Figure 5. Mitochondrial OXPHOS dysfunction causes upregulation of de novo serine synthesis in both non-transformed crypts and adenomas from mice.

a: Heat map showing differential gene expression in non-transformed crypt homogenates from the small intestines of PolgA^+/+ and PolgA^mut/mut mice (n=4 mice per group). b: Mean relative expression of the SSP genes by RT-PCR which were identified to be upregulated by RNASeq analysis in normal crypts. n=7 mice per group, one-way Mann-Whitney U test, p=0.0003 for all genes except Slc1a4 where p=0.0035 c: Mean relative expression of the SSP genes by RT-PCR which were identified to be upregulated by RNASeq analysis in laser-microdissected adenomas. n=6 mice per group, one-way Mann-Whitney U test, p values as follows: Phgdh p=0.0325, Psat1 p=0.066, Psph p=0.0130, Aldh1l2 p=0.1548, Mthfd2 p=0.0043, error bars show s.e.m. d-e: Immunohistochemistry images showing in situ levels of SSP proteins in the non-transformed normal small intestinal mucosa (d), and adenomas (e). Immunohistochemistry was performed on n=4 mice per genotype and representative images are shown. Scale bars 50µm. f: Organoids were generated from n=3 PolgA^mut/mut;Apc^fl/fl and n=3 Apc^fl/fl mice. Representative images of adenoma organoids are shown. Scale bars 100µm. g: Oxygen consumption rates measured by Seahorse analysis in adenoma organoids (n=3 mice per genotype, 8 technical replicates per mouse, mean ±s.e.m per mouse are shown). h: Quantification of major mass isotopomers detected in adenoma organoids following growth in the presence of ¹³C₆-glucose for 24 hours. ¹³C labelling is shown as M+3 (serine) or M+2 (glycine). n=3 mice per group with 3 technical replicates performed per mouse (one-way unpaired t-test, p=0.0143 for labelled serine and p=0.0151 for labelled glycine, data are mean per mouse ±s.e.m). i: Quantification of the growth of adenoma organoids in medium with (+SG) or without (-SG) serine and glycine for 5 days. Data are normalised to organoid area on day 0. Mean organoid size per mouse relative to day 0 ± s.e.m is shown, n=3 mice per group, unpaired, two-tailed t-test, p=0.4140 for Apc^fl/fl and p=0.0021 for PolgA^mut/mut;Apc^fl/fl. For all panels: * p<0.05, **p<0.01, ***p<0.001.