cpla2 pab  (Bioss)

Journal: Advanced Science

Article Title: Geometric Constraints Regulate Energy Metabolism and Cellular Contractility in Vascular Smooth Muscle Cells by Coordinating Mitochondrial DNA Methylation

doi: 10.1002/advs.202203995

Figure Lengend Snippet: Cytosolic phospholipase A2 (cPLA2), a nuclear mechanosensor, responds to changes in cell geometry. A) The nuclear shape index and nuclear perimeter in micropatterned SMCs were calculated. B) Representative three‐dimensional reconstruction images of the nucleus of micropatterned cells and the quantification of the nuclear surface area. Cells were infected with pSIN‐H2B‐tagGFP viruses. C) Representative fluorescent images of cPLA2‐EYFP fusion proteins in micropatterned cells. Cells were transfected with the cPLA2‐EYFP plasmids before seeding. D) Representative immunofluorescent staining of cPLA2 and nuclear envelope protein lamin B1 in micropatterned cells. Z1–Z3 indicate three different scanning layers. The distribution of cPLA2 and lamin B1 and the degree of overlap between them are indicated by fluorescence intensity profiling (right panels). E) Production of arachidonic acid (ArAc) in the micropatterned cells was assayed using ELISA. Data were obtained from eight biological repeats. In (A, B) (right panel), each dot represents a single cell. In (E), data were from eight biological repeats. Significance was assessed using a Student's t ‐test. The error bars show ± SD. The exact P values between the indicated groups are presented.

Article Snippet: Rabbit pAb against cPLA2, mouse mAb against AKT1, and mouse mAb against Myc‐tag were from Bioss.

Techniques: Infection, Transfection, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: Geometric Constraints Regulate Energy Metabolism and Cellular Contractility in Vascular Smooth Muscle Cells by Coordinating Mitochondrial DNA Methylation

doi: 10.1002/advs.202203995

Figure Lengend Snippet: cPLA2 inhibition blocks the geometry‐regulated DNMT1 redistribution and mtDNA methylation. A) Representative immunofluorescent staining (upper panels) and quantification (lower panel) of p‐AKT in micropatterned cells treated with a cPLA2 inhibitor (CAY10650, 12 nmol L −1 ) or the control reagent (DMSO) for 1 h. B) Representative immunofluorescence staining (upper panels) and quantification (lower panel) of the nuclear/cytoplasmic ratio of DNMT1 in micropatterned cells treated with CAY10650 or DMSO. C) Representative agarose gel electrophoresis of the products of MSP for the D‐loop of bisulfite‐modified DNA isolated from micropatterned cells treated with CAY10650 or DMSO. Semi‐quantification of the MSP results is presented in the right panel. M: methylated, U: unmethylated. Data were obtained from four biological repeats. Fluorescence images were acquired using a confocal microscope. In (A, B), each dot represents a single cell. In (C), data were from four biological repeats. Significance was assessed using two‐way ANOVA with Tukey's post hoc analysis. The error bars show ± SD. The exact P values between the indicated groups are presented.

Article Snippet: Rabbit pAb against cPLA2, mouse mAb against AKT1, and mouse mAb against Myc‐tag were from Bioss.

Techniques: Inhibition, Methylation, Staining, Immunofluorescence, Agarose Gel Electrophoresis, Modification, Isolation, Fluorescence, Microscopy

Journal: Advanced Science

Article Title: Geometric Constraints Regulate Energy Metabolism and Cellular Contractility in Vascular Smooth Muscle Cells by Coordinating Mitochondrial DNA Methylation

doi: 10.1002/advs.202203995

Figure Lengend Snippet: Intervention of the cPLA2‐AKT1‐DNMT1 pathway affects ATP production and cell contractility. A,B) Measurement of the intracellular ATP content of micropatterned SMCs subjected to the indicated treatments. Data were obtained from five biological repeats. C) Schematic representation of the MTS‐DNMT1 construct. MTS, mitochondrial targeting sequence, CMV cytomegalovirus promoter, ORF open reading frame. D) Representative immunofluorescent staining of Discosoma sp. red fluorescent protein (DsRed). Cells were transfected either with pcDNA (CL vector) or with MTS‐DNMT1. E) Representative immunofluorescence staining of DNMT1 in micropatterned cells transfected with the CL vector or MTS‐DNMT1. F) Measurement of the intracellular ATP content of micropatterned cells with pcDNA or MTS‐DNMT1 transfection. Data were obtained from five biological repeats. G) Gel contraction assay with cells with the indicated treatments. Quantifications from four biological repeats are shown in the lower panels. H) Gel contraction assay with cells subjected to DNMT1‐AA or DNMT1‐DD transfection. Quantifications from six biological repeats are shown in the lower panels. I) Traction force microscopy assay to detect the cellular contractility of micropatterned cells treated with CAY10650 (a cPLA2 inhibitor) or DMSO. Representative bright field images and cell force images are presented in the left panel. Quantification of the traction is shown in the right panel. Each dot represents a single cell. Fluorescence images were acquired using a confocal microscope. Significance was assessed using one‐way or two‐way ANOVA with Tukey's post hoc analysis. The error bars show ± SD.

Article Snippet: Rabbit pAb against cPLA2, mouse mAb against AKT1, and mouse mAb against Myc‐tag were from Bioss.

Techniques: Construct, Sequencing, Staining, Transfection, Plasmid Preparation, Immunofluorescence, Collagen Gel Contraction Assay, Microscopy, Fluorescence