Journal: The Journal of Infectious Diseases

Article Title: Interferon-Induced Transmembrane Protein–Mediated Inhibition of Host Cell Entry of Ebolaviruses

doi: 10.1093/infdis/jiv255

Figure Lengend Snippet: Interferon-induced transmembrane (IFITM) proteins inhibit entry driven by glycoproteins representing all viral species within the genus Ebolavirus. A , 293T cells, transduced to express human IFITM1, IFITM2, IFITM3, or chloramphenicol acetyltransferase (Cat), were subsequently transduced with vectors encoding luciferase and bearing the indicated viral entry proteins. Means are shown for 3 independent experiments performed with triplicate samples. Transduction of Cat-expressing cells (control) was set as 100%. Error bars indicate standard errors of the mean (SEMs). B , Experiment conducted as described in A but with examination of cells expressing human ( top ) or rhesus macaque ( bottom ) IFITM proteins. Means and SEMs are shown for 3 independent experiments performed with triplicate samples. Transduction of Cat-expressing cells (control) was set as 100%. C , Expression of myc-tagged IFITM proteins in transduced 293 T cells was analyzed by Western blot using anti-myc antibody. D , Expression of IFITM-cyan fluorescent protein fusion proteins in transduced 293T cells was analyzed by flow cytometry. Single representative experiments are shown in C and D and were confirmed in ≥1 separate experiment. Abbreviations: BDBV, Bundibugyo virus; EBOV, Ebola virus; FLUAV, influenza A virus; GP, glycoprotein; HA, hemagglutinin; LASV, Lassa virus; MACV, Machupo virus; MARV, Marburg virus; MLV, murine leukemia virus; RESTV, Reston virus; SUDV, Sudan virus; TAFV, Taï Forest virus; VSV, vesicular stomatitis virus.

Article Snippet: For production of EBOV-like particles, plasmids encoding the viral envelope protein and GLucN/VP40 were cotransfected into 293T cells employing TransIT-2020 (Mirus).

Techniques: Transduction, Luciferase, Expressing, Control, Western Blot, Flow Cytometry, Virus

Journal: The Journal of Infectious Diseases

Article Title: Interferon-Induced Transmembrane Protein–Mediated Inhibition of Host Cell Entry of Ebolaviruses

doi: 10.1093/infdis/jiv255

Figure Lengend Snippet: Stimulation with type I interferon (IFN) induces IFN-induced transmembrane (IFITM) protein expression in human macrophages. Monocyte-derived macrophages were incubated with the indicated IFNs for 24 hours or left untreated and expression of STAT1, phosphorylated STAT1, β-actin, IFITM1, and IFITM2/3 was assessed by Western blot analysis. As controls, 293T cells were transfected with empty plasmid or expression plasmids for IFITM1–3. Similar results were obtained in 3 separate experiments. Abbreviation: MDM, monocyte-derived macrophages.

Article Snippet: For production of EBOV-like particles, plasmids encoding the viral envelope protein and GLucN/VP40 were cotransfected into 293T cells employing TransIT-2020 (Mirus).

Techniques: Expressing, Derivative Assay, Incubation, Western Blot, Transfection, Plasmid Preparation

Journal: The Journal of Infectious Diseases

Article Title: Interferon-Induced Transmembrane Protein–Mediated Inhibition of Host Cell Entry of Ebolaviruses

doi: 10.1093/infdis/jiv255

Figure Lengend Snippet: Amphotericin B does not rescue Ebola virus (EBOV) glycoprotein (GP)–driven entry from inhibition by interferon-induced transmembrane (IFITM) proteins. After 293T cells were transduced to express IFITM1, IFITM2, and IFITM3 or chloramphenicol acetyltransferase (control) and treated with 10 µmol/L amphotericin B for 1 hour, they were then transduced with luciferase-encoding murine leukemia virus particles bearing EBOV-GP or influenza A virus (FLUAV) hemagglutinin (HA). Means are shown for 3 independent experiments; error bars represent standard errors of the mean. Transduction of control cells was set as 100%.

Article Snippet: For production of EBOV-like particles, plasmids encoding the viral envelope protein and GLucN/VP40 were cotransfected into 293T cells employing TransIT-2020 (Mirus).

Techniques: Virus, Inhibition, Control, Transduction, Luciferase

Journal: The Journal of Infectious Diseases

Article Title: Interferon-Induced Transmembrane Protein–Mediated Inhibition of Host Cell Entry of Ebolaviruses

doi: 10.1093/infdis/jiv255

Figure Lengend Snippet: Inhibition of Lloviu virus (LLOV) glycoprotein (GP)–driven entry by interferon-induced transmembrane (IFITM) proteins. A , 293T cells transduced to express human IFITM1, 2, 3 or chloramphenicol acetyltransferase (Cat) as a control were subsequently transduced with vectors bearing the indicated GPs and luciferase activity in cell lysates was determined. Means and standard errors of the mean (SEMs) are shown for 3 independent experiments performed with triplicate samples. Transduction of control cells was set as 100%. Abbreviations: FLUAV-HA, influenza A virus hemagglutinin; MLV-Env, murine leukemia virus . B , RD/ Gaussia luciferase (GLucC) cells transduced to express human IFITM 3 or Cat as control were spin-infected with VP40/N-terminal portion of GLuc virus-like particles (VLPs) bearing the indicated GPs, and the luciferase activity in cell lysates was determined. Means and SEMs are shown for 2 experiments conducted with quintuplicate samples. Infection of control cells was set as 100%. Similar results were obtained when 293T cells transfected to express IFITM3 were analyzed. C , Western blot analysis of IFITM expression in transduced 293T cells. The expression of wild type-IFITM3 and an IFITM3 mutant in which the amino acids SVKS were mutated to alanine was analyzed. D , Experiment was conducted as described for A , but the SVKS-AAAA mutant was analyzed. Means and SEMs are shown for 3 independent experiments. Transduction of control cells was set as 100%. Abbreviation: MARV, Marburg virus. E , Experiment was carried as described for B, but the IFITM3 SVKS-AAAA mutant was included, and VLPs bearing no GP served as negative control. Results of a single experiment conducted with quintuplicate samples are shown and were confirmed in a separate experiment; error bars indicate standard deviations.

Article Snippet: For production of EBOV-like particles, plasmids encoding the viral envelope protein and GLucN/VP40 were cotransfected into 293T cells employing TransIT-2020 (Mirus).

Techniques: Inhibition, Virus, Control, Transduction, Luciferase, Activity Assay, Infection, Transfection, Western Blot, Expressing, Mutagenesis, Negative Control

Journal: The Journal of Infectious Diseases

Article Title: Interferon-Induced Transmembrane Protein–Mediated Inhibition of Host Cell Entry of Ebolaviruses

doi: 10.1093/infdis/jiv255

Figure Lengend Snippet: Synergistic inhibition of Ebola virus (EBOV) glycoprotein (GP)–driven entry by interferon-induced transmembrane (IFITM) proteins and GP-specific antiserum. 293T cells transduced to express IFITM1, IFITM2, and IFITM3 or chloramphenicol acetyltransferase as a control were transduced with vectors bearing the indicated GPs, which were preincubated with medium alone (control), 1:500 diluted rabbit preimmune serum, or 1:500 diluted rabbit anti–EBOV-GP antiserum for 30 minutes. The means and standard errors of the mean for 2 (Lassa virus [LASV]–GPC) or 4 (EBOV-GP and influenza A virus [FLUAV] hemagglutinin [HA]) experiments are shown.

Article Snippet: For production of EBOV-like particles, plasmids encoding the viral envelope protein and GLucN/VP40 were cotransfected into 293T cells employing TransIT-2020 (Mirus).

Techniques: Inhibition, Virus, Control, Transduction

Journal: Molecular Cancer Therapeutics

Article Title: TUB-040, a Homogeneous and Hydrophilic NaPi2b-Targeting ADC with Stably Linked Exatecan, Exhibits Long-lasting Antitumor Activity and a Well-Tolerated Safety Profile

doi: 10.1158/1535-7163.MCT-25-0254

Figure Lengend Snippet: TUB-040 shows high-affinity binding, fast internalization, potent target-specific cytotoxicity and bystander activity in vitro . A, Binding ELISA of increasing concentrations of the NaPi2b mAb and TUB-040 ADC to purified recombinant NaPi2b antigen (amino acids 235–361, NaPi2b MBP-TST-6xHis ). B, Binding of NaPi2b mAb and TUB-040 ADC to NaPi2b High OVCAR-3 cells by flow cytometry. Graphs show means of n = 2 ± SEM. C, Internalization of the NaPi2b mAb, TUB-040 ADC, isotype mAb (Iso mAb), and isotype ADC (Iso ADC) in NaPi2b-expressing OVCAR-3 and NaPi2b-negative (NaPi2b - ) SW-620. Cells were incubated for indicated time points with pHrodo-conjugated mAbs and ADCs, and fluorescence was analyzed by flow cytometry. Graphs show means of n = 2 ± SEM. D, NaPi2b-expressing OVCAR-3 or HCC-78 cells were incubated with increasing concentrations of TUB-040 ADC or Iso ADC for 7 days. Cell viability was measured via Alamar Blue/resazurin assay. E, Healthy (noncancerous) NaPi2b-negative human umbilical vein endothelial cells (HUVEC) were incubated with increasing concentrations of TUB-040 or Iso ADC for 7 days. Cell viability was measured via Alamar Blue/resazurin assay. Graphs show means of n = 2 ± SEM. F, TUB-040 bystander activity was measured via coculture assay. Cocultures of NaPi2b-overexpressing HEK293 cells and NaPi2b-negative SW-620 cells were incubated with TUB-040 ADC for 5 days, and the bystander effect was analyzed by flow cytometry. The graph shows means of n = 2 ± SEM.

Article Snippet: Cell lines were purchased from the “German Collection of Microorganisms and Cell Cultures” (DSMZ, Leibniz Institute), ATCC, Japanese Collection of Research Bioresources via Tebubio, Merck, or Thermo Fisher Scientific: OVCAR-3 (HTB-161, ATCC; RRID:CVCL_0465; purchased: July 2021; sex: female), HCC-78 (ACC 563, DSMZ; RRID:CVCL_2061; purchased: April 2021; sex: male), HEK293 (ACC 305, DSMZ; RRID:CVCL_0045; purchased December 2020; sex: female), IGROV-1 (SCC203, Merck; RRID:CVCL_1304; purchased: October 2022; sex: female), SW-620 (300466, Tebubio; RRID:CVCL_0547; purchased: September 2021; sex: male), ExpiCHO-S (A29127, Thermo Fisher Scientific; RRID:CVCL_5J31; purchased: March 2020; sex: female), and Expi293F (A14527, Thermo Fisher Scientific; RRID:CVCL_D615; purchased: July 2021; sex: female).

Techniques: Binding Assay, Activity Assay, In Vitro, Enzyme-linked Immunosorbent Assay, Purification, Recombinant, Flow Cytometry, Expressing, Incubation, Fluorescence, Resazurin Assay, Co-culture Assay