Extended Data Fig. 4: Vitamin B12 and 1-carbon (1 C) metabolism markers during in vivo reprogramming.

(a) Schematic of methylmalonyl-coA mutase (MUT) enzymatic activity in the mitochondria, Figure adapted from ref. ²⁶, Springer Nature Limited. TCA: tricarboxylic acid. (b) Metabolites of the Gly/Ser/Thr metabolism pathway (KEGG map00260) identified in the serum of WT and OSKM mice (n = 6 mice per genotype; 3 M 3 F) during a time course of doxycycline administration. Fold change (FC) of the peak area of the indicated metabolites as detected by mass spectrometry is shown relative to (D0) for the respective groups (that is WT and OSKM). P-values represent significant difference between OSKM and WT mice on the indicated day. Serum was collected repeatedly from the same mice. (c) WT and OSKM mice (n = 5 per group; 5 M) were treated with doxycycline and given a bolus of vitamin B12 on day 6, as schematized in Fig. 2f. Serum (repeated measurements) was analyzed by mass spectrometry prior to doxycycline (day 0), prior to the B12 bolus (day 6), and 24 hours after the bolus (day 7). The ratio of SAM/SAH abundance (upper) and total abundance of homocysteine (Hcy, lower) are shown. P-value represents comparison of WT vs. OSKM by two-way ANOVA. (d) Expression of Bhmt and Cbs in previously published RNA sequencing data from n = 2 paired parental MEFs and induced pluripotent stem cells (iPSC), generated by doxycycline-induced OSKM expression. Data source: 40. (e) Serum analysis of betaine in the mice described in panel (b). (f) Expression of methylmalonyl-coA mutase (Mmut) in WT (n = 4) and OSKM (n = 8) mice from Fig. 1b. Mice were treated with doxycycline for 7 days and RT-qPCR was performed in tissues that do (pancreas, stomach, colon; green) and do not (kidney; orange) exhibit histologic signs of reprogramming. Bar graphs represent average ± SD; p-values by two-tailed Student’s t-test unless otherwise indicated.

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