Autophagy related RHEB-CSF1R complex promotes tumor metastasis via advancing phosphorylation levels of PI3K, AKT, mTOR in pancreatic cancer

Figure 8 Ras homolog enriched in brain-colony stimulating factor 1 receptor complex may inhibit autophagy by advancing phosphorylation levels of phosphatidylinositol 3-kinase, AKT serine/threonine kinase 1, and mammalian target of rapamycin. A and B: Representative images and quantitative analyses of autophagic vacuoles (autophagosomes and autolysosomes) in subsets of 3 randomly-selected cells of the Capan-1 and CFPAC-1 cells transfected with the short hairpin negative control or the short hairpin Ras homolog enriched in brain (RHEB); in Colo357 cells transfected with the vector or the RHEB by using transmission electron microscopy; C-F: Immunofluorescence staining of RHEB with microtubule-associated protein 1 light chain 3 in Capan-1, CFPAC-1, and Colo357 cells; G-J: Western blot analyses of phosphatidylinositol 3-kinase (PI3K), p-PI3K, AKT serine/threonine kinase 1 (AKT), p-AKT (p-PI3K corresponds to the Tyr524 phosphorylation site of PI3K; p-AKT corresponds to the Ser473 phosphorylation site of AKT), mammalian target of rapamycin and p-mammalian target of rapamycin, and the autophagy biomarkers, Beclin 1, autophagy related 5 and microtubule-associated protein 1 light chain 3 in the short hairpin negative control and the short hairpin RHEB transfected the Capan-1 and CFPAC-1 cells with PI3K activator 740 Y-P, and the control vector and the RHEB overexpression vector transfected the Colo357 cells with PI3K inhibitor LY294002. DMSO was used as controls. ^aP < 0.05, ^bP < 0.01, and ^cP value < 0.001. shNC: Short hairpin negative control; shRHEB: Short hairpin Ras homolog enriched in brain; LC3: Microtubule-associated protein 1 light chain 3; RHEB: Ras homolog enriched in brain; PI3K: Phosphatidylinositol 3-kinase; AKT: AKT serine/threonine kinase 1; mTOR: Mammalian target of rapamycin; ATG5: Autophagy related 5; LC3: Microtubule-associated protein 1 light chain 3; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; shNC: Short hairpin negative control; shRHEB: Short hairpin Ras homolog enriched in brain; ns: No significance.