murine bend3 cerebral endothelial cells  (ATCC)

Journal: Journal of Ginseng Research

Article Title: Panaxadiol promotes angiogenesis against chronic cerebral hypoperfusion injury through the VEGF-A/p38 MAPK/Src signaling pathway

doi: 10.1016/j.jgr.2025.10.001

Figure Lengend Snippet: PD treatment promotes the cell proliferation, migration, and tube formation in vitro . (A) The bEnd.3 cells viability after PD treatment for 24 h. (B) The cytotoxicity of PD treatment for 24 h. (C and D) Representative immunofluorescence images and quantitative graph of bEnd.3 cells. The ratio of BrdU positive cells was determined by immunofluorescence staining and expressed as a percentage of total cells. Proliferation cells are labeled with BrdU (green), and nuclei are labeled with DAPI (blue). Scale bar: 100 μm. (E and F) Representative transwell migration images and quantitative graph of bEnd.3 cells. Scale bar: 250 μm. (G and H) Representative horizontal migration images and quantitative graph of bEnd.3 cells. The wound healing of bEnd.3 cells was expressed as a percentage of the 16 h wound area compare to 0 h wound area. Scale bar: 150 μm. (I–K) Representative tube formation images and quantitative graph of bEnd.3 cells. Scale bar: 250 μm. One-way ANOVA with Tukey post hoc test was used. Each data represent mean ± SEM, n is 3–5 in per group, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 vs PD (0 μM) group or control group.

Article Snippet: bEnd.3 cells (ATCC, CRL-2299) were cultured in complete medium at 37 °C with 5 % CO 2 [ ].

Techniques: Migration, In Vitro, Immunofluorescence, Staining, Labeling, Control

Journal: mBio

Article Title: Toxoplasma effector GRA15-driven CCL5 secretion enhances brain parasite load through microvascular sequestration of phagocytes

doi: 10.1128/mbio.03444-25

Figure Lengend Snippet: Effectors and cell signaling implicated in Ccl5 expression and CCL5 secretion by endothelial cells upon challenge with T. gondii. ( A ) Representative micrographs of confluent bEnd.3 monolayers unchallenged or challenged with GFP-expressing T. gondii tachyzoites (RH-LDM, green) and stained for the tight junction protein ZO-1 (red), with DAPI-stained nuclei (blue). Scale bar = 20 μm. ( B–F ) Relative mRNA expression (quantitative polymerase chain reaction [qPCR]) of Ccl5 in confluent bEnd.3 cell monolayers challenged with freshly egressed T. gondii tachyzoites (indicated line/mutant, MOI 2, 24 h). The unchallenged condition indicates cells left untreated in the complete medium (CM) during the duration of the assay. ( B ) T. gondii type I (RH-LDM) and tachyzoite lysate (MOI 2-equivalent). ( C ) T. gondii type I (RH-LDM) and type II (PRU A7) in the presence of NF-kB inhibitors (NF-kBi) JSH-23 (9 μM) and TPCA-1 (3 μM), or dimethyl sulfoxide (DMSO) as a vehicle control. ( D ) T. gondii type I (RH, parental wild type) and MYR1-deficient mutant (Δ myr1 ); T. gondii type II (PRU Ku80, parental wild type) and MYR1-deficient mutant (Δ myr1 ). ( E ) T. gondii type II (PRU Ku80, parental wild type) and TEEGR-deficient mutant (Δ TEEGR ). ( F ) T. gondii type II (PRU A7, parental wild type), GRA15-deficient mutant (Δ gra15 ), and reconstituted mutant (Δ gra15 +GRA15). ( G ) Relative mRNA expression (qPCR) of Ccl5 in confluent primary MBEC monolayers challenged with T. gondii tachyzoites (PRU A7 wild type or Δ gra15 , MOI 2, 24 h). ( H ) Abundance of CCL5 (pg/mlL) in supernatants from MBECs challenged as in panel ( G ) for 24 h, determined by ELISA. For each condition, data are from 3 to 5 independent experiments (n = 3–5). In graphs, means (± SEM) are indicated. qPCR data are displayed as fold change (2 -ΔΔCt ) in relation to the unchallenged condition. Statistical comparisons were performed with the mixed-effects model ( B, C ) and one-way analysis of variance (ANOVA), with Sidak's post hoc test ( D–H ) ;* P < 0.05, ** P < 0.01, *** P < 0.001, ns: non significant.

Article Snippet: Human foreskin fibroblasts (HFF-1 SCRC-1041, American Type Culture Collection) and bEnd.3 cells (CRL-2299, American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium, high glucose (DMEM; HyClone), with 10% FBS (HyClone), 20 μg/mL gentamicin (Sigma-Aldrich) and 2 mM L-glutamine at 37°C/5% CO 2 .

Techniques: Expressing, Staining, Real-time Polymerase Chain Reaction, Mutagenesis, Control, Enzyme-linked Immunosorbent Assay

Journal: mBio

Article Title: Toxoplasma effector GRA15-driven CCL5 secretion enhances brain parasite load through microvascular sequestration of phagocytes

doi: 10.1128/mbio.03444-25

Figure Lengend Snippet: Chemotactic responses and transmigration of T. gondii -challenged DCs toward rCCL5. ( A, B ) Representative motility plots of T. gondii (PRU A7)-infected or bystander DCs in a collagen matrix with CM ( A ) or with a rCCL5 gradient ( B ), as detailed in Materials and Methods. Scales are expressed in μm. Tracks of cells migrating to the left side from their initial position are shown in black, and the opposite in gray. The red dot represents the center of mass. For each condition, a rose plot diagram depicts the chemotactic tendency of the cell population. P -values indicate the chemotaxis effect compared to hypothetical uniformity of a circular distribution of cell endpoints (Rayleigh test as described in Methods). Plots depict 60 tracked cells per condition from 1 representative experiment. ( C ) Mean forward migration index (FMI) for T. gondii -infected or bystander DCs in a collagen matrix with CM (mock) or with a rCCL5 gradient, as described under Materials and Methods. Data are from 168 to 220 analyzed cells per condition from 4 independent experiments. ( D ) Mean FMI for DCs infected with T. gondii wild type II (PRU A7) or GRA15 mutant (PRUΔ gra15 ), in CM (mock) or with a rCCL5 gradient. Data are from 77 to 89 analyzed cells per condition from 3 independent experiments. ( E ) Schematic representation of the experimental setup in a transwell filter system with polarized endothelial monolayers grown in the upper compartment of the culture insert: (1) TCER, (2) DCs challenged with T. gondii tachyzoites added to the upper compartment, and (3) FITC-dextran (3 kDa) fluorescent permeability tracer added to the upper compartment and measured in the lower compartment, as detailed under Materials and Methods. ( F ) Relative transmigration of DCs challenged with T. gondii tachyzoites across polarized bEnd.3 cell monolayers in the presence of rCCL5 (250 ng/mL) added to the lower compartment. When indicated, DCs were pretreated with mvc (10 μM) 1 h prior to the start of the assay. Transmigration frequency indicates the ratio of transmigrated DCs (counted in a lower chamber) related to the number of DCs initially added to the upper chamber. Shown as fold change in relation to the control condition (unchallenged mock-treated DCs). ( G ) TCER of bEnd.3 cell monolayers after DCs transmigration. Data are shown as % TCER values (Ω•cm 2 ) relative to TCER values at the initiation of the assay (100%), as detailed in Materials and Methods. ( H ) Permeability to FITC-dextran (3 kDa) of bEnd.3 cell monolayers after DCs transmigration. Signal, measured as arbitrary fluorescence units (AU), from leaked FITC-dextran in the lower compartment was recorded at the end of the transmigration assay and related to the signal of the culture insert in the absence of a polarized monolayer (100%). Data are from 3 ( D ), 4 ( C ), or 6 ( E–G ) independent experiments. Means (±SEM) are indicated. Statistical comparisons were performed with one-way ANOVA, with Sidak’s post hoc test ( C, D ) and mixed-effects model ( F–H ) ;* P < 0.05; ** P < 0.01, **** P < 0.0001, ns: nonsignificant.

Article Snippet: Human foreskin fibroblasts (HFF-1 SCRC-1041, American Type Culture Collection) and bEnd.3 cells (CRL-2299, American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium, high glucose (DMEM; HyClone), with 10% FBS (HyClone), 20 μg/mL gentamicin (Sigma-Aldrich) and 2 mM L-glutamine at 37°C/5% CO 2 .

Techniques: Transmigration Assay, Infection, Chemotaxis Assay, Migration, Mutagenesis, Permeability, Control, Fluorescence