine uptake. g, Serine levels in HCT116 NTC (control) or PHGDH-depleted cells showing [¹³C₃,¹⁵N]l-serine-derived labeled carbon and nitrogen incorporation following 15 min and 180 min incubation in medium supplemented with [¹³C₃,¹⁵N]l-serine. Values are mean ± s.d. from n = 3 biological replicates. h, Messenger RNA expression levels of indicated SLC-encoding genes in HCT116 PHGDH-depleted cells versus HCT116 NTC control. Values are mean ± s.e.m. from n = 3 independent experiments. i, Cell number (ratio to NTC) of HCT116 parental, NTC (CRISPR control) and two PHGDH-depleted cell lines upon knockdown of indicated SLC-encoding genes, after 72 h of growth (left). Values are mean ± s.d. from n = 3 biological replicates. Heat map indicating P values for each condition compared to NTC (control) (right). Statistical significance was assessed by two-tailed one-sample t-test on natural log-transformed values.

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