Extended Data Fig. 7: Assessing the symmetry of g6p labeling pattern using GC-MS.

a, GC-MS fragments of g6p were identified using various g6p standards. The m/z 271 peak was identified to be a fragment encompassing all six carbons of g6p (M + 0). The m/z 204 was determined to be a fragment containing the second and third carbons ([C₂-C₃]) of g6p (M + 0). Meanwhile, m/z 370 was pinpointed as a fragment covering the fourth to the sixth carbons ([C₄-C₆]) of g6p (M + 0). These fragment identities were confirmed based on the mass shifts of various isotope-labeled standards as shown in different peak colors. b, Isotopologue distributions of various metabolites from identical [U-¹³C]glucose tracing samples (collected at 6, 9 and 12 h post tracing) were compared using LC-MS and GC-MS analyses. Each bar represents the mean ± s.d. of experimental data and each dot represents a single time point in a biological replicate (n = 3). Shapes indicate biological replicates. The results show that the data obtained from GC-MS are consistent with data from LC-MS, evidenced by the similar levels in average labeling and their variances across replicates. c, The left cartoon illustrates that in the absence of cyclic PPP flux, the fraction of [C2-C3] (M1) is expected to be lower than or equal to that of [C4-C6] (M1 + M2). Detailed reasoning can be found in Supplementary Methods. The bar plot shows that gene knockdown in the PPP, but not in the glycolysis/gluconeogenesis pathway, markedly affects the asymmetric labeling pattern of g6p. Each bar represents the mean ± s.d. of experimental data and each dot indicates a biological replicate collected at 9 h post tracing. Sample sizes: n = 10 (vector), n = 4 (enol-1), n = 6 (dlat-1), n = 6 (T25B9.9) and n = 8 (tald-1). The P values were derived by comparing data between each red and blue bar (P = 5.50 × 10⁻¹² (vector), P = 4.74 × 10⁻⁴ (enol-1), P = 4.15 × 10⁻¹⁰ (dlat-1), P = 2.38 × 10⁻³ (tald-1), and P = 9.29 × 10⁻¹ (T25B9.9)).

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