normal colon epithelial cell line ncm460  (ATCC)

Journal: Advanced Science

Article Title: DR5 Governs Compound Exocytosis in Colonic Goblet Cells via TATA‐Box Binding Protein‐Dependent Bestrophin‐2 Transcriptional Regulation

doi: 10.1002/advs.202516789

Figure Lengend Snippet: DR5 modulates intracellular pH via regulating BEST2 expression A) qRT‐PCR analysis of DR5 knockdown efficiency in CCD 841 CoN cells transfected with si‐ DR5 or NC ( n = 4). B) The effect of DR5 knockdown on intracellular pH in CCD 841 CoN cells. Cells were transfected with si‐ DR5 or NC and then incubated with BCECF AM (1 µ m ) probe for 30 min. The lower the fluorescence intensity, the stronger the acidity. C) The effect of DR5 activation on intracellular pH in CCD 841 CoN cells. Cells were stimulated with Bioymifi (100 n m ) for 48 h and then incubated with BCECF AM (1 µ m ) probe for 30 min. D) qRT‐PCR analysis of BEST2 mRNA expression in CCD 841 CoN cells transfected with BEST2 overexpression plasmid or empty vector ( n = 4). E) The effect of BEST2 overexpression on intracellular pH. CCD 841 CoN cells were transfected with BEST2 overexpression plasmid or vector and then incubated with BCECF AM probe (1 µ m ) for 30 min. F) qRT‐PCR analysis of BEST2 mRNA expression in CCD 841 CoN cells treated with Bioymifi (100 n m , 48 h) ( n = 4). G) Immunofluorescence images of BEST2 protein in CCD 841 CoN cells stimulated with Bioymifi (100 n m , 48 h). H) qRT‐PCR analysis of BEST2 mRNA expression in CCD 841 CoN cells transfected with si‐ DR5 or NC ( n = 4). I) Immunofluorescence images of BEST2 protein in CCD 841 CoN cells transfected with si‐ DR5 or NC. Data are expressed as mean ± SD. All data were analyzed by an unpaired t ‐test. * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: The human normal colon epithelial cell line CCD 841 CoN (Research Resource Identifiers, RRID: CVCL_2871) was acquired from ATCC.

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Transfection, Incubation, Fluorescence, Activation Assay, Over Expression, Plasmid Preparation, Immunofluorescence

Journal: Advanced Science

Article Title: DR5 Governs Compound Exocytosis in Colonic Goblet Cells via TATA‐Box Binding Protein‐Dependent Bestrophin‐2 Transcriptional Regulation

doi: 10.1002/advs.202516789

Figure Lengend Snippet: DR5 driving BEST2 transcription requires its DD activity. A) Dual‐luciferase reporter assay assessing the effect of DR5 knockdown on the BEST2 promoter activity in CCD 841 CoN cells ( n = 4). B) Mutation sites of the DR5 DD. Red refers to the mutated amino acid. C) qRT‐PCR analysis of DR5 mRNA expression in HEK293T cells transfected with DR5 WT plasmid or vector ( n = 4). D) qRT‐PCR analysis of DR5 mRNA expression in HEK293T cells transfected with DR5 Mut plasmid or vector ( n = 4). E) qRT‐PCR analysis of TRAIL mRNA expression in HEK293T cells transfected with TRAIL plasmid or vector ( n = 4). F) Dual‐luciferase reporter assay showing the effect of DR5 WT plasmid on BEST2 promoter activity in HEK293T cells ( n = 4). G) qRT‐PCR analysis of BEST2 mRNA expression in HEK293T cells transfected with DR5 WT plasmid or vector ( n = 4). H) qRT‐PCR analysis of BEST2 mRNA expression in HEK293T cells co‐transfected with DR5 WT and TRAIL plasmids or DR5 Mut and TRAIL plasmids ( n = 4). I) Dual‐luciferase reporter assay showing the effect of co‐transfection with DR5 WT and TRAIL plasmids or DR5 Mut and TRAIL plasmids on BEST2 promoter activity in HEK293T cells ( n = 4). Data are expressed as mean ± SD. Data in Figure were analyzed by one‐way ANOVA followed by Tukey's post hoc test. Other data were analyzed by an unpaired t ‐test. Ns indicates not significant. ** p <0.01, *** p <0.001, **** p <0.0001.

Article Snippet: The human normal colon epithelial cell line CCD 841 CoN (Research Resource Identifiers, RRID: CVCL_2871) was acquired from ATCC.

Techniques: Activity Assay, Luciferase, Reporter Assay, Knockdown, Mutagenesis, Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation, Cotransfection