sc 29228 5 gcaguuguaugcugugguu 3 sirnas  (Santa Cruz Biotechnology)

Journal: Frontiers in Cell and Developmental Biology

Article Title: The short-chain fatty acid butyrate exerts a specific effect on VE-cadherin phosphorylation and alters the integrity of aortic endothelial cells

doi: 10.3389/fcell.2023.1076250

Figure Lengend Snippet: Perturbation of FFAR2/3 expression and function diminishes BUT-induced VEC phosphorylation. (A) Detection of FFAR2/3 expression by RT-PCR and immunohistochemistry (IHC). IHC staining was performed on fixed and agarose-embedded HAOEC (left panel) and of FFPE samples of human thoracic aorta (right panel). Images below represent zoomed-in areas indicated by black rectangles with red arrowheads pointing to specific receptor staining. Scale bar (100 μm). (B) BUT-induced VEC phosphorylation analyzed as shown in upon pre-incubation overnight with 0.1 µM of FFAR3 antagonist GLPG0974 (left panel) and co-incubation with FFAR2 antagonist ß-HB (0.1mM, right panel). For the GLPG0974 approach, cells were treated either with a vehicle alone (DMSO) or in combination with BUT and GLPG0974. Data represent n ≥ 5 experiments. (C) Validation of FFAR3 KD by RT-qPCR. Relative expression of FFAR3 normalized to GAPDH between CTRL (=1) and FFAR3 siRNA-transfected HAOEC of n = 3 independent transfection experiments. (D) BUT-induced VEC phosphorylation analyzed as shown in in the presence and absence of FFAR3 KD of n = 6 experiments. In FFAR3 KD cells, Y731 phosphorylation in response to butyrate treatment was decreased by 40.91% compared to scrambled siRNA. (E) Immunofluorescence microscopy images of non-permeabilized HAOEC stained for junctional VEC (antibody clone BV6) after 1 mM BUT or medium-only (-BUT) treatment for 1 h. Cells were transfected with control or FFAR3 siRNA under the same conditions used in (C , D) . Small images represent enlarged areas of the sections marked by white rectangles. Images show representative data of n = 3 independent experiments. Scale bar (100 μm).

Article Snippet: The following reagents were used for transfection: control siRNA-A (Santa Cruz, sc-37007), siRNA transfection medium (Santa Cruz, sc-36868), siRNA transfection reagent (Santa Cruz, sc-29528), siRNA dilution buffer (sc-29517), VE-cadherin siRNA (h) (Santa Cruz, sc-36814), GPR41 siRNA(h) (Santa Cruz, sc-97148), and siRNA c-Src (h) (Santa Cruz, sc-29228).

Techniques: Expressing, Phospho-proteomics, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry, Staining, Incubation, Biomarker Discovery, Quantitative RT-PCR, Transfection, Immunofluorescence, Microscopy, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: The short-chain fatty acid butyrate exerts a specific effect on VE-cadherin phosphorylation and alters the integrity of aortic endothelial cells

doi: 10.3389/fcell.2023.1076250

Figure Lengend Snippet: BUT engages Src kinases for specific tyrosine phosphorylation at VEC. (A) HAOECs were treated for 15 min before and during BUT incubation with PP2 (10 μM) or vehicle (DMSO). Subsequently, phospho-VECY731 levels were analyzed by western blotting. Phospho-signals of n = 4 experiments were quantified by densitometry. (B) Immune fluorescent microscopy of HAOECs treated with the vehicle (DMSO) or PP2 and BUT (5 mM) for 1 h and subsequently stained for VEC and general phospho-tyrosine residues. Scale bar (10 μm). (C) Validation of Src KD by RNAi. HAOECs were transfected with Src-specific siRNA or control-siRNA. Cells were lysed and analyzed for Src expression 12 h later by western blotting. (D) Stimulation of Src KD and control cells with BUT (1 mM) for 1 h and analysis of phosho-VECY731 expression as shown in . Quantification corresponds to n = 6 experiments. (E) Phospho-SrcY416 western blotting of BUT-treated or -untreated cells, transfected with Ctrl or FFAR3 siRNA. Data display densiometric quantification of relative phospho-Src signals normalized to the level in BUT-untreated Ctrl siRNA-transfected cells (dashed red line) of n = 3 experiments.

Article Snippet: The following reagents were used for transfection: control siRNA-A (Santa Cruz, sc-37007), siRNA transfection medium (Santa Cruz, sc-36868), siRNA transfection reagent (Santa Cruz, sc-29528), siRNA dilution buffer (sc-29517), VE-cadherin siRNA (h) (Santa Cruz, sc-36814), GPR41 siRNA(h) (Santa Cruz, sc-97148), and siRNA c-Src (h) (Santa Cruz, sc-29228).

Techniques: Phospho-proteomics, Incubation, Western Blot, Microscopy, Staining, Biomarker Discovery, Transfection, Control, Expressing