Fig 2: Inhibitory effect of everolimus and ABT-737 combination on cell cycle and mTORC1 complex. (A) A-498 cells were treated either with 1 µM everolimus and/or 5 µM ABT-737 and Caki-1 cells were exposed to 1 µM everolimus and/or 10 µM ABT-737. After 24 hours of treatment, protein extracts of A-498 and Caki-1 cells were used to determine the protein levels of CDK4, Cyclin D1, Cyclin D3, CDK2, Cyclin E1, and p27Kip1 by Western blot analysis. ß-Actin was used as loading control. (B) Effect of the combination treatment on mTOR pathway was investigated by the determination of protein levels of p-mTOR (Ser2448), mTOR, p-p70S6K (Thr389), p70S6K, p-S6 (Ser235/236), p-S6 (Ser240/244), S6, p-4E-BP1 (Ser65), and 4E-BP1 in A-498 cells treated with 1 µM everolimus and/or 5 µM ABT-737, and Caki-1 cells exposed to 1 µM everolimus and/or 10 µM ABT-737 for 24 hours. ß-Actin was used as loading control.

Fig 3: Proposed Model of the CXCR4-LASP1-eIF4F Axis. An illustration of CXCR4 and its relation to the eIF4F complex upon stimulation with CXCL12. This relationship is occurring through two distinctive mechanisms. First, LASP1 dissociates from CXCR4 and directly interacts with eIF4A and eIF4B. Second, phosphorylation of PDCD4, 4E-BP1, and eIF4B is promoted through G protein-coupled receptor signaling. As a result, both complex formation increases along with the function of eIF4A. Consequently, the translation of oncogenic proteins is promoted.

Fig 4: Protein kinases phosphorylating 4E-BP1 in the oocytes. (A) Detection of 4E-BP1 by immunoblotting in the oocytes treated with specific inhibitors Rapa (100 nM), Rosco (10 µM), or BI2536 (100 nM) post-NEBD. Arrowhead marks the presence of upper band (phosphorylation shift) of 4E-BP1 in the oocytes treated for 2 h post-NEBD, GAPDH was used as a loading control, a typical experiment from at least 3 replicates is shown. (B) Quantification of non-phosphorylated and phosphorylated form of 4E-BP1 in the post NEBD oocytes. Data are presented as mean ± SD, Student's t-test, NS = non-significant. (C) CDK1 effect on mTOR phosphorylation in the oocytes treated by Rosco (10 µM) or OA (1 µM). Immunoblot was probed with mTOR(Ser2448) and control (mTOR and GAPDH) antibodies. Twenty oocytes were used per sample. (D) Presence of mTOR(Ser2448) normalized to the mTOR in the Rosco or OA treated oocytes. Data are presented as mean ± SD, Student's t-test. (E) Localization of mTOR(Ser2448) and CDK1 in the post NEBD and pro-MI stage oocytes, n = 30, phospho-specific antibody (green) and DNA (blue). Scale bar = 20 µm.

Fig 5: Corosolic acid activates AMPK signaling in SNU-620/5-FUR cells. (A) Structure of corosolic acid [(2a,3ß)-2,3-Dihydroxyurs-12-en-28-oic acid, 2a-Hydroxyursolic acid]. (B) Cells were incubated with corosolic acid for 24 h and equal amounts of protein samples were resolved by SDS-PAGE followed by immunoblotting using antibodies against AMPK, p-AMPK, mTOR, p-mTOR, 4EBP1, and P-4EBP1. GAPDH expression served as an internal control. (C) Cell growth inhibition was determined by Cell Counting Kit-8 assay. Values are presented as the mean ± standard deviation of three independent experiments. *P<0.05, as indicated. AMPK, adenosine monophosphate-activated protein kinase; 5-FU, 5-fluorouracil; SNU-620/5-FUR, 5-FU-resistant gastric cancer cell line; TS, thymidine synthase; p-, phosphorylated; mTOR, mammalian target of rapamycin; 4EBP1, 4E-binding protein 1.