Fig 2: Loss of INPPL1, PIK3R2 and TSC2 cause drug resistance through re-activation of PI3K-AKT-mTOR pathway signaling.A Bar chart of results from the competitive proliferation assay for INPPL1, TSC2 and PIK3R2 KO and vector control cells. Data are mean ± SD; n = 3. Statistical significance was determined using Dunnett’s multiple comparisons test (****p < 0.0001, ***p < 0.001). B Crystal violet staining of EVSA-T INPPL1, PIK3R2 and TSC2 KO cells treated with DMSO, 250 nM AZD8186 or 1 μM capivasertib for 10 days. Data are representative of three independent experiments. C Western blot analysis of PI3K pathway effectors (pAKT, pPRAS40, pS6, p4E-BP1) in EVSA-T INPPL1, TSC2 and PIK3R2 KO cells treated with AZD8186, capivasertib or DMSO control for 4 h. Data are representative of three independent experiments. D Control and KO cells as indicated were treated with AZD8186 and capivasertib alone and with the addition of 1μM capivasertib, 100 nM Rapamycin, 500 nM AZD2014 and 500 nM AZD8835 combinations. Data are representative of three independent experiments and two independent gRNAs. E, F Plates from (D) were quantified using ImageJ software and % confluency of each well calculated. Data are mean of 3 independent experiments ± SD. All images shown are from the same blots at the same exposures, panels for each marker have been separated to aid visualisation, but images for each marker are comparable between KO cell lines and treatments.