Journal: Gland Surgery

Article Title: Silencing of AJAP1 expression by promoter methylation activates the Wnt/β-catenin signaling pathway to promote tumor proliferation and metastasis in salivary adenoid cystic carcinoma

doi: 10.21037/gs-23-127

Figure Lengend Snippet: AJAP1 reduces the nuclear localization of β-catenin by forming the AJAP1/E-cadherin/β-catenin complex. (A-C) The co-IP assays showed that endogenous protein of AJAP1 interacted with β-catenin and E-cadherin; (D) immunofluorescence displayed the co-localization among AJAP1 (45 kDa), E-cadherin (97 kDa), and β-catenin (85 kDa) (×200); (E) AJAP1 can bind to β-catenin in the cytoplasm, thereby reducing the content of β-catenin in the nucleus; (F) AJAP1 upregulation reduced the β-catenin/TCF/LEF-mediated transcription activity in SACC-LM cells; (G) AJAP1 depletion in SACC-83 cells increased the β-catenin/TCF/LEF-mediated transcription activity; (H-J) overexpression/knockdown of AJAP1 significantly up-regulated/down-regulated the expression of β-catenin downstream genes, respectively (AJAP1, 45 kDa; C-myc, 49 kDa; CyclinD1, 33 kDa; MMP1, 54 kDa; GAPDH, 36 kDa). *, P<0.05. IB, immunoblotting; IP, immunoprecipitation; IgG, immunoglobulin G; AJAP1, adherens junctions associated protein 1; DAPI, 4’,6-diamidino-2-phenylindole; SACC, salivary adenoid cystic carcinoma; LM, SACC-LM; NC, normal control; co-IP, co-immunoprecipitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Next, to conduct dual luciferase reporter gene analysis, 2.5×10 5 SACC cells were inoculated into 24-well plates and cultured for 24 h. When the concentration reached 70–80%, the cells were transfected with β-catenin reactive double luciferin reporter plasmid Top-Flash or a negative control FOPFLASH (Merck-Millipore, USA) using the fugene6 (Invitrogen, USA) kit.

Techniques: Co-Immunoprecipitation Assay, Immunofluorescence, Activity Assay, Over Expression, Knockdown, Expressing, Western Blot, Immunoprecipitation, Control

Journal: Gland Surgery

Article Title: Silencing of AJAP1 expression by promoter methylation activates the Wnt/β-catenin signaling pathway to promote tumor proliferation and metastasis in salivary adenoid cystic carcinoma

doi: 10.21037/gs-23-127

Figure Lengend Snippet: Regulation of Wnt/β-catenin signaling pathway could reverse the effect of AJAP1 on the function of SACC cells. (A,B) Wnt/β-catenin agonist 2 could reverse the downregulation of invasion ability of SACC-LM cells induced by AJAP1 overexpression (crystal violet staining, ×200); (C,D) LF3 could reverse the upregulation of invasion ability of SACC-83 cells induced by the knockdown of AJAP1 (crystal violet staining, ×200); (E,F) Wnt/β-catenin agonist 2 could reverse the migration ability downregulation of SACC-LM cells induced by AJAP1 overexpression (white light, high contrast resolution, ×100); (G,H) LF3 could reverse the migration ability upregulation of SACC-83 cells induced by AJAP1 knockdown (white light, high contrast resolution, ×100); (I) Wnt/β-catenin agonist 2 could reverse the proliferative ability downregulation of SACC-LM cells induced by AJAP1 overexpression; (J) LF3 could reverse the proliferative ability upregulation of SACC-83 cells induced by AJAP1 knockdown. *, P<0.05. SACC, salivary adenoid cystic carcinoma; AJAP1 , adherens junctions associated protein 1; Wnt/β-catenin signaling pathway, Wingless/Integrated/β-catenin signaling pathway; OD, optical density.

Article Snippet: Next, to conduct dual luciferase reporter gene analysis, 2.5×10 5 SACC cells were inoculated into 24-well plates and cultured for 24 h. When the concentration reached 70–80%, the cells were transfected with β-catenin reactive double luciferin reporter plasmid Top-Flash or a negative control FOPFLASH (Merck-Millipore, USA) using the fugene6 (Invitrogen, USA) kit.

Techniques: Over Expression, Staining, Knockdown, Migration

Journal: Gland Surgery

Article Title: Silencing of AJAP1 expression by promoter methylation activates the Wnt/β-catenin signaling pathway to promote tumor proliferation and metastasis in salivary adenoid cystic carcinoma

doi: 10.21037/gs-23-127

Figure Lengend Snippet: AJAP1 inhibited the growth and metastasis of SACC tumors in vivo . (A-C) AJAP1 overexpression in SACC-LM cells significantly reduced the subcutaneous tumorigenicity of SACC-LM cells in nude mice; (D-F) AJAP1 knockdown in SACC-83 cells significantly increased subcutaneous tumorigenesis of SACC-83 cells in nude mice; (G,H) immunohistochemical showed AJAP1 overexpression in SACC-LM cells significantly down-regulated the expression of β-catenin downstream genes (×200); (I,J) immunohistochemical showed AJAP1 knockdown significantly up-regulated the expression of β-catenin downstream genes in SACC-83 cells (×200). *, P<0.05. AJAP1 , adherens junctions associated protein 1; SACC, salivary adenoid cystic carcinoma; NC, normal control.

Article Snippet: Next, to conduct dual luciferase reporter gene analysis, 2.5×10 5 SACC cells were inoculated into 24-well plates and cultured for 24 h. When the concentration reached 70–80%, the cells were transfected with β-catenin reactive double luciferin reporter plasmid Top-Flash or a negative control FOPFLASH (Merck-Millipore, USA) using the fugene6 (Invitrogen, USA) kit.

Techniques: In Vivo, Over Expression, Knockdown, Immunohistochemical staining, Expressing, Control

Journal: Cancer Cell International

Article Title: P53 and Beta-Catenin Activity during Estrogen treatment of Osteoblasts

doi: 10.1186/1475-2867-5-24

Figure Lengend Snippet: Increase in beta-catenin expression during E2 treatment does not result in beta-catenin signaling through TCF/LEF response elements in ROS-PG13 cells. Cells were transfected with TopFlash (wild type promoter) and FopFlash (mutant promoter) luciferase reporters in 2% media, and E2 treatment was started three hours later. E2 treatments were staggered during the 48 h interval and all cells were harvested after 48 h after the indicated time of exposure to the hormone. Mutant FopFlash activity was unchanged during the treatment and is not shown. LiCl treatment was carried out to demonstrate the validity of the assay (Inset). Cells were exposed to LiCl or NaCl 24 h after transfection for 16 h. In both these experiments luciferase activity was measured in cell lysates using equal amounts of protein. Experiments represent average ± SEM of triplicate measurements.

Article Snippet: For reporter assays, cells were transfected with the beta-catenin responsive firefly luciferase reporter plasmids TopFlash (wild type promoter) or FopFlash (Mutant promoter) (Upstate Biotechnologies) for 48 h. Three hours after transfection, cells received 17-beta estradiol to a concentration of 10–11 M for the times indicated.

Techniques: Expressing, Transfection, Mutagenesis, Luciferase, Activity Assay

Journal: PLoS Computational Biology

Article Title: Efficient algorithms to discover alterations with complementary functional association in cancer

doi: 10.1371/journal.pcbi.1006802

Figure Lengend Snippet: Comparison of UNCOVER with REVEALER on REVEALER’s datasets.

Article Snippet: In particular we used the following files available through the Supplementary Material of [ ]: CTNBB1_transcriptional _reporter.gct , which consists of measurements of a β -catenin reporter in 81 cell lines; NFE2L2_activation_profile.gct , which includes NFE2L2 enrichment profiles for 182 lung cell lines; MEK_inhibitor_profile.gct , which contains MEK-inhibitor PD-0325901 sensitivity profile in 493 cancer cell lines from the Broad Novartis CCLE14l; and KRAS_essentiality_profile.gct , which corresponds to the feature KRAS from a subset of 100 cell lines from the Achilles project dataset.

Techniques: Activation Assay

Journal: PLoS Computational Biology

Article Title: Efficient algorithms to discover alterations with complementary functional association in cancer

doi: 10.1371/journal.pcbi.1006802

Figure Lengend Snippet: (a) Solution found by ILP and greedy for KRAS essentiality target. (b) Solution found by ILP and greedy for β -catenin activation target. (c) Solution found by ILP for MEK inhibitor target. (d) Solution found by greedy for MEK inhibitor target. (e) Solution found by ILP and greedy for NFE2L2 activation target. Each panel shows the value of the target (top row) for various samples (columns), with yellow being negative and blue being positive values. For each gene in the solution, alterations in each sample are shown in dark blue, while samples not altered are in yellow. The last row shows the alteration profile of the entire solution.

Article Snippet: In particular we used the following files available through the Supplementary Material of [ ]: CTNBB1_transcriptional _reporter.gct , which consists of measurements of a β -catenin reporter in 81 cell lines; NFE2L2_activation_profile.gct , which includes NFE2L2 enrichment profiles for 182 lung cell lines; MEK_inhibitor_profile.gct , which contains MEK-inhibitor PD-0325901 sensitivity profile in 493 cancer cell lines from the Broad Novartis CCLE14l; and KRAS_essentiality_profile.gct , which corresponds to the feature KRAS from a subset of 100 cell lines from the Achilles project dataset.

Techniques: Activation Assay

Journal: Iubmb Life

Article Title: Hypoxia inducible factor 3‐alpha promotes a malignant phenotype in colorectal cancer cells

doi: 10.1002/iub.70007

Figure Lengend Snippet: HIF‐3α modulates the canonical Wnt/β pathway in a cell context‐dependent manner. Stable scrambled‐sh control and HIF‐3α‐sh RKO (A) or SW480 cells (B) were transiently transfected with the pTOPFlash or pFOPFlash (control) reporter plasmids. Twenty‐four hours posttransfection, SW480 cells were washed and lysed, and the luciferase activity was assayed (B). For RKO cells, which display normal canonical Wnt that needs to be activated by Wnt3a ligand (A), cells were serum starved (2%) for 10 h, and then Wnt3a (100 ng/mL) or vehicle was added to the medium to stimulate cells for 12 h period; the cells were then washed and lysed, and the luciferase activity was assayed. The activity in both cell types was normalized with respect to the activity of Renilla luciferase or with respect to the protein content in each sample. The data represent the means ± SEM from at least five independent assays. *** p < .001.

Article Snippet: The β‐catenin transcriptional activity‐reporter plasmids pTOPFlash and pFOPFlash (control) were obtained from Upstate Biotechnology (Lake Placid, NY, USA).

Techniques: Control, Transfection, Luciferase, Activity Assay