Journal: bioRxiv

Article Title: The proton-sensing GPR4 receptor regulates paracellular gap formation and permeability of vascular endothelial cells

doi: 10.1101/601088

Figure Lengend Snippet: Plasma membrane staining and paracellular gap area quantitation of ECs treated for up to 5 hours under physiological or acidic pH. Acidosis increases EC gap formation when compared to physiological pH treatment conditions. (A) Representative pictures of plasma membrane staining in Human Umbilical Vein Endothelial Cells (HUVECs) at 0, 3, and 5 hours treated under physiological or acidic pH. Quantitative analysis of gap formation in (B) HUVECs, (C) Human Pulmonary Artery Endothelial Cells (HPAECs), (D) Human Colon Microvascular Endothelial Cells (HMVEC-Colon), and Human Lung Microvascular Endothelial Cells (HMVEC-Lung) over 5 hours. All experiments were performed in triplicate and are representative of four experiments. Data at each time point are presented as mean ± SEM and analyzed for statistical significance between the pH 7.4 group and the pH 6.4 group using the unpaired t -test where **p<0.01 and ***p<0.001. White arrows point to paracellular gaps. Scale bar = 100µm.

Article Snippet: Primary human umbilical vein endothelial cells (HUVEC), human pulmonary artery endothelial cells (HPAEC), and human lung microvascular endothelial cells (HMVEC-Lung) (Lonza, Walkersville, MD, USA), and primary human colon microvascular endothelial cells (HMVEC-Colon) (Cell Biologics Inc., Chicago, IL, USA) were cultured at 37°C and 5% CO 2 in a humidified incubator for experimentation.

Techniques: Staining, Quantitation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: ER stress inhibition enhances formation of triacylglcerols and protects endothelial cells from lipotoxicity

doi: 10.1186/s12964-024-01682-y

Figure Lengend Snippet: ER stress inhibition restores palmitate-dependent impairment of autophagy and induction of UPR in macro- and microvascular endothelial cells. ( a ) Immunofluorescence of the ER morphology in HUVEC treated with 150 μm of oleate (OA) or palmitate (PA) in combination with DMSO or 2.5 mM 4-PBA. Prior to treatment the cells were transfected with the ER-scarlet probe. Lipid droplets were stained using BODIPY 493/503. The samples were imaged using Zeiss AxioObserver. Scale bar represents 15 μm. (b) Immunoblot analysis of HUVEC treated for 16 h with 150 μm PA in combination with DMSO or 2.5 mM 4-PBA. Cells were lysed in 2x sample buffer and analysed for expression of LC3B, p62, CHOP and Grp78. GAPDH immunblot was used as a loading control. (c) Quantification of immunoblot results from (b). 20 images per condition were analyzed in total in 3 independent experiments. (d) Immunoflourescence analysis of CHOP and p62 expression in HUVEC. The cells were treated as in ( b ), fixed and stained with anti-CHOP and anti-p62 antibodies and DAPI. Scale bar represents 50 μm. (e) Quantification of immunofluorescence analysis depicted in ( d ). For CHOP quantification percentage of CHOP positive cells is displayed and for p62 mean fluorescence intensity (MFI) normalized to control sample. 5 images per condition were analyzed in each of 3 independent experiments. (f) Immunofluorescence analysis of CHOP and p62 expression in HMVEC. Sample preparation and imaging was performed as in ( d ). (g) Quantification of CHOP and p62 staining in HMVEC depicted in ( f ) was performed as described for HUVEC in ( e ). ( i ) FITC labelled dextran leakage assay was performed in HUVEC. Cells were seeded on Transwell inserts and treated for 16 h with BSA + DMSO as a control, 150 µM PA + DMSO or 150 µM PA + 2.5 mM 4-PBA, 150 µM OA or 10 ng/ml TNFa as positive control. The leakage of FITC-labelled 75 kDa Dextran into lower compartment was measured by reading of mean fluorescence intensity at 488 nm using the microplate reader. 3 technical replicates were measured for each condition in 3 independent experiments. GraphPad Prism software and one-way ANOVA with Tukey’s multiple comparison test were used for statistical analysis. Error bars represent standard deviation (SD)

Article Snippet: Human Lung Microvascular Endothelial Cells (HMVEC) were obtained from Sciencell Research Laboratories (#sc-3000) and cultured in Endothelial Cell Medium (ECM)(#sc-1001).

Techniques: Inhibition, Immunofluorescence, Transfection, Staining, Western Blot, Expressing, Control, Fluorescence, Sample Prep, Imaging, Positive Control, Software, Comparison, Standard Deviation