Extended Data Fig. 3. Additional data on 1938-driven signalling.

a, MEFs were stimulated at different time points with 1938 (5 μM) or for 2 min with PDGF (20 ng/ml), followed by lipid extraction and PI(3,4)P₂ measurement by mass spectrometry. b, MEFs were stimulated for 2 min with 1938 (30 μM) or PDGF (0.5 or 1 ng/ml), followed by lipid extraction and PI(3,4)P₂ measurement by mass spectrometry. (a,b) n=independent experiments, shown in figure. Error bars represent SD. c, Control TIRF microscopy data from DMSO-treated HeLa cells expressing the PIP₃ or the PI(3,4)P₂ reporter. HeLa cells expressing the EGFP-tagged PIP₃ reporter PH-ARNO-I303Ex2 (ARNO) (black lines) or the PI(3,4)P₂ reporter mCherry-cPH-TAPP1x3 (blue lines) were stimulated with DMSO as indicated. Overlay plots (mean ± SEM) were generated by scaling to minimum and maximum values of the normalised fluorescence intensity for each time point (Fn(t)). PIP₃ reporter data are representative of 2 experiments and 16 single cells. PI(3,4)P₂ reporter data are representative of 4 experiments and 28 single cells. Individual measurements were acquired every 2 min. d, pAKT^S473 induction by 1938 in PI3Kα-KO MEFs transiently transfected with p110α-WT or p110α-mutants. Blot representative of n=2 experiments. e, Time course analysis of 1938-induced pAKT^S473 in A549 by 1938+BYL719 or a saturating insulin concentration. Blot representative of n=3 experiments. f, Time course analysis of 1938-induced pAKT^S473 and pS6^S240/244 in MCF10A cells in the presence or absence of BYL719. Shown is a representative blot of n=2 independent experiments. g, Time course analysis of insulin- or 1938-induced PI3K/AKT/mTORC1 signalling in A549, n=2 experiments.