Fig. 4: NESG1 stimulated the expression of vacuolar protein sorting 33B (VPS33B) via epidermal growth factor receptor (EGFR)/phosphoinositide-3 kinase (PI3K)/AKT/c-Jun signaling. | Cell Death & Disease

Fig. 4: NESG1 stimulated the expression of vacuolar protein sorting 33B (VPS33B) via epidermal growth factor receptor (EGFR)/phosphoinositide-3 kinase (PI3K)/AKT/c-Jun signaling.

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. 4

a The mRNA and protein levels of VPS33B were upregulated in the NESG1-overexpressed HONE1 and SUNE1 cells by quantitative polymerase chain reaction (qPCR). Student’s t test, mean ± SD, *P < 0.05, **P < 0.01. b Bioinformatics analysis revealed the promoter regions of VPS33B with the putative c-Jun-binding site. c qPCR assay indicated that VPS33B was downregulated in the c-Jun-overexpressing HONE1 and SUNE1 cells. Student’s t test, mean ± SD, *P < 0.05, **P < 0.01. d qPCR and Gel electrophoresis confirmed the amplification of c-Jun-binding sites after chromatin immunoprecipitation using antibody against c-Jun. IgG antibody was used as the negative control. Student’s t test, mean ± SD, *P < 0.05, **P < 0.01. e Electrophoretic mobility shift assay and supershift assay of c-Jun binding to VPS33B promoter in HONE1 and SUNE1 cells. Labeled wild-type probe was incubated without (lane 1) or with (lane 4) cell nuclear proteins in the absence or presence of unlabeled probe (lanes 2–3). Unlabeled wild-type probe (lane 2) and mutant c-Jun probe (lane 3) were used to compete with c-Jun binding, each at 100-fold excess. Supershift assay (lane 5) was performed using an anti-c-Jun antibody. f Luciferase reporter assay demonstrated the luciferase activities of the wild type and Mut VPS33B promoter in HONE1 and SUNE1 cells transfected with c-Jun plasmid. Student’s t test, mean ± SD, *P < 0.05, **P < 0.01. g Changes in PI3K/AKT/p-PI3K/p-AKT, c-Jun, and VPS33B expression were detected by western blot analysis in NESG1-overexpressed HONE1 and SUNE1 cells after transduction of EGFR plasmids. β-Actin was used as a loading control. h qPCR assay indicated that VPS33B was downregulated in the NESG1-overexpressing HONE1 and SUNE1 cells after transduction of EGFR plasmids. Student’s t test, mean ± SD, *P < 0.05, **P < 0.01. i The binding of c-Jun with VPS33B promoter was enhanced in the NESG1-overexpressed HONE1 and SUNE1 cells after transfecting with EGFR plasmids. Student’s t test, mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001

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