(A) Representative image from western blot analysis of PDGFRa/PI3K/mTOR pathway in HSJD-DIPG007 cells treated with PENAO, temsirolimus and dual therapy for 48 h. Significant decrease is observed in protein levels in PDGFR, PI3K subunits, mTOR and its targets (B) Expression analysis of a panel of genes involved in AMPK pathway in HSJD-DIPG007 cells treated with PENAO, temsirolimus and dual therapy for 48h. Significant increase in mRNA levels of AMPK subunits and its targets Tsc1, and Deptor is observed, (p values for: PRKAB1: V vs C < 0.05, P vs C < 0.05, T vs C < 0.01, PRKAB2: V vs C < 0.01, P vs C < 0.05, T vs C< 0.05; PRKAG1, V vs C ns, P vs C ns, T vs C ns; PRKAG2, V vs C < 0.01, P vs C < 0.05, T vs C < 0.01; PRKAG3, V vs C < 0.05, P vs C < 0.05, T vs C < 0.05; TSC1, V vs C < 0.05, P vs C < 0.05, T vs C < 0.05; DEPTOR, V vs C < 0.01, P vs C < 0.05, T vs C < 0.001; TEL2, V vs C < 0.05, P vs C < 0.05, T vs C < 0.05); ULK1 V vs C < 0.05, P vs C < 0.05, T vs C < 0.05; (C) Representative image from western blot analysis of mTOR regulators AMPK and HSP90 in HSJD-DIPG007 cells treated with PENAO, temsirolimus and dual therapy for 48 h. Significant decrease is observed in phosphorylation of AMPK indicating activation and protein levels of HSP90. (D) ATP determination in HSJD-DIPG007 cells treated with PENAO, temsirolimus and dual therapy for 48h. Significant decrease in ATP levels is observed in PENAO and combination treatments; Experiment performed twice each time N = 5; P vs C p < 0.001, T vs C p < 0.001.