Journal: bioRxiv

Article Title: ApoE4 disrupts intracellular trafficking and iron homeostasis in an improved iPSC-based model of human brain endothelial cells

doi: 10.1101/2024.09.03.610802

Figure Lengend Snippet: A, Quantification of relative apparent permeability (P app ) for dextrans of different molecular weights of cells generated with the protocols described in using a transwell system. Values were normalized to the apparent permeability of iECs. Graph shows boxplots with interquartile ranges and median. Lines show the 5th and 95th percentiles, data from n = 4 independent differentiations with 8 technical replicates per condition. Differences in apparent permeability are statistically significant as evaluated by Two-way ANOVA with Sidak multiple comparisons. ns, not statistically significant; ***, p <0.001. B, Haematoxylin and Eosin staining of iCE-BECs grown on a transwell filter showing a cell monolayer. Scale bar, 50 µm. C, Representative fluorescence images after immunostaining with endothelial-specific markers pseudo-colored showing PECAM1, VE-Cadherin, ZO-1 and Claudin-5 of iCE-BECs grown in a MIMETAS OrganoPlate® 2-lane 96, Scale bar, 100 µm. D, Representative bright field (grey left panels) and fluorescence images of MIMETAS OrganoPlate® 2-lane chambers comparing iEC (top) and iCE-BECs (bottom) after incubation with 3 kDa or 40 kDa dextran for 2 or 40 minutes. The ratio between fluorescent intensity between the upper (vascular cell compartment) and lower gel chamber is used for the estimation of apparent permeability, Scale bar, 500 µm. E, Quantification of apparent permeability (P app ) of iCE-BECs to 3, 40 or 70 kDa dextran in basal conditions or after stimulation with 200 ng/mL VEGF-A for 24 hours. Graph shows boxplots with interquartile ranges and median. Lines show the 5th and 95th percentiles, data from one differentiation with at least 7 technical replicates per condition. F, Representative fluorescent images of iCE-BECs grown on MIMETAS OrganoPlate® 2-lane chambers comparing iEC (top) and iCE-BECs (bottom) after incubation with 200 nM non-targeting IgG (NT IgG) or a Brainshuttle™ antibody. Images were acquired immediately after incubation (0 hours) or after 2 hours. Scale bar, 500 µm. G, Quantification of relative antibody transcytosis across iCE-BECs (see Methods for details). Values are normalized relative to the NT-IgG condition. Graph shows boxplots with interquartile ranges and median. Lines show the 5th and 95th percentiles, data from 3 independent differentiations, each at least 7 technical replicates per condition. Differences in transcytosis are statistically significant as evaluated by Two-way ANOVA with Sidak multiple comparisons. **, p <0.01; ***, p <0.001.

Article Snippet: To assess transcytosis of Brainshuttle TM molecules across iCE-BECs, cells were seeded in an OrganoPlate 2-lane (9605-400-B, MIMETAS) as described above.

Techniques: Permeability, Generated, Staining, Fluorescence, Immunostaining, Incubation

Journal: bioRxiv

Article Title: Guard-cell phytosterol homeostasis is critical for proper stomatal development

doi: 10.1101/2024.08.29.610202

Figure Lengend Snippet: Overexpressed GSEase in an Escherichia coli or in a Pichia pastoris system was not effective in our experimental setup. Therefore, we generated a GSEase CDS ( S ) overexpression line using the Pro35S::GSEase CDS-6x histidine in A. thaliana . GSEase recombinant proteins were then purified with diethylaminoethanol (DEAE) sepharose and the histidine-tag-column procedure and verified by western blot assay. The purified protein was then used for characterising its substrate specificity in vitro . (A-B) Purification of histidine-tagged GESase (GSEase-6xHis). SDS-PAGE (A) and western blot (B) detection of the GSEase-6xHis during purification steps. M, Precision Plus Protein Standards Dual Color, BIO-RAD; lane 1, crude protein extract collected from DEAE column elution; lane 2, flow through; lane 3, 20 mM imidazole; lane 4, 40 mM imidazole; lane 5, 60 mM imidazole; lane 6, 80 mM imidazole; lane 7, 100 mM imidazole; lane 8, 120 imidazole; lane 9, 140 mM imidazole. Western blot analysis involved using a monoclonal 6X His tag antibody (GT359, GeneTex, USA). (C) Enzyme kinetic analysis of GSEase - 6xHis indicating that GSEase processed a higher (≈2 times) maximum rate of production (Vmax) and turnover number (kcat) in cholesteryl n-stearates than in p -NP stearate. Furthermore, the higher substrate affinity of cholesteryl n-stearate is indicated by its lower Km value than that of p -NP stearate. Lipids possess an acyl and alcohol group, and both fit in their respective binding tunnel in a lipase . p -NP stearate and cholesteryl n-stearate have the same acyl group but different alcohol groups. Thus, the substrate affinity of GSEase is possibly determined on the alcohol group binding tunnel but not the acyl group binding tunnel. In addition, we assumed that the alcohol group binding tunnel explains why GSEase does not prefer triacylglycerol (TAG) substrates such as tristearate because the alcohol group of TAGs may not be suitable for GSEase binding. The kinetic parameters were calculated with the Lineweaver–Burk equation in a nonlinear regression, with cholesteryl n-stearate or p -NP stearate used as substrates at room temperature at pH 8.0. Data are mean ± SD of three independent enzyme reactions.

Article Snippet: M, Precision Plus Protein Standards Dual Color, BIO-RAD; lane 1, crude protein extract collected from DEAE column elution; lane 2, flow through; lane 3, 20 mM imidazole; lane 4, 40 mM imidazole; lane 5, 60 mM imidazole; lane 6, 80 mM imidazole; lane 7, 100 mM imidazole; lane 8, 120 imidazole; lane 9, 140 mM imidazole.

Techniques: Generated, Over Expression, Recombinant, Purification, Western Blot, In Vitro, SDS Page, Binding Assay