u118 brain cells  (ATCC)

Journal: iScience

Article Title: Phosphatidylserine receptors TIM-1 and AXL mediate tick-borne Powassan virus entry

doi: 10.1016/j.isci.2025.113930

Figure Lengend Snippet: Genomic sequence analysis and infectious U118 neuronal cell culture system (A) Phylogenetic analysis of five Powassan viral genome sequences from known lineage I and II strains. GenBank accession numbers are provided along with strain information including location and year of isolation. Phylogenetic tree was constructed using the neighbor-joining method with pairwise distance estimation following ClustalW alignment program. (B) Matrix panel shows the percent identity in the upper right and nucleotide divergence in the lower left of aligned Powassan viral genomic sequences (ClustalW program, DNASTAR Lasergene MegAlign 15). (C) Graph shows the RT-qPCR analysis of POWV (LB), POWV ( M11665 ), and DTV (SPO) genome replication in U118 cells 24 and 48 hpi. The y axis shows genome copies per 50 ng of total RNA in log scale. Error bars represent the SD of three biological replicates. Student’s t test. ∗ p < 0.01, ∗∗ p < 0.001, ∗∗∗ p < 0.0001, ∗∗∗∗ p < 0.00001. (D) Representative immunofluorescence images of mock and LB-, DTV- ( M11665 ), and DTV- (SPO) infected U118 neuronal cells detected by viral NS1 at 24 and 48 hpi (scale bars, 25 μm). Red, NS1 viral protein; blue, DAPI. (E) Western blot analysis of infected cell lysates probed with the anti-M protein antibody targeting LB, DTV ( M11665 ), and DTV (SPO) at 24 and 48 hpi. Cells were infected with each virus at an MOI of 1. Two independent experiments were conducted. Representative data are presented.

Article Snippet: U118 brain cells (HTB-15, ATCC) were cultured as monolayers in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1X non-essential amino acids (NEAAs), penicillin (100 units/ml), and streptomycin (100 units/ml).

Techniques: Sequencing, Cell Culture, Isolation, Construct, Genomic Sequencing, Quantitative RT-PCR, Immunofluorescence, Infection, Western Blot, Virus

Journal: iScience

Article Title: Phosphatidylserine receptors TIM-1 and AXL mediate tick-borne Powassan virus entry

doi: 10.1016/j.isci.2025.113930

Figure Lengend Snippet: Expression analysis of TIM-1, TIM-4, AXL, TYRO3, and MERTK on Vero E6 and U118 cells and viral entry-neutralization assay (A) FSC/SSC plot with gating and histograms from flow analysis are shown. Black line shows cell staining by control goat antibody, while the red line represents staining by antibodies against specific PtdSer receptors. The value indicated in the histogram corresponds to the expression level of each receptor protein calculated by subtracting the mean fluorescent intensity of the control antibody-stained cells from that of the cells stained with an antibody specific to a PtdSer receptor. ND, not detected. Note: TIM-1 and AXL expression on the Vero E6 cell surface was found. TIM-4, TYRO3, and MERTK were not expressed. On the U118 cell surface, only AXL expression was found. (B) Schematic representation of neutralization assay using 100 plaque-forming units of virus incubated with soluble forms of TIM-1 receptor Fc-sTIM1 (sTIM1dMLDR801) or control protein Fc-NS (NCFcCQ R801). (C) Graphs show fold inhibition of indicated viral replication following neutralization with sTIM1dMLDR801. Error bars represent the SD using three biological replicates. Student’s t test. ∗ p < 0.01, ∗∗ p < 0.001, ∗∗∗ p < 0.0001. Representative data from two independent experiments were provided.

Article Snippet: U118 brain cells (HTB-15, ATCC) were cultured as monolayers in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1X non-essential amino acids (NEAAs), penicillin (100 units/ml), and streptomycin (100 units/ml).

Techniques: Expressing, Neutralization, Staining, Control, Virus, Incubation, Inhibition