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