EF2: Impact of the feedback levels of insulin observed in figure 1 upon BKM120 efficacy in vitro.

(A) Proliferation in minimal growth media of cells whose growth is partially rescued by the addition of the observed feedback levels of insulin (10ng/ml) induced by BKM120 in mice. N=3 biologically independent samples per arm, graphed as mean number of cells with standard deviation. (B) Cell viability assay demonstrating the effects that these feedback levels of insulin have upon 2 patient (Pt)-derived organoid cultures (A and B) being treated in a dose response with BKM120 as measured by cell titer-glo at 96 hours. N = 3 biologically independent samples per treatment. (C) Proliferation in minimal growth media of murine TNBC cells treated with PI3K inhibitors partially rescued by the addition of the observed feedback levels of insulin induced by BKM120 in mice as observed in figure 1. N =6 biologically independent samples per treatment graphed as mean confluence with standard deviation. (D) Proliferation of HCT116-neo cells and (E) HCT116 PTEN knockout (KO) cells with and without treatment with the physiologically observed levels of insulin (10ng/ml) and treatment with clinically relevant PI3K inhibitors GDC-0032 and BYL-719. N = 4 biologically independent samples per treatment graphed as mean confluence with standard deviation. (F) Proliferation of DLD1-Neo and (G) DLD-1 PTEN Knockout cells under the same treatment conditions as in F and G. Of note, the loss of PTEN in these isogenic sets of colon cancer lines does not uniformly alter the response to insulin in the setting of PI3K inhibition. In the context of PTEN loss, physiologic levels of insulin can restore normal proliferation in HCT116s despite the presence of PI3K inhibitors. N = 4 biologically independent samples per treatment graphed as mean confluence with standard deviation.