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

Journal: Nature Communications

Article Title: The Bub1–Plk1 kinase complex promotes spindle checkpoint signalling through Cdc20 phosphorylation

doi: 10.1038/ncomms10818

Figure Lengend Snippet: ( a , b ) Anti-Myc blot of the in-vitro ubiquitination reactions of APC/C Cdc20 using cyclin B1 1–97 –Myc as the substrate and UbcH10 ( a ) or both UbcH10 and Ube2S ( b ) as ubiquitin-conjugating enzymes. Recombinant Cdc20 WT or S92E at different concentrations (16.6, 66.4 and 332 nM) were incubated with APC/C isolated from Xenopus egg extract. ( c ) Quantification of the mitotic index of HeLa Tet-On parental cells and cells stably expressing Flag-Cdc20 WT or S92E that were treated with or without Cdc20 siRNA or Dox (mean±s.d.; n =3 independent experiments). ( d ) Quantification of the mitotic index of HeLa Tet-On cells stably expressing Flag-Cdc20 WT or S92E that were treated with Cdc20 siRNA, Dox and 200 nM taxol in the presence or absence of Bub1 siRNA or BI 2536 (BI) (mean±s.d.; n =3 independent experiments). ( e ) HeLa Tet-On parental cells and cells stably expressing Flag-Cdc20 WT or S92E were treated with the indicated siRNAs and 500 nM nocodazole. Cell lysates were blotted with the indicated antibodies. ( f ) Quantification of the mitotic index of cells in e (mean±s.d.; n =3 independent experiments).

Article Snippet: For protein-binding assays, purified His 6 –Cdc20 ΔN60 WT or the S92E mutant were immobilized on Ni 2+ -NTA beads (QIAGEN).

Techniques: In Vitro, Recombinant, Incubation, Isolation, Stable Transfection, Expressing

Journal: Nature Communications

Article Title: The Bub1–Plk1 kinase complex promotes spindle checkpoint signalling through Cdc20 phosphorylation

doi: 10.1038/ncomms10818

Figure Lengend Snippet: ( a ) HeLa Tet-On cells stably expressing Flag-Cdc20 WT or 5A were arrested in mitosis by 5 μM nocodazole. The cell lysates and the anti-Flag immunoprecipitates (IP) of these cells were blotted with the indicated antibodies. The graphs at the bottom show the quantification of the relative BubR1 and Mad2 signals normalized to that of total Cdc20 in the IP (mean±range; n =2 independent experiments). ( b ) Blots of the input and beads-bound proteins of the binding reactions among the indicated proteins. The graphs at the bottom show the quantification of the relative BubR1 and Mad2 signals normalized to Cdc20. ( c ) Schematic drawing of the experimental design in d and . APC/C is pre-activated with Cdc20 WT or S92E and then incubated with mini-MCC comprising BubR1N, Mad2 in the closed conformation and Cdc20 (WT or S92E). ( d ) Anti-Myc blot of the in-vitro ubiquitination reactions of the indicated APC/C Cdc20 incubated with the indicated proteins and using securin-Myc as the substrate. The asterisk indicates a nonspecific cross-reacting band.

Article Snippet: For protein-binding assays, purified His 6 –Cdc20 ΔN60 WT or the S92E mutant were immobilized on Ni 2+ -NTA beads (QIAGEN).

Techniques: Stable Transfection, Expressing, Binding Assay, Incubation, In Vitro

Journal:

Article Title: C-terminal-binding protein corepresses epithelial and proapoptotic gene expression programs

doi: 10.1073/pnas.0830998100

Figure Lengend Snippet: Epithelial phenotype-related proteins are regulated by CtBP. Equal amounts of total cellular protein from CtBP1,2−/− cells (−/−), cells rescued with vector alone (vec−/−), cells rescued with wild-type CtBP1 [res.(wt)] or the H315Q mutant [res.(H315Q)], or heterozygous control cells (−/+) were assayed for expression of desmoglein-2 (DSG-2), plakoglobin (PG), keratin-8 (K8), E-cadherin (E-cad), or occludin (Occl) by Western blotting. Confirmation of mouse CtBP (left) or human CtBP (right) expression in the mixed populations of CtBP-rescued cells and an actin-loading control is also shown; CtBP expression in >95% of the rescued cells was additionally verified by immunofluorescence (data not shown). (Lower) Epithelial genes indicated by the microarray analysis to be both down-regulated in the CtBP1,2−/+ and the CtBP-rescued cells, relative to the CtBP1,2−/− cells, are shown. The complete microarray results are published as supporting information on the PNAS web site.

Article Snippet: His-tagged CtBP and H315Q mutant were expressed in BL21 (DE3) and purified by Ni-NTA affinity (Qiagen).

Techniques: Plasmid Preparation, Mutagenesis, Control, Expressing, Western Blot, Immunofluorescence, Microarray

Journal:

Article Title: C-terminal-binding protein corepresses epithelial and proapoptotic gene expression programs

doi: 10.1073/pnas.0830998100

Figure Lengend Snippet: Dehydrogenase activity of CtBP is not required for transcriptional repression. (a) The PERP or β-actin control promoters linked to luciferase (1.5 μg) were cotransfected with the indicated CtBP mutants, and activity of the luciferase reporters was assayed. (b) GST-E1a fusion proteins coupled to glutathione-Sepharose beads were incubated with His-tagged human CtBP or H315Q mutant. The bound material was dissolved in sample buffer and analyzed by Western blotting by using an antibody against the His-tag. Equal amounts of GST and GST-E1a proteins were used in each pull-down assay, as shown (Lower). (c) An SV40 promoter adjacent to four gal4-binding sites (1.5 μg) was assayed for repression by the gal4-CtBP fusion proteins (0.5 μg), as indicated.

Article Snippet: His-tagged CtBP and H315Q mutant were expressed in BL21 (DE3) and purified by Ni-NTA affinity (Qiagen).

Techniques: Activity Assay, Control, Luciferase, Incubation, Mutagenesis, Western Blot, Pull Down Assay, Binding Assay