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. 2019 Jul 9;9(18):5246–5260. doi: 10.7150/thno.34713

Figure 5.

Figure 5

APLN activates PI3K/Akt signaling. (A) Nine cancer pathway- related firefly luciferase reporter constructs were co-transfected with renilla luciferase reporter (internal control) in HKCI-10 cells. (B) BYL-719 (4uM) and GDC-0941 (1uM), two specific PI3K/Akt Inhibitors, were added to HKCI-2 or HKCI-10 cells. Control cells were treated with an equivalent dilution of DMSO. MTT assays were performed. (C) Upregulation of APLN increased phospho-PI3K p85, phospho-Akt, phospho-GSK3β and Cyclin D1 expression by western blot analysis. (D) APLN knockdown decreased the expression of phospho-PI3K p85, phospho-Akt, phospho-GSK3β and Cyclin D1 by western blot analysis. (E) Top panel showed APLN overexpression was associated with the increased expression of phospho-Akt and phospho-GSK3β in human HCC specimens. Bottom panel demonstrated positive correlation of APLN with PI3K-AKT pathway in TCGA-LIHC cohort by gene set enrichment analysis. (F) HKCI-2 and HKCI-10 cells were serum starved overnight and stimulated with 1uM Apelin-13 for indicated time. For ML221 treatment, HKCI-2 and HKCI-10 cells were pretreated with 50µM ML221 for 30min before Apelin-13 stimulation. Cell lysates were blotted with indicated antibodies. The images are representative of two independent experiments. Error bars in A and B represent means ± sd from three independent experiments, (* P < 0.05, ** P < 0.01, *** P < 0.001).