human immortalized endothelial cells  (ATCC)

Journal: bioRxiv

Article Title: Plasmodium falciparum diacylglycerol acyltransferase maintains phospholipid homeostasis to regulate sexual differentiation, ER stress, and cytoadhesion

doi: 10.1101/2025.10.10.681613

Figure Lengend Snippet: (a) Schematics of cytoadhesion assay under static and flow conditions. (b, c) Dot plots showing the number of erythrocytes infected with Pfdgat :LoxPint:HA parasites that adhered to 100 human brain microvascular endothelial cells (HBMECs) under static (b) and flow (c) conditions after 6 days of rapamycin treatment. Data are shown as mean ± SD from n = 3 independent biological replicates (b) and from n = 2 independent biological replicates from 3 independent experiments (c). P values were calculated using one-way ANOVA followed by Tukey–Kramer test. BFA, brefeldin. (d) Dot plots showing the number of SBP1 puncta on infected erythrocytes with Pfdagt :LoxPint:HA on 6 and 8 days of rapamycin treatment. P values were calculated using one-way ANOVA followed by Tukey–Kramer test. Representative immunofluorescence images are shown above the corresponding plots. Samples were stained for SBP1 with rabbit anti-SBP1 antibody (red) and for nuclei with DAPI (blue). Scale bars = 2 μm. was created with Biorender.com. Source data are provided as a Source Data file.

Article Snippet: For the static cytoadherence assay, immortalized human brain microvascular endothelial cells (HBMEC) (P10361-IM; Innoprot, Dario, Spain) were cultivated on 13-mm coverslips (Matsunami Glass, Osaka, Japan) in 24-well plates containing Endothelial Cell Medium (P60104; Innoprot, hereafter referred to as HBMEC medium) supplemented with 5% fetal bovine serum (175012; Nichirei Biosciences, Tokyo, Japan).

Techniques: Infection, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: BMP9 regulates the endothelial secretome to drive pulmonary hypertension

doi: 10.1101/2025.08.29.673113

Figure Lengend Snippet: (A) SDS-PAGE analysis of recombinant pro-complex BMP9 protein from different sources under reducing and non-reducing conditions reveals prodomain of ∼40 Kd, and covalently linked homodimer of ∼25 Kd, and homodimeric from of ∼12.5 Kd. Lanes 1-2 reveal two preparations containing nearly 100% disulfide-linked BMP9 homodimer under non-reducing conditions, both produced in CHO cells; lane 3 reveals ∼25% disulfide-linked homodimer and ∼75% non-disulfide linked BMP9 monomers, also produced in CHO cells and used in reference . (B) BRE-luciferase assay showing relative BMP-mediated transcriptional activity in telomerase-immortalized microvascular endothelial cells stimulated for 24 h with 40 pM of 100% disulfide-linked BMP9 dimer, mixed 70%/30% disulfide-linked/non-disulfide linked BMP9 dimer, and 100% non-disulfide linked mutant BMP9 C329S protein. (C) Immunoblot analysis (biotinylated polyclonal anti- BMP9 BAF3209, followed by streptavidin-HRP) of BMP9 immunoprecipitated from 10 mL pooled human AB donor serum (HS) using anti-BMP9 (MAB3209, α-BMP9, 1 µg/mL, 4C x 12h). Serum immunoprecipitated BMP9 migrated as a ∼25 Kd dimer under non-reducing conditions and as a ∼12.5 Kd monomer under reducing conditions (R), similar to control (CTRL) recombinant mature BMP9 homodimer (1 μg). Anti-VEGFA antibody (α-VEGF) was used as a non-specific immunoprecipitation control antibody. (D) SDS-PAGE analysis under non-reducing conditions of recombinant BMP9 produced as 70%/30% disulfide linked/non-linked BMP9 (“70/30”), 100% non-disulfide linked mutant BMP9 C329S protein (“0/100”), 100% disulfide linked pro-complex BMP9 (“100/0”), and 100% disulfide-linked mature BMP9. (E) Immunoblot demonstrates the ability of different BMP9 preparations to elicit activation of SMAD1 and SMAD3 in cultured TIME cells. A 70%/30% disulfide linked/non-linked BMP9, 100% disulfide linked pro-complex BMP9, and disulfide-linked mature BMP9 elicit similar activation of SMAD1 and SMAD3, whereas non-disulfide linked mutant BMP9 C329S (“0/100”) protein elicited attenuated activation of SMAD1.

Article Snippet: Human telomerase immortalized microvascular endothelial (TIME) cells were obtained from ATCC (CRL-4025).

Techniques: SDS Page, Recombinant, Produced, Luciferase, Activity Assay, Mutagenesis, Western Blot, Immunoprecipitation, Control, Activation Assay, Cell Culture