armadillo repeats mutant jup  (Addgene inc)

Journal: Journal of Advanced Research

Article Title: Effects of differential distributed-JUP on the malignancy of gastric cancer

doi: 10.1016/j.jare.2020.06.026

Figure Lengend Snippet: Expression and localization of JUP in three differently differentiated gastric cancer cells and tissues. (A) Western blot was done to show protein levels of JUP in NCI-87 (well differentiated GC cells), NUGC-3 (moderately differentiated GC cells), MGC-803 (poorly differentiated GC cells) cells. (B) Subcellular expression levels of JUP and β -catenin in different differentiated gastric cancer cells were analyzed by western blotting. PCNA is the loading control for nuclear proteins. β-tubulin, a loading control of the cytoplasmic proteins. (C) Immunofluorescence staining was performed to detect the expression and localization of JUP in GC cells (Scale bars, 50 µm). (D) IHC staining to detect expression and location of JUP and β -catenin in gastric cancer tissues with different degrees of differentiation (G1, well differentiated, n = 7; G2, moderately differentiated, n = 13; G3, poorly differentiated, n = 10). Representative images of IHC staining are presented in left panel (Scale bars, 100 µm). The ratio of nuclear/cytoplasmic JUP and β -catenin in right panel (* P < 0.05, ** P < 0.01). (E) Western blot to show subcellular levels of JUP and β -catenin in gastric cancer tissues with different degrees of differentiation (G1, G2, and G3). PCNA is the loading control for nuclear proteins. β-tubulin, a loading control of the cytoplasmic proteins. (F) The protein levels of E-cadherin, Vimentin and Fibronectin were detected by Western blotting. GAPDH is the loading control. (G) Cell migration was determined by Transwell assay for NCI-87, NUGC-3 and MGC-803 cells. The experiment was repeated three times (** P < 0.01, vs NCI-87 cells).

Article Snippet: The construct encoding JUP were purchased from Addgene (Plasmid #32228), and the corresponding 11-13 armadillo repeats mutant JUP (Mut JUP ARM11-13) and pcDNA-β-catenin constructs were generated by GenePharma (Shanghai, China).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Immunohistochemistry, Migration, Transwell Assay

Journal: Journal of Advanced Research

Article Title: Effects of differential distributed-JUP on the malignancy of gastric cancer

doi: 10.1016/j.jare.2020.06.026

Figure Lengend Snippet: JUP mediates MMP7 expression via the interaction of JUP/β -catenin/TCF4. (A, B) Levels of phosphorylated β-catenin (p-β-catenin), nuclear β-catenin (n-β-catenin), total β-catenin (T-β-catenin) and MMP7 were determined by Western blot in the JUP knocked-down (A) and overexpressing (B) gastric cancer cells and its control cells. Histone H3 and GAPDH are the loading control. (C, D) Whole-cell lysates from WT MGC-803 (C) and JUP ARM11-13 mutant MGC-803 cell (D) were immunoprecipitated with anti-JUP and anti-β-catenin antibodies. Western blot showed the interaction of JUP, β-catenin and TCF4. IgG was used as a control antibody. (E) 293 T cells were co-transfected with control luciferase reporter or TOP-Flash reporter and indicated constructs, relative reporter activity were measured for TCF4 (a, P < 0.01 vs vector; b, P < 0.01, β -catenin/JUP vs β -catenin alone; c, P < 0.01, β -catenin/mutant JUP vs β -catenin/JUP). (F) NUGC-3 and MGC-803 and their engineered cells (JUP-knocked down cells and ectopic JUP-overexpressed cells) were transfected with control luciferase reporter or TOP-Flash reporter. Endogenous TCF4 transcript activity was detected using luciferase assay (* P < 0.05, ** P < 0.01 vs Control reporter; a, P < 0.01, TOP-Flash reporter vs control reporter; b, P < 0.01, MGC-803 vs NUGC-3).

Article Snippet: The construct encoding JUP were purchased from Addgene (Plasmid #32228), and the corresponding 11-13 armadillo repeats mutant JUP (Mut JUP ARM11-13) and pcDNA-β-catenin constructs were generated by GenePharma (Shanghai, China).

Techniques: Expressing, Western Blot, Mutagenesis, Immunoprecipitation, Transfection, Luciferase, Construct, Activity Assay, Plasmid Preparation

Journal: Journal of Advanced Research

Article Title: Effects of differential distributed-JUP on the malignancy of gastric cancer

doi: 10.1016/j.jare.2020.06.026

Figure Lengend Snippet: JUP regulates the activity of EGFR/AKT/GSK3β to stable β-catenin. (A) A network to show the interaction of JUP and other proteins identified by TOF-MS. Red represents JUP, blue represents EGFR, green represents GSK3β. (B) The correlation coefficient of JUP and EGFR was calculated by Pearson’s correlation analysis. (C) JUP co-localizing with EGFR at cellular membrane in NCI-87 was determined by immunofluorescence (Green: JUP; Red: EGFR; Blue: DAPI. Scale bars, 50 µm). (D) IP-Western blot to show the interaction of JUP and EGFR in cell lysates of NCI-87. IgG was used as a control antibody. (E, F) Western blotting was used to determine the protein levels of p-EGFR, T-EGFR, p-AKT, T-AKT, p-GSK3 β and T-GSK3 β in the indicated cells. NCI-87 with shJUP was treated with EGFR inhibitor, PD153035 (10 μmol/L); NUGC-3 and MGC-803 with ectopic JUP were treated with IGF-1 (100 ng/mL). GAPDH was used as a loading control. (G) The total and phosphorylated EGFR, AKT, and GSK3β levels in representative gastric cancer tissues with different degrees of differentiation (G1, G2 and G3) were analyzed by western blotting. GAPDH was used as a loading control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The construct encoding JUP were purchased from Addgene (Plasmid #32228), and the corresponding 11-13 armadillo repeats mutant JUP (Mut JUP ARM11-13) and pcDNA-β-catenin constructs were generated by GenePharma (Shanghai, China).

Techniques: Activity Assay, Immunofluorescence, Western Blot

Journal: Journal of Advanced Research

Article Title: Effects of differential distributed-JUP on the malignancy of gastric cancer

doi: 10.1016/j.jare.2020.06.026

Figure Lengend Snippet: A working model of different distributed-JUP in regulating cell invasion via activation of β-catenin in gastric cancer cells. The proposed model shows the role of JUP in differently differentiated GC cells. In the well differentiated GC cells (left panel), JUP locates at cell membrane and links with E-cadherin and -catenin to block activation of EGFR and its downstream signaling. In moderately differentiated GC cells (middle panel), loss of partial membrane JUP leads phosphorylated EGFR and activation of downstream AKT/GSK3β/β-catenin signaling. In the poorly differentiated GC cells (right panel), complete loss of membrane JUP triggers an enhanced EGFR/AKT/GSK3β/β-catenin signaling, and location of JUP in nuclear, which collaborates with nuclear β-catenin, further promotes MMP7 expression and cell invasion potential.

Article Snippet: The construct encoding JUP were purchased from Addgene (Plasmid #32228), and the corresponding 11-13 armadillo repeats mutant JUP (Mut JUP ARM11-13) and pcDNA-β-catenin constructs were generated by GenePharma (Shanghai, China).

Techniques: Activation Assay, Blocking Assay, Expressing