(F) Transmission electron microscopy of purified virus. oncolytic vaccinia viruses that can mimic complement-resistant extracellular enveloped virions by incorporating human CRP CD55 on the intracellular mature virion (IMV) membrane. Methods The N-terminus of the Cetilistat (ATL-962) human CD55 protein was fused to the transmembrane domains of the six type I membrane proteins of the IMV; the resulting recombinant viruses were named SJ-600 series viruses. The SJ-600 series viruses also expressed human granulocyte-macrophage colony-stimulating factor (GM-CSF) to activate dendritic cells. The viral thymidine kinase (and genes were placed under the control of the VACV synthetic early/late promoter and synthetic late promoter, Cetilistat (ATL-962) respectively.32 All viruses were amplified in HeLa cells and purified by 36% sucrose cushion centrifugation, in accordance with the standard protocol.33 Flow cytometry U-2-OS cells were infected at a multiplicity of infection (MOI) of 0.5 and harvested 16?hours post-infection. The infected cells were fixed, permeabilized, and stained with anti-CD55 antibody (#67; Invitrogen, Carlsbad, California, USA) (1:250); they were then incubated with Alexa Fluor 594-conjugated secondary antibody (Invitrogen) (1:1000). CD55 expression was detected by LSRFortessa (BD Biosciences, Franklin Lakes, New Jersey, USA) and analyzed with FlowJo software. Western blotting analysis Protein from purified virions was separated in pre-casted SDS-PAGE 4C15% gels (Bio-Rad, Hercules, California, USA) by electrophoresis and transferred onto nitrocellulose membranes in Trans-Blot SD Cell (Bio-Rad). The membranes were stained with anti-CD55 (#NaM16-4D3; Santa Cruz Biotechnology, Santa Cruz, California, USA) (1:200), anti-VACV A27 (#NR-627; BEI Resources, Manassas, Virginia, USA) (1:5000), and anti–actin (#BA3R; Invitrogen) (1:1000) antibodies, then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1:5000). In deglycosylation experiments, lysed virions were treated with Protein Deglycosylation Mix (New England Biolabs, Ipswich, Massachusetts USA) and analyzed with antibodies to CD55, VACV A27, and -actin. Blots were imaged using Davinch-Chemi CAS-400 (Davinch-K, Seoul, Korea). Immunofluorescence imaging U-2-OS cells were infected at an MOI of 5 for 12?hours; they were then fixed, permeabilized, and incubated with anti-CD55 (#67; Invitrogen) (1:1000) and anti-VACV A27 (#ab35219; Abcam, Cambridge, UK) (1:1000) antibodies. Next, cells were incubated with Alexa Fluor 488-conjugated (Abcam) (1:1000) and Alexa Fluor 594-conjugated (Jackson ImmunoResearch, West Grove, Pennsylvania, USA) (1:1000) secondary antibodies, as well as 4,6-diamidino-2-phenylindole (DAPI) (Invitrogen); images were obtained by confocal microscopy (STELLARIS 8; Leica Microsystems, Wetzlar, Germany). Transmission electron microscopy Purified virions were incubated with anti-CD55 antibody (#NaM16-4D3; Santa Cruz) (1:50), then with 12?nm colloidal gold-conjugated antibody (Jackson ImmunoResearch) (1:20). Grids were negatively stained with NanoVan (Nanoprobes, Yaphank, New York, USA) and imaged by transmission Mouse monoclonal to PSIP1 electron microscopy at 120 kV (JEM-1400; JEOL, Tokyo, Japan). In vitro viral stability in human serum SJ-600 series viruses were mixed with commercially available normal human serum (HS) (Sigma-Aldrich, Darmstadt, Germany) to a final serum dilution of 20%. U-2-OS cells were incubated with virus for 3?days (until plaques formed). In subsequent experiments, cells were infected with virus in the presence of 20% or 50% HS. The numbers of plaques were compared with the numbers obtained from control cells. In vitro cytotoxicity Ten human cancer cell lines were infected with serially diluted virus for 72?hours. Cell Counting Kit-8 (CCK-8) solution (Dojindo, Kumamoto, Japan) was added, and the absorbance at 450?nm (A450) was measured using a Microplate Absorbance Reader (Tecan, Mannedorf, Switzerland). Experimental animals NOD.Cg-PrkdcIL2rggene was fused with the transmembrane domain of one of the vaccinia virus membrane proteins, as indicated. GFP, green fluorescent protein; GM-CSF, granulocyte-macrophage colony-stimulating factor; LacZ, -galactosidase; Luc, luciferase; N/A, not applicable; TM, transmembrane domain; VV, vaccinia virus. Supplementary data jitc-2022-006024supp002.pdf Confirmation of the expression of human CD55 Cetilistat (ATL-962) on SJ-600 series OVs The expression of CD55 by SJ-600 series recombinant VACVs was evaluated by flow cytometry analysis of virus-infected U-2-OS cells. Cells infected with the control JX-594 virus, in which only GM-CSF was recombined with the J2R region, were used as negative controls (table 1).32 Although CD55 was.