Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: (A) A mammalian two-hybrid assay was performed by transfecting Vero cells with the indicated combinations of pACT and pBIND plasmids along with the pG5luc firefly luciferase reporter plasmid. At 48 h after transfection, the normalized firefly luciferase activity (firefly luciferase activity/Renilla luciferase activity) was measured and represented as fold of the control activity, which was obtained by combination with the empty pACT or empty pBIND plasmid. Data are the mean ± SD of at least three independent experiments. (B) 293T cells were transfected for 24 h with FLAG-tagged IFITM1 and HA-tagged 3Cm, 2B, 2BC, 2C, 3A, or 3AB, or HA-tagged IFITM1 and FLAG-tagged TGN46 expression plasmids, as indicated, followed by coimmunoprecipitation (IP) with an anti-FLAG, -HA or control IgG antibody. The resulting immunoprecipitates and whole-cell lysates were subjected to immunoblotting (IB) with anti-FLAG and anti-HA antibodies. (C) Vero cells were transfected with FLAG-IFITM1. At 24 h after post-transfection, the cells were fixed and double stained with anti-IFITM and anti-EEA1, anti-CD63, or anti-LBPA antibodies, as indicated. Pearson correlation coefficient analyses for data were obtained from ≥10 cells. Correlation coefficients are presented as the mean and standard deviation. (D) Vero cells was transfected with FLAG-IFITM1 and HA-2B, HA-2BC, HA-2C, HA-3A, or HA-3AB. At 24 h after transfection, cells were fixed and stained with anti-FLAG and anti-HA antibodies. Bars, 4 μm. Pearson correlation coefficient analyses for data were obtained from 4–8 cells.

Article Snippet: Mouse monoclonal anti-CD63 antibody was from Novus Biologicals.

Techniques: Two Hybrid Assay, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Control, Expressing, Western Blot, Staining, Standard Deviation

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: (A) A mammalian two-hybrid analysis was carried out to examine interactions between IFITM1 and ACBD3, PI4KB, OSBP, or CERT, and the results are shown as described in . Data are the mean ± SD of at least three independent experiments. (B) HA-tagged IFITM1 and FLAG-tagged ACBD3, PI4KB, OSBP, or CERT were cotransfected into 293T cells, and the cell lysates were subjected to immunoprecipitation with anti-HA antibody or control IgG. The resulting immunocomplexes and whole-cell lysates were detected by anti-FLAG and anti-HA antibodies. (C) Vero cells were transfected with FLAG-IFITM1. At 24 h, the cells were labeled with anti-FLAG and anti-ACBD3 (top), anti-PI4KB (middle), or anti-OSBP (bottom) antibodies. (D) Vero IFITM1 cells were incubated with Tet (−) or Tet (+) for 72 h, and the cells were fixed and double stained with the indicated antibodies. (E) Vero cells were transfected with HA-IFITM1. At 24 h, the cells were labeled using anti-HA, anti-OSBP and anti-EEA1, or anti-CD63 antibodies. Bars, 4 μm. Pearson correlation coefficient analyses for data were obtained from ≥10 cells. Correlation coefficients are presented as the mean and standard deviation (C-E).

Article Snippet: Mouse monoclonal anti-CD63 antibody was from Novus Biologicals.

Techniques: Immunoprecipitation, Control, Transfection, Labeling, Incubation, Staining, Standard Deviation

Journal: Journal of Extracellular Vesicles

Article Title: Lipid profile of circulating placental extracellular vesicles during pregnancy identifies foetal growth restriction risk

doi: 10.1002/jev2.12413

Figure Lengend Snippet: Characterization and validation of placental sEVs isolated from maternal plasma. (a) Representative NTA measurements of P‐sEVs isolated from maternal plasma of normal and SGA pregnancies. (b) Immunoblots for CD63, ALIX, TSG101, PLAP and CALNEXIN (negative control) of P‐sEVs isolated from maternal plasma of normal and SGA pregnancies at gestational windows G1, G2, G3 and G4 of pregnancy ( n = 3 different sEV isolations). (c) Representative electron micrographs of P‐sEVs isolated from maternal plasma of normal pregnancy. Bar = 100 nm. (d) Immunoblots for ApoB and ALIX of total (T‐sEV) and placental (P‐sEV) extracellular vesicles isolated from maternal plasma of normal pregnancy ( n = 3 different sEV isolations). (e‐upper panels) Representative flow cytometry density plots for CD63 and ApoB in T‐sEVs and P‐sEVs isolated from maternal plasma of normal pregnancy. (e‐lower panel, left) Representative cytometry density plots for CD63, PLAP and ApoB in P‐sEVs from maternal plasma of normal pregnancy. (e‐lower panel, right) Percentage of double positive CD63/ApoB EVs in total and placental sEVs ( n = 4 different sEV isolations). EV, extracellular vesicles; NTA, nanoparticle tracking analysis; sEV, small EV; SGA, small‐for‐gestational age.

Article Snippet: Latex beads were resuspended in 100 μL of PBS for staining with combinations of 1:100 diluted PE‐conjugated mouse anti‐human CD63 (Novus #NBP2‐42225PE), APC‐conjugated mouse anti‐human PLAP (Novus #NB110‐3638APC) and AF488‐conjugated mouse anti‐human ApoB (Santa Cruz #sc13538) monoclonal antibodies for 30 min at 4°C.

Techniques: Biomarker Discovery, Isolation, Clinical Proteomics, Western Blot, Negative Control, Flow Cytometry, Cytometry

Journal: iScience

Article Title: Identification of optimal conditions for human placental explant culture and extracellular vesicle release

doi: 10.1016/j.isci.2023.108046

Figure Lengend Snippet:

Article Snippet: Human anti-CD81 PE-Cy7 , Cellarcus Biosciences , Cat# CBS12-PC7-100T.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Proliferation Assay, LDH Cytotoxicity Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: microRNA Expression of Renal Proximal Tubular Epithelial Cells and Their Extracellular Vesicles in an Inflammatory Microenvironment In Vitro

doi: 10.3390/ijms241311069

Figure Lengend Snippet: Characterization of PTCs ( A , B ) and EVs isolated from PTC supernatant ( C , D ). ( A ) Characteristic phase contrast microscopy of confluent PTCs cultured in standard cell culture. ( B ) Representative flow cytometric overlay histograms of characteristic PTC marker expression (CD13, CD26, and EpCAM), and of CD63, a characteristic marker of exosomes. The black histograms represent isotype controls. ( C ) Representative nanoparticle tracking analysis (NTA) of EVs isolated from unstimulated PTC after standard cell culture for 48 h. ( D ) Representative flow cytometric overlay histogram of PTC-EVs isolated using EpCAM-beads and immunostained with CD63-APC. The black histogram represents an unstained control. ( E ) Representative nanoparticle tracking analysis (NTA) of EVs isolated from PTCs after culture in an inflammatory microenvironment for 48 h.

Article Snippet: The bead-EV solution (100 µL) was then stained with 20 µL of APC-labeled anti-CD63 detection antibody (ImmunoTools, Friesoythe, Germany) and incubated under rotation at RT in the dark for 45 min and washed and re-suspended in a 300 µL isolation buffer.

Techniques: Isolation, Microscopy, Cell Culture, Marker, Expressing