Fig. 3: Vacuolar protein sorting 33B (VPS33B) interacted with NESG1 through suppressing epidermal growth factor receptor (EGFR)/phosphoinositide-3 kinase (PI3K)/AKT/c-Jun pathway in nasopharyngeal carcinoma cells. | Cell Death & Disease

Fig. 3: Vacuolar protein sorting 33B (VPS33B) interacted with NESG1 through suppressing epidermal growth factor receptor (EGFR)/phosphoinositide-3 kinase (PI3K)/AKT/c-Jun pathway in nasopharyngeal carcinoma cells.

From: VPS33B interacts with NESG1 to modulate EGFR/PI3K/AKT/c-Myc/P53/miR-133a-3p signaling and induce 5-fluorouracil sensitivity in nasopharyngeal carcinoma

Fig. 3

a Confirming the effectiveness of transfection of HA-VPS33B and MYC-NESG1 and the interaction of VPS33B and NESG1 after co-immunoprecipitation in 293T cells transfected with HA-VPS33B and MYC-NESG1 by western blot. b VPS33B co-located with NESG1 in cytoplasm by laser confocal assay. Scale bar: 50 μm. c, d Quantitative polymerase chain reaction (qPCR) (c) and Gel electrophoresis (d) confirmed the amplification of c-Jun-binding sites of NESG1 after chromatin immunoprecipitation using antibody against c-Jun. IgG antibody was used as the negative control. Student’s t test, mean ± SD, **P < 0.01, ***P < 0.001. e Electrophoretic mobility shift assay and supershift assay of c-Jun binding to NESG1 promoter in A549 and H1975 cells. Labeled wild-type probe was incubated without (lane 1) or with (lane 5) cell nuclear proteins in the absence or presence of unlabeled probe (lanes 2–4). Unlabeled wild-type probe (lane 2) and mutant c-Jun probe (lanes 3 and 4) were used to compete with c-Jun binding, each at 100-fold excess. Supershift assay (lane 6) was performed using an anti-c-Jun antibody. f Changes in EGFR, PI3K/AKT/p-PI3K/p-AKT, c-Jun, and NESG1 expression were detected by western blot analysis in VPS33B-overexpressed HONE1 and SUNE1 cells after transfection of EGFR plasmids. β-Actin was used as a loading control. g, h 3-[4,5-Dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (g) and 5-ethynyl-2′-deoxyuridine incorporation (h) assays were performed to demonstrate the impact of silencing NESG1 on the proliferation of VPS33B-overexpressing HONE1 and SUNE1 cells. Student’s t test, one-way analysis of variance, mean ± SD, *P < 0.05, **P < 0.01. Scale bar: 100 μm. i Changes in EGFR, PI3K/AKT/p-PI3K/p-AKT, c-Jun, c-Myc, p53, CCND1, and E2F1 expression were detected by western blot analysis in VPS33B-overexpressed HONE1 and SUNE1 cells after silencing NESG1. β-Actin was used as a loading control. j miR-133a-3p was downregulated in the VPS33B-overexpressing HONE1 and SUNE1 cells after silencing NESG1 by qPCR assay. Student’s t test, mean ± SD, **P < 0.01, ***P < 0.001

Back to article page