Journal: Antiviral Research

Article Title: Bioluminescence technologies to detect calicivirus protease activity in cell-free system and in infected cells

doi: 10.1016/j.antiviral.2011.02.002

Figure Lengend Snippet: Luminescence signals in FCV-infected cells. CellGlo-FCV-Cut- (light-gray bar) and CellGlo-FCV-Uncut-expressing CRFK cells (white bar) in a 96-well plate were infected with FCV at the indicated MOI, and incubated for 20 h. The ratios of the intensity of the signal between the incubation with FCV and the incubation without FCV are shown. Error bars represent the standard error of the mean ( n = 4). At least two separate experiments were performed.

Article Snippet: Crandell Rees feline kidney (CRFK) cells were obtained from the Japanese Collection of Research Bioresources and cultured in Eagle's minimal essential medium (MEM) (Sigma–Aldrich, St. Louis, MO) supplemented with 5% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) (Invitrogen).

Techniques: Infection, Expressing, Incubation

Journal: Antiviral Research

Article Title: Bioluminescence technologies to detect calicivirus protease activity in cell-free system and in infected cells

doi: 10.1016/j.antiviral.2011.02.002

Figure Lengend Snippet: BRET 2 assays in infected cells. (a) BRET 2 signals in FCV-infected cells. BRET 2 -FCV-Cut- and BRET 2 -FCV-Uncut-expressing CRFK cells in a 96-well plate were infected with FCV at the indicated MOI, and incubated for 20 h. The BRET 2 ratio was defined as the intensity of GFP 2 emission at 515 nm divided by that of RLuc at 410 nm. Error bars represent the standard error of the mean ( n = 4). At least two separate experiments were performed. (b) Cleavage products of BRET 2 -FCV-Cut- (lanes 1, 3, 5, 7, and 9) and BRET 2 -FCV-Uncut-expressing (lanes 2, 4, 6, 8, and 10) CRFK cells detected by Western blotting. M: molecular size marker (MagicMark XP: Invitrogen).

Article Snippet: Crandell Rees feline kidney (CRFK) cells were obtained from the Japanese Collection of Research Bioresources and cultured in Eagle's minimal essential medium (MEM) (Sigma–Aldrich, St. Louis, MO) supplemented with 5% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) (Invitrogen).

Techniques: Infection, Expressing, Incubation, Western Blot, Marker

Journal: Antiviral Research

Article Title: Bioluminescence technologies to detect calicivirus protease activity in cell-free system and in infected cells

doi: 10.1016/j.antiviral.2011.02.002

Figure Lengend Snippet: Effect of ribavirin in cell-free system and in FCV-infected cells. (a) InvitroGlo-FCV-Cut was expressed by in vitro translation and incubated with FCV protease diluted 50-fold in the presence of ribavirin at final concentrations of 50, 100, 200, and 400 μM. After 1 h incubation, bioluminescent signals were measured and the inhibitory effect relative to the ribavirin-untreated control was calculated. (b) CellGlo-FCV-Cut protein-expressing CRFK cells in a 96-well plate were infected with FCV at an MOI of 0.01. The medium was aspirated and replaced with medium containing ribavirin at a concentration of 50, 100, 200, or 400 μM. After 20 h, the bioluminescent signals were measured. Inhibition was calculated as the percentage decrease of luminescence relative to the ribavirin-untreated control. Error bars represent the standard error of the mean ( n = 4). At least two separate experiments were performed. (c) BRET 2 -FCV-Cut protein-expressing CRFK cells in a 96-well plate were infected with FCV at an MOI of 1. The medium was aspirated and replaced with medium containing ribavirin at a concentration of 50, 100, 200, or 400 μM. After 20 h, the bioluminescent signals were measured. Inhibition was calculated as the percentage decrease of the multiplicative inverse of the recovered BRET 2 signal ratio relative to the ribavirin-untreated control. Error bars represent the standard error of the mean ( n = 4). At least two separate experiments were performed. (d) Cleavage of BRET 2 -FCV-Cut protein in FCV-infected cells with ribavirin (lanes 1–5: 0, 50, 100, 200, and 400 μM) and uninfected cells (lane 6). M: molecular size marker (MagicMark XP; Invitrogen).

Article Snippet: Crandell Rees feline kidney (CRFK) cells were obtained from the Japanese Collection of Research Bioresources and cultured in Eagle's minimal essential medium (MEM) (Sigma–Aldrich, St. Louis, MO) supplemented with 5% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) (Invitrogen).

Techniques: Infection, In Vitro, Incubation, Control, Expressing, Concentration Assay, Inhibition, Marker

Journal: bioRxiv

Article Title: Conserved yet Divergent Smc5/6 Complex Degradation by Mammalian Hepatitis B Virus X Protein

doi: 10.1101/2024.10.28.620692

Figure Lengend Snippet: Mammalian hepatitis B virus X proteins exhibit differential Smc5/6 complex degradation activities depending on the host species. Lenti-X 293T (human), COS-7 (African green monkey), Fcwf-4 (feline), and CRFK (feline) cells were transfected with plasmids encoding mNeonGreen-HBV(A) X, mNeonGreen-DCHBV(KT116) X, or mNeonGreen or only pCAGGS. The levels of Smc6 and mNeonGreen at 2 days after transfection were analyzed by Western blotting. The results are representative of at least 3 independent experiments.

Article Snippet: Lenti-X 293T cells ( Homo sapiens or human; TaKaRa, Kusatsu, Japan, Cat# Z2180N), COS-7 ( Cercopithecus aethiops or African green monkey [AGM]; Japanese Collection of Research Bioresources Cell Bank [JCRB], Ibaraki, Japan, Cat# JCRB9127), Fcwf-4 ( Felis catus or cat; American Type Culture Collection, Manassas, VA, USA, Cat# CRL-2787), and CRFK (cat; JCRB, Cat# JCRB9035) cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Nacalai Tesque, Kyoto, Japan, Cat# 08458-16) supplemented with 10% fetal bovine serum and 1 × penicillin-streptomycin (Nacalai Tesque, Cat# 09367-34).

Techniques: Virus, Transfection, Western Blot

Journal: bioRxiv

Article Title: Conserved yet Divergent Smc5/6 Complex Degradation by Mammalian Hepatitis B Virus X Protein

doi: 10.1101/2024.10.28.620692

Figure Lengend Snippet: Subcellular localization of HBV(A) X and DCHBV(KT116) X. (A) Lenti-X 293-T cells were transfected with pCAGGS plasmids encoding mNeonGreen-HBV(A) X, mNeonGreen-DCHBV(KT116) X, mNeonGreen or only pCAGGS. (B) The nuclear fraction, cytosolic fraction, and whole-cell lysates (Total) were analyzed by Western blotting using anti-lamin A/C (nuclear), anti-α-tubulin (cytosolic), and anti-mNeonGreen (X protein) antibodies. The results are representative of at least 3 independent experiments.

Article Snippet: Lenti-X 293T cells ( Homo sapiens or human; TaKaRa, Kusatsu, Japan, Cat# Z2180N), COS-7 ( Cercopithecus aethiops or African green monkey [AGM]; Japanese Collection of Research Bioresources Cell Bank [JCRB], Ibaraki, Japan, Cat# JCRB9127), Fcwf-4 ( Felis catus or cat; American Type Culture Collection, Manassas, VA, USA, Cat# CRL-2787), and CRFK (cat; JCRB, Cat# JCRB9035) cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Nacalai Tesque, Kyoto, Japan, Cat# 08458-16) supplemented with 10% fetal bovine serum and 1 × penicillin-streptomycin (Nacalai Tesque, Cat# 09367-34).

Techniques: Transfection, Western Blot

Journal: bioRxiv

Article Title: Conserved yet Divergent Smc5/6 Complex Degradation by Mammalian Hepatitis B Virus X Protein

doi: 10.1101/2024.10.28.620692

Figure Lengend Snippet: A flow cytometry–based SMC6 degradation assay using sfCherry3C-Smc6. (A) Lenti-X 293T cells were transfected with pCAGGS plasmids encoding SpyCatcher-sfCherry3C(1-10) only, SpyTag-sfCherry3C(11)-mNeonGreen only, or in combination of SpyCatcher-sfCherry3C(1-10), SpyTag-sfCherry3C(11)-mNeonGreen, and SpyTag-sfCherry3C(11)-HA-Smc6 (human). The signals of mNeonGreen and sfCherry3C were measured 2 days after transfection. (B) Lenti-X 293T cells were co-transfected with pCAGGS plasmids encoding SpyCatcher-sfCherry3C(1-10) only, SpyTag-sfCherry3C(11)-HA-Smc6 (human) only, or in combination with mNeonGreen-HBV(A) X, mNeonGreen-DCHBV(KT116) X, or mNeonGreen. Smc6 degradation was evaluated by measuring sfCherry3C levels using flow cytometry. The results, presented as the mean and SD of sextuplicate measurements from 1 assay, are representative of at least 3 independent experiments. The differences in sfCherry3C positivity between the cells producing HBV(A) X, DCHBV(KT116) X, and mNeonGreen were evaluated using one-way ANOVA, followed by the Tukey test. * p < 0.05, *** p < 0.001, **** p < 0.0001, ns (not significant).

Article Snippet: Lenti-X 293T cells ( Homo sapiens or human; TaKaRa, Kusatsu, Japan, Cat# Z2180N), COS-7 ( Cercopithecus aethiops or African green monkey [AGM]; Japanese Collection of Research Bioresources Cell Bank [JCRB], Ibaraki, Japan, Cat# JCRB9127), Fcwf-4 ( Felis catus or cat; American Type Culture Collection, Manassas, VA, USA, Cat# CRL-2787), and CRFK (cat; JCRB, Cat# JCRB9035) cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Nacalai Tesque, Kyoto, Japan, Cat# 08458-16) supplemented with 10% fetal bovine serum and 1 × penicillin-streptomycin (Nacalai Tesque, Cat# 09367-34).

Techniques: Flow Cytometry, Degradation Assay, Transfection

Journal: bioRxiv

Article Title: Conserved yet Divergent Smc5/6 Complex Degradation by Mammalian Hepatitis B Virus X Protein

doi: 10.1101/2024.10.28.620692

Figure Lengend Snippet: DCHBV(KT116) X degrades the Smc5/6 complex independently of DDB1. (A) Lenti-X 293-T cells were transfected with DsiRNA targeting human Ddb1 or non-targeting control DsiRNA. The level of DDB1 in Lenti-X 293-T cells was determined using Western blotting. (B) After 2 days of DsiRNA transfection, the cells were co-transfected with pCAGGS plasmids encoding SpyCatcher-sfCherry3C(1-10), SpyTag-sfCherry3C(11)-HA-Smc6 (human, feline, or feline with KVRNT insertion), or in combination with mNeonGreen-HBV(A) X, mNeonGreen-DCHBV(KT116) X, or mNeonGreen. Smc6 degradation was evaluated by measuring sfCherry3C levels with flow cytometry. The results, presented as the mean and SD of sextuplicate measurements from 1 assay, are representative of at least 3 independent experiments. The differences in sfCherry3C positivity between the cells producing HBV(A) X, DCHBV(KT116) X, and mNeonGreen were evaluated using one-way ANOVA, followed by the Tukey test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns (not significant). (C) Fcwf-4 cells were transfected with DsiRNA targeting cat Ddb1 or non-targeting control DsiRNA. The level of Ddb1 in Fcwf-4 cells was determined by Western blotting. (D) After 2 days of DsiRNA transfection, the cells were co-transfected with pCAGGS plasmids encoding SpyCatcher-sfCherry3C(1-10), SpyTag-sfCherry3C(11)-HA-Smc6 (human, feline, or feline with +KVRNT insertion), in combination with mNeonGreen-HBV(A) X, mNeonGreen-DCHBV(KT116) X, or mNeonGreen. Smc6 degradation was evaluated by measuring sfCherry3C levels with flow cytometry. The results, presented as the mean and SD of sextuplicate measurements from 1 assay, are representative of at least 3 independent experiments. The differences in sfCherry3C positivity between cells producing HBV(A) X, DCHBV(KT116) X, and mNeonGreen were evaluated by one-way ANOVA, followed by the Tukey test. *** p < 0.001, **** p < 0.0001, ns (not significant).

Article Snippet: Lenti-X 293T cells ( Homo sapiens or human; TaKaRa, Kusatsu, Japan, Cat# Z2180N), COS-7 ( Cercopithecus aethiops or African green monkey [AGM]; Japanese Collection of Research Bioresources Cell Bank [JCRB], Ibaraki, Japan, Cat# JCRB9127), Fcwf-4 ( Felis catus or cat; American Type Culture Collection, Manassas, VA, USA, Cat# CRL-2787), and CRFK (cat; JCRB, Cat# JCRB9035) cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Nacalai Tesque, Kyoto, Japan, Cat# 08458-16) supplemented with 10% fetal bovine serum and 1 × penicillin-streptomycin (Nacalai Tesque, Cat# 09367-34).

Techniques: Transfection, Control, Western Blot, Flow Cytometry

Journal: International Journal of Antimicrobial Agents

Article Title: Fighting viruses with antibiotics: an overlooked path

doi: 10.1016/j.ijantimicag.2016.07.004

Figure Lengend Snippet: Main findings of recent studies on the antiviral activity of teicoplanin and ivermectin.

Article Snippet: , Coronaviruses: SARS-CoV and feline infectious peritonitis virus (FIPV) , ca. 180 , Modified glycopeptide derivatives including teicoplanin derivatives , Vero E6 (SARS-CoV) and CRFK (FIPV) cells , Between 5.4 ± 3.1 μM and 64 ± 10 μM for SARS-CoV and 1.6 ± 0.3 μM and 62 ± 29 μM for FIPV , HIV-1 (T lymphoblastoid cell line CEM).

Techniques: Activity Assay, Virus, Concentration Assay, Inhibition, Modification, Glycoproteomics, In Vitro, Transfection

Journal: Virology Journal

Article Title: Apoptosis is mediated by FeHV-1 through the intrinsic pathway and interacts with the autophagic process

doi: 10.1186/s12985-023-02267-w

Figure Lengend Snippet: FeHV-1 induces apoptosis in a time-dependent manner. FeHV-1 triggers apoptosis in permissive CRFK cells. Images indicated with B , C and D showed how the cells appears after 24, 48 and 72 h from the infection ( A indicated the control cells). The red arrows indicate the typical blebbing of apoptotic cells while the yellow arrows indicate the formation of pseudopods which connect the few cells that survived after the infection. The letter E indicates the expression of caspase 3 and its cleaved form at the various times of infection. The letters F and G indicated quantization after normalization. Full blots are available as supplementary file

Article Snippet: Crandell-Rees Feline Kidney Cells (CRFK) cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Corning) were infected using MOI 1 of FeHV-1 strain Ba/91 at different time points (3, 6, 12, 24, 48, and 72 h) to reveal the time dependence activation of apoptosis during FeHV-1 infection (cell characteristics were observed using the light microscopy ZOE Cell Imager, Bio-Rad Laboratories).

Techniques: Infection, Control, Expressing