Journal: Journal of Molecular Endocrinology

Article Title: CEBPB/POU2F2 modulates endothelin 1 expression in prehypertensive SHR vascular smooth muscle cells

doi: 10.1530/JME-22-0178

Figure Lengend Snippet: Different transcriptional regulation of the endothelin (ET)-1 promoter enhancer region. (A) Nuclear and cytoplasmic proteins were isolated from vascular smooth muscle cells (VSMCs) of spontaneously hypertensive rates (SHRs) and Wistar-Kyoto (WKY) rats. Western blot analysis of nuclear protein-poly(ADP ribose) polymerase (PARP) to confirm isolation purity. Coomassie blue staining of SDS-PAGE was used as an indicator of protein loading. (B) The DNA of the enhancer region was used as a probe and was incubated with SHR and WKY VSMC nuclear proteins for the EMSA analysis. Competing DNA was a 100-fold concentrated probe. Arrows indicate differences between probe-bound SHR and WKY VSMC nucleoproteins. (C) An RT-PCR analysis of the expressions of Pou1f1 in a pituitary cell line (GH3), smooth muscle cell line (A10), pituitary gland (from SHRs and WKY rats), and VSMCs (from SHRs and WKY rats). (D) Pou2f2 proteins were incubated with POU2F2 consensus sequence probes and enhancer region probes, and an EMSA was used to analyze protein–DNA interactions. Arrows indicate specific binding sites after mixing with competitor DNA of the POU2F2 consensus sequence. Arrowheads indicate specific binding sites after mixing with competitor DNA of the enhanced region sequence. (E) The POU2F2 and CEBPB consensus sequences of DNA sequences indicated on the bottom line were mutated in the 1309prET1 construct and co-transfected with Renilla reporter plasmids in SHR or WKY VSMCs. Statistical data are shown as the mean ± s.e.m. , * P < 0.05, ** P < 0.01. Student's t -test from three independent experiments. Smooth muscle cells were collected from the aortas of three SHR or WKY rats, and each data set consists of three experiments. *vs 1309prET1 in SHR rats. A full color version of this figure is available at https://doi.org/10.1530/JME-22-0178 .

Article Snippet: The rat VSMC line (A10) was obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Isolation, Western Blot, Staining, SDS Page, Incubation, Reverse Transcription Polymerase Chain Reaction, Sequencing, Binding Assay, Construct, Transfection

Journal: Journal of Molecular Endocrinology

Article Title: CEBPB/POU2F2 modulates endothelin 1 expression in prehypertensive SHR vascular smooth muscle cells

doi: 10.1530/JME-22-0178

Figure Lengend Snippet: Overexpression of POU2F2 and CEBPB affects endogenous endothelin (ET)-1 expression. Total RNA or culture medium from Wistar-Kyoto (WKY) vascular smooth muscle cells (VSMCs) was collected from cells transfected with pcDNA3.1A (3.1A), POU2F2, CEBPB, and POU2F2 + CEBPB expression vectors. (A) Edn1 mRNA expression was examined by a qPCR. Data shown were normalized to the expression of the α-actin reference gene, and the expression level with 3.1A empty vector transfection was set to 1. (B) EDN1 ELISA for measurement of EDN1 secretion in VSMC culture medium. * P < 0.01. *vs 3.1A empty vector transfection. (C and D) Cells were infected with lentivirus-expressing POU2f2 shRNAs (sh- Pou2f2 -1 and sh- Pou2f2 -2) for 24 h. As a negative control, cells were infected with a luciferase shRNA-containing lentivirus (sh-Luc). POU2F2 shRNA infection was confirmed by an RT-qPCR (C) and western blotting (D). Smooth muscle cells were collected from the aortas of three SHR or WKY rats, and each data set consists of three experiments.

Article Snippet: The rat VSMC line (A10) was obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Infection, Negative Control, Luciferase, shRNA, Quantitative RT-PCR, Western Blot