Extended Data Fig. 1: Generation of mice expressing a Pik3ca^H1047R mutation.

a, Schematic of the conditional targeted Pik3ca allele. Pik3ca exon 20 was flanked by loxP sites (triangles). The duplicate region of exon 20 encodes the H1047R mutation, a self-cleaving T2A peptide and an enhanced Yellow Fluorescent Protein (EYFP), followed by a nuclear localization signal (NLS). Wild-type p110α protein is expressed until Cre mediated recombination, after which the allele co-expresses p110α^H1047R mutant protein and EYFP-NLS. Cre recombination was mediated by crossing the conditional mutant strain with AhCre^ERT mice which express the Cre recombinase upon induction with β-naphthoflavone (BNF) and tamoxifen (TAM). b, Typical top-down confocal image of esophageal epithelial whole-mount from an Ahcre^ERTPik3ca^H1047R/wt mouse, 3 months post-induction. Optical section through basal cell layer is shown. Pik3ca^H1047R/wt clones, showing nuclear EYFP (green) indicated by white arrows. Nuclei are stained with DAPI (blue). Clones are delimited by dashed white lines. Scale bar, 20 μm. c, Immunoblots of protein expression of p110α, GFP and AKT (total and, phosphorylated Ser⁴⁷³ (top) or Thr³⁰⁸ (bottom)) in NIH3T3 cells transfected with empty vector, or vector carrying wild-type p110α, p110α^H1047R or p110α^H1047R -P2A-GFP. Cells were starved for 24 h and lysed. Images are representative of 3 separate experiments. d-f, Signaling in induced mutant cells. Protocol (d): primary esophageal keratinocytes from Pik3ca^H1047R/wt mice were infected either with null-adenovirus (uninduced Pik3ca^wt/wt controls) or Cre-adenovirus (induced Pik3ca^H1047R/wt). Cells were treated in medium containing either 0.1% serum and no added growth factors (Starved, STV), 20% serum and growth factors (FCS) or FCS and the PI3K inhibitor LY294002 (50 µM), lysed and the pAKT(Ser473)/Total-AKT (e) and p-PRAS40/Total-PRAS40 (f) analyzed by immune capillary electrophoresis. Two-tailed ratio paired t-test. n = 4 paired (Ad-Null and Ad-Cre infected) biological replicates (mice) per condition (paired samples are linked by lines). Each dot is a biological replicate culture, lines link uninduced and induced cultures derived from the same mouse.

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