PIK3CD mutant transfectants treated with leniolisib or an mTOR inhibitor. Rat-1 fibroblasts were transfected with human WT (wt PI3K) p110δ or p110δ carrying published APDS mutations N334K, E525K, E1021K, and C416R. The pAKT(S473) levels in the presence of titrated concentrations of leniolisib (A) and the mTOR inhibitor everolimus (B) are shown as individual data points with interpolated data for leniolisib and linear regression for everolimus. (C) Western blot for PI3K, pAKT, and β-actin as loading control. (D-E) Quantification of the western blot from panel C. Arbitrary units of scanning density for PI3K or pAKT were calibrated for loading control by division with the corresponding scanning density of β-actin. Average variation over 6 independent experiments is <5% pAKT arbitrary units (AU) for mutant and wt PI3K. (F) Fifty percent inhibitory concentration (IC₅₀) values with SD derived from 6 independent experiments.