Extended Data Fig. 6: Pik3ca^H1047R/wt cells activate Hif1α pathway and glycolysis.

a-b, RNA-seq analysis comparing Pik3ca^wt/wt (WT, uninduced) and Pik3ca^H1047R/wt mutant cultures from the same animal, in minimal medium. Heatmap shows altered transcripts (adjusted p < 0.05). n = 4 mice per group, paired induced-uninduced samples. Wald test corrected for multiple testing. c-f, Gene set enrichment analysis histograms of PI3K/Akt/mTOR (c), mTOR (d) Hypoxia (e) and myc targets (f) signaling Hallmark gene sets in WT and mutant cultures. Nominal p-value, normalized enrichment score (NES) and false discovery rate (FDR) q-value are indicated. g, KEGG pathway enrichment analysis for up-regulated genes in mutant vs WT (p-value adjusted for multiple hypotheses testing using the Benjamini-Hochberg method<0.01). Intensity indicates pathway enrichment. h, Proportion of HIF1α target genes significantly up-regulated (adjusted p-value (as in g)<0.01) in mutant vs WT. i, Transcripts per million (TPM) relative to WT CTL condition of Hif1a and Vegfa in Pik3ca^wt/wt and Pik3ca^H1047R/wt cells in control (CTL) versus cultures treated with 5 μg/ml insulin (INS). n = 4 biological replicates per condition. Red lines, median values. Wald test corrected for multiple testing. j, Uninduced (Pik3ca^wt/wt) and induced (Pik3ca^H1047R/wt) cells were cultured in starvation media (STV) +/− PI3K inhibitor LY294002 (0.5 µM), and HIF1α/α-Tubulin analyzed by immune capillary electrophoresis. HIF1α/α-Tubulin ratio versus the average of the WT STV shown. Two-tailed ratio paired t-test. n = 5 paired biological replicates per condition (lines link paired samples). k, GSEA histogram of Glycolysis Hallmark gene set comparing induced Pik3ca^H1047R/wt and WT cells in minimal medium. n = 4 biological replicates per condition. Statistics as in c-f. l, Heatmaps comparing WT and Pik3ca^H1047R/wt cultures in CTL and INS conditions. Two-tailed Wald test corrected for multiple testing comparing WT and Pik3ca^H1047R/wt CTL conditions. ***p < 0.001, n.s.= not significant. m, GSEA histograms of Glycolysis Hallmark set comparing control (CTL) and 5 μg/ml insulin (+INS) treated wild-type cells. n = 4 biological replicates per condition. Statistics as in c-f. n, Basal OCR to ECAR ratios of WT and Pik3ca^H1047R/wt cultures in CTL or INS conditions. Dots, average per animal (n = 4 mice). OCR/ECAR ratios are mean ± SD. Two-tailed paired t-test.

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