passages 3 4  (Thermo Fisher)

Journal: Cardiovascular Research

Article Title: Endothelial insulin-like growth factor-1 signalling regulates vascular barrier function and atherogenesis

doi: 10.1093/cvr/cvaf055

Figure Lengend Snippet: IGF-1 receptor over-expression reduces paracellular leakage and LDL-cholesterol uptake in human endothelial cells via its kinase domain. ( A ) Schema of lentiviral constructs used to achieve doxycycline-inducible WT or kinase-dead (K1003R) IGF-1R over-expression. ( B ) WT and K1003R IGF-1R protein expression are increased approximately three-fold by doxycycline. ( C ) Representative images of HUVEC VE-Cadherin with either WT or K1003R IGF-1R over-expression (VE-Cadherin—red, DAPI—blue). D ) VE-Cadherin junction thickness is reduced in confluent HUVEC exposed to 100 μM hydrogen peroxide over-expressing WT IGF-1R, but remains unchanged with cells over-expressing K1003R IGF-1R. ( E ) VE-Cadherin junction area is similar in confluent HUVEC over-expressing WT or K1003R IGF-1R. ( F ) Paracellular leakage of 40 kDa FITC-Dextran is reduced in HUVEC over-expressing WT IGF-1R, but not K1003R, IGF-1R. G ) Uptake of BODIPY-labelled LDL-cholesterol is reduced in HUVEC over-expressing WT IGF-1R, but not K1003R, IGF-1R (representative images on left; BODIPY—green, DAPI—blue). ( H ) Representative immunoblots of YAP/TAZ and loading controls in nuclear and cytosolic lysates, accompanied by quantification of normalized nuclear TAZ expression and nuclear:cytosolic TAZ ratio, showing reduced nuclear TAZ in HUVEC over-expressing WT vs. K1003R IGF-1R. ns denotes non-specific band in α-tubulin blot. Data expressed as mean (SEM). * P < 0.05; n = 3, 3 in a–g and n = 4, 4 in h. All statistical comparisons are made with unpaired Student’s t -tests.

Article Snippet: Passage 3–4 HUVEC (PromoCell) from at least three different donors were cultured in Endothelial Cell Growth Medium 2 (PromoCell) supplemented with 1× Antibiotic Antimycotic Solution (Merck) at 37°C in 5% CO 2 .

Techniques: Over Expression, Construct, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Perfluorooctanoic acid in indoor particulate matter triggers oxidative stress and inflammation in corneal and retinal cells

doi: 10.1038/s41598-020-72600-8

Figure Lengend Snippet: Effects of perfluorooctanoic acid (PFOA) on human corneal cells. ( a ) PFOA showed minimal cytotoxicity against human corneal epithelial cells (HCEpiC) treated with different concentrations of PFOA for 72 h. Cell viability was evaluated using the MTT assay. ( b ) PFOA induced the production of reactive oxygen species (ROS) in HCEpiC treated with different concentrations of PFOA for 6 h. Levels of ROS were determined using 2,7-dichlorofluorescin diacetate (DCFH-DA). Experimental groups were compared using ANOVA ( P < 0.0001), and Dunnett’s multiple comparisons tests were used for paired comparisons between phosphate-buffered saline (PBS; control) and 0.5% (v/v) DMSO, 400 ppm PFOA, 200 ppm PFOA or 100 ppm PFOA-treated HCEpiC. P values below 0.05 were considered to indicate statistical significance. ( c ) PFOA altered tight junctions in HCEpiC. Relative transepithelial electrical resistance (TEER) was measured in HCEpiC incubated with PBS (control), 0.5% DMSO, 400 ppm PFOA, or 200 ppm PFOA for 24 h. Experimental groups were compared using ANOVA ( P < 0.0001), and Dunnett’s multiple comparisons tests were used for paired comparisons between control and 0.5% DMSO, 400 ppm PFOA or 200 ppm PFOA-treated HCEpiC. P values below 0.05 were considered to indicate statistical significance. ( d ) PFOA treatment decreased the expression level of claudin-1. Cells were treated with different concentrations of PFOA for 24 h, and the level of claudin-1 was determined by western blotting. β-actin was used as an endogenous reference protein. The expression levels of claudin-1 were normalized against β-actin and the relative expression levels compared with the control were noted above each lane. ( e ) Levels of IL-6 and IL-8 in HCEpiC. Cells were seeded on transwell inserts with a 0.4-µm pore size incubated with PBS (control) or 400 ppm PFOA for 16 h, and the levels of IL-6 and IL-8 in apical and basolateral compartments were determined using enzyme-linked immunosorbent assays. Unpaired t-test was used to determine significant differences between control and PFOA treated groups. P values below 0.05 were considered to indicate statistical significance. Asterisks denote statistical significance.

Article Snippet: Human corneal epithelium cells (HCEpiC; passages 3–5) and retinal pigment epithelial cells (HREpiC; passages 3–4) were obtained from ScienCell Research Laboratories (San Diego, CA, USA) and cultured according to the instructions provided by the supplier.

Techniques: MTT Assay, Saline, Control, Incubation, Expressing, Western Blot, Pore Size

Journal: Scientific Reports

Article Title: Perfluorooctanoic acid in indoor particulate matter triggers oxidative stress and inflammation in corneal and retinal cells

doi: 10.1038/s41598-020-72600-8

Figure Lengend Snippet: Perfluorooctanoic acid (PFOA) induced the secretion of proinflammatory cytokines by corneal epithelial cells, which affected inflammatory responses of corneal endothelial cells. ( a ) Inflammatory cytokines in the corneal epithelial cells (HCEpiC) basolateral media may alter the tight junction integrity of corneal endothelial cells (HCEC). Relative transepithelial electrical resistance of HCEC incubated with PBS (control), interleukin (IL) 6 (20 ng/mL), or HCEpiC basolateral media for 24 h. Experimental groups were compared using ANOVA ( P = 0.0014), and Dunnett’s multiple comparisons tests were used for paired comparisons between control and IL-6- or HCEpiC basolateral media-treated HCEC. P values below 0.05 were considered to indicate statistical significance. ( b ) Inflammatory cytokines in the corneal epithelial cells (HCEpiC) basolateral media may induce the secretion of inflammatory cytokines by HCEC. Basolateral media of HCEpiC cells treated with PBS (control) or PFOA for 16 h were collected and used to treat HCEC for 16 h. The levels of IL-6 and IL-8 were determined using enzyme-linked immunosorbent assays. Unpaired t-test was used to determine significant differences between control and PFOA treated groups. P values below 0.05 were considered to indicate statistical significance. Asterisks denote statistical significance.

Article Snippet: Human corneal epithelium cells (HCEpiC; passages 3–5) and retinal pigment epithelial cells (HREpiC; passages 3–4) were obtained from ScienCell Research Laboratories (San Diego, CA, USA) and cultured according to the instructions provided by the supplier.

Techniques: Incubation, Control

Journal: Scientific Reports

Article Title: Perfluorooctanoic acid in indoor particulate matter triggers oxidative stress and inflammation in corneal and retinal cells

doi: 10.1038/s41598-020-72600-8

Figure Lengend Snippet: Perfluorooctanoic acid (PFOA) induced inflammation in retinal pigment epithelial cells. ( a ) Cytotoxicity of different concentrations of N-retinylidene-N-retinylethanolamine (A2E) towards retinal pigment epithelial cells (ARPE-19). Cell viability was determined by the MTT assay. ( b ) Inflammatory cytokines in the HCEC basolateral media may disrupt tight junctions in ARPE-19 cells. Relative transepithelial electrical resistance (TEER) of ARPE-19 cells incubated with PBS (control), interleukin (IL) 6 (20 ng/mL), or human corneal endothelial cells (HCEC) basolateral media for 24 h. Experimental groups were compared using ANOVA ( P = 0.0001), and Dunnett’s multiple comparisons tests were used for paired comparisons between control and IL-6- or HCEC basolateral media-treated ARPE-19 cells. P values below 0.05 were considered to indicate statistical significance. ( c ) Basolateral media of HCEC treated with PBS (control) or corneal epithelial cells (HCEpiC) basolateral media for 16 h were collected and used to treat ARPE-19 cells for 16 h. ARPE-19 cells were also treated with 50 μM A2E for 16 h. Levels of IL-6 and IL-8 were determined using enzyme-linked immunosorbent assays. Experimental groups were compared using ANOVA, and Dunnett’s multiple comparisons tests were used for paired comparisons between control and HCEC basolateral media- or HCEC basolateral media + A2E-treated ARPE-19 cells. P values below 0.05 were considered to indicate statistical significance. ( d ) Same experiment as in Fig. 3c, using human primary retinal epithelial cells instead of ARPE-19 cells. Asterisks denote statistical significance.

Article Snippet: Human corneal epithelium cells (HCEpiC; passages 3–5) and retinal pigment epithelial cells (HREpiC; passages 3–4) were obtained from ScienCell Research Laboratories (San Diego, CA, USA) and cultured according to the instructions provided by the supplier.

Techniques: MTT Assay, Incubation, Control

Journal: Nature Communications

Article Title: Japanese encephalitis virus neuropenetrance is driven by mast cell chymase

doi: 10.1038/s41467-019-08641-z

Figure Lengend Snippet: Inhibition of chymase abolishes JEV-induced breakdown of the BBB. a bEND.3 cell monolayers were treated with media alone or media containing JEV or supernatants of MCs only, JEV-stimulated MCs, or JEV-stimulated MCs treated with either TY-51469 (chymase inhibitor) or nafamostat mesylate (tryptase inhibitor). Supernatants from JEV-stimulated BMMCs reduced the TEER, which was reversed by TY-51469 (100 µM), but not nafamostat mesylate (10 µM); analyzed by two-way ANOVA. b JEV-activated BMMC supernatants increased FITC-dextran leakage across transwells 24 h post-exposure. TY-51469, but not nafamostat mesylate, reduced FITC-dextran leakage; n = 3. c Supernatants from JEV-stimulated WT BMMCs reduced TEER of b.END3 monolayers, but control media, JEV, supernatants from WT or MCPT4-KO BMMCs or JEV-stimulated MCPT4-KO BMMCs did not. d JEV-activated WT BMMC supernatants increased FITC-dextran leakage (24 h), but controls or supernatants from JEV-activated MCPT4-KO BMMCs did not; n = 3. e Claudin-5, ZO-1, ZO-2, and occludin levels in bEND.3 cells exposed to JEV-activated MC supernatants were reduced compared to media, JEV, and unstimulated MC supernatant-treated groups, by western blotting, which was inhibited by TY-51469. GAPDH blotting served as loading controls. Quantification is provided (Supplementary Figure ). f TY-51469 reduced EBD leakage into brains of JEV-infected WT mice, to levels similar to JEV-infected Sash mice, 5 days post-i.p. infection with Nakayama (2 × 10 7 PFU); n = 5. g Representative images from f . h IgG (red) of brain sections of JEV-infected WT, Sash, and TY-51469-treated WT mice, 5 days post-i.p. infection with Nakayama. During JEV infection, IgG was detected in the brain parenchyma of WT mice, but not Sash or TY-51469-treated WT mice. Scale bar = 50 μm. i TY-51469 reduced the JEV titers in brains, 6 days post-i.p. Nakayama infection, compared to vehicle-treated WT mice, to levels similar to Sash mice. Chymase injection (30 ng, i.p.) of Sash mice led to increased j EBD leakage ( n = 5) and k brain JEV titers, to levels similar to WT JEV-infected mice (5 days post-infection); n = 8; representative of two experiments. Error bars represent the SEM. Unless indicated, all data are representative of three independent experiments, were analyzed by one-way ANOVA with Holm-Sidak’s multiple comparison test; * denotes P < 0.05 and ** denotes P < 0.01. Chymase inhibition limits JEV penetration of the brain by preventing BBB leakage

Article Snippet: Mouse brain endothelial bEND.3 cells (5 × 10 5 cells in 500 µL DMEM media, passage number 4 or 5) were grown for 1 day on 3 μM transwell inserts (BD Biosciences; Corning, #353492) until confluency and tight junctions were well-established.

Techniques: Inhibition, Western Blot, Infection, Injection