Characterization of Zeb1 Promoter Activity

Human embryonic kidney cells (HEK293) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS, HyClone) as previously described^{27 (link),28 (link)}. Primary murine renal fibroblasts were isolated and cultured as previously described^{29 (link)}. TheZeb1 promoter–luciferase construct was generated by amplifying genomic DNA spanning the proximal promoter and the first exon of the Zeb1 gene (-1000/+100) and ligating it into a pGL3-basic vector (Promega). The MRTF-A^{30 (link)} and Zeb1^{31 (link)} expression vectors have been previously described. Truncation mutants were made using a QuikChange kit (Thermo Fisher Scientific, Waltham, MA, United States) and were verified by direct sequencing. Small interfering RNAs were purchased from Dharmacon. Transient transfections were performed with Lipofectamine 2000. Luciferase activities were assayed 24-48 hours after transfection using a luciferase reporter assay system (Promega) as previously described^{32 (link)}.

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Zhao Q., Shao T., Zhu Y., Zong G., Zhang J., Tang S., Lin Y., Ma H., Jiang Z., Xu Y., Wu X, & Zhang T. (2023). An MRTF-A–ZEB1–IRF9 axis contributes to fibroblast–myofibroblast transition and renal fibrosis. Experimental & Molecular Medicine, 55(5), 987-998.

Publication 2023

pGL3-basic vector QuikChange kit Small interfering RNAs luciferase reporter assay system

Assay Cells Embryonic Exon Fibroblasts Genomic Human Lipofectamine 2000 Luciferase Murine Pgl3 Promega Renal Small interfering rnas Transfection Transient Vectors

Corresponding Organization : Nanjing Sport Institute

Other organizations : Nanjing Medical University, China Pharmaceutical University

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Variable analysis

independent variables

Zeb1 promoter–luciferase construct
MRTF-A expression vector
Zeb1 expression vector
Truncation mutants
Small interfering RNAs

dependent variables

Luciferase activities

control variables

HEK293 cells maintained in DMEM supplemented with 10% FBS
Primary murine renal fibroblasts isolated and cultured

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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