Figure 3.

Total lysates of LAPC9 and BM18 organoids treated with rapalink-1 (RL, red bars), rapamycin (RM, blue bars), everolimus (EV, black bars), disulfiram (DS, green bars), or vehicle (V, DMSO, open bars), at the reported concentrations (μM) for 48 h were fractionated by SDS-PAGE followed by Western blotting. Phosphorylated sites or total proteins were detected by immunoblotting using antibodies against the indicated phosphosites or protein. Molecular weight (MW) marker sizes are indicated on the left. Beta-actin was used as loading control. (A) The activation status of mTOR was assessed by analyzing the expression of the mTORC1 downstream targets ULK (p-ULK1, S757) and S6 (pS6, S240/244) and of the mTORC2 downstream target Akt (p-Akt, S473). (B) Quantification of the phosphosites analyzed in (A). (C) The lysates analyzed in (A) were further assayed for the activation of the hexosamine biosynthesis pathway (GFAT1; p-GFAT1, S243; NAGK) and of glutamine (GS, GLS), nucleotide (CAD), glucose (HK II), and lipid (ACC) metabolism. (D) Quantification of the targets assayed in (C). Glutamine:fructose 6-phosphate Amidotransferase, GFAT1; Glutamine synthetase, GS; Hexokinase II, HK II; acetyl-CoA carboxylase, ACC; Glutaminase, GLS; carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, dihydroorotase, CAD; N-acetyl-Dglucosamine kinase, NAGK.