Fig 2: Response to dactolisib is dependent upon TP53 mutation status.(A) Western blotting on lysates from NCI-H358 and NCI-H1299 cells following a cisplatin pulse, with the addition of dactolisib (1 µM), as indicated. (B) Quantification of effects of dactolisib upon cisplatin induced p63 and p21 expression (n = 3, mean ± SD). (C) Western blotting on lysates from NCI-H358 cells, treated with P70S6K or control siRNA, as indicated, prior to and following a cisplatin pulse (n = 3, mean ± SD). (D) Apoptosis measured by propidium iodide staining for the sub-G1 population performed 72 hr following a cisplatin pulse with the addition of dactolisib (1 µM) as indicated (n = 3, mean ± SD). (E) Western blotting on lysates from A549 and NCI-H1573 cells, 48 hr following a cisplatin pulse, with the addition of dactolisib (1 µM), as indicated (n = 3, mean ± SD). (F) Western blotting across a second panel of lung adenocarcinoma cell lines. (G) Correlation between P70S6K phosphorylation and apoptosis, as measured by propidium iodide staining for the sub-G1 population, performed 72 hr following a cisplatin pulse (n = 3, mean ± SD). For all panels the statistical significance was determined by one-way ANOVA (***p<0.001, **p<0.01, *p<0.05).

Fig 3: P70S6K in lung adenocarcinoma.(A) The frequency of somatic mutations and mRNA over-expression for the RPS6KB1, RPS6KB2 and TP53 genes in a publically available cohort of lung adenocarcinoma patients (cBioPortal). (B) The association between RPS6KB1 and RPS6KB2 mRNA expression and overall patient survival in a publically available patient cohort (KM Plotter). Statistical significance was determined by log rank test. (C) Representative images of P70S6K immuno-histochemistry staining from a cohort of 52 lung adenocarcinoma patients (Scale bar = 100 µM). (D) Survival analysis based upon P70S6K staining in this cohort. Statistical significance was determined by log rank test. (E) The association between tumour P70S6K staining intensity (H-Score) and patients that underwent a partial response (PR), or presented with stable disease (SD) or progressive disease (PD). Statistical significance was determined by one-way ANOVA, (F) The association between tumour P70S6K staining intensity and tumour stage. Statistical significance was determined by one-way ANOVA. (G) P70S6K staining in eight matched patient samples from diagnosis and relapse (n = 8). Statistical significance was determined by t-test (*p<0.05).