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ATCC american mink lung epithelial mv1lu ccl 64 cells

Optimizing hCoV-OC43 NG-ns12.9 ds-circDNA transfection and co-cultivation for infectious NG virus production. ( A ) The N protein promotes virus production from CPER-derived ds-circDNAs. HEK293T cells were co-transfected with an FL hCoV-OC43 NG-ns12.9 ds-circDNA along with an hCoV-OC43 N expression vector (pCOC42) or a control vector (pFLAG-CMV-5.1) and then co-cultivated with HCT-8 for 8 days. The numbers of NG + HCT-8 cells were counted and averaged from 10 random microscopic fields on days 6, 7, and 8 (D6–D8) post-transfection. ( B ) A representative microscopic field showing the NG + HCT-8 cells co-transfected by an FL hCoV-OC43 NG-ns12.9 ds-circDNA with an hCoV-OC43 N expression vector (pCOC42) or a control vector pFLAG-CMV-5.1 on D6 and D8. ( C, D ) Fetal bovine serum (FBS), but not new calf serum (NCS), is required for efficient virus production in HCT-8 cells. HEK293T cells were co-transfected with an FL hCoV-OC43 NG-ns12.9 ds-circDNA along with an N expression vector pCOC42 and maintained in DMEM supplemented with 2% FBS overnight. The transfected cells were then co-cultivated by the addition of HCT-8 cells with cell passage every 3 days for a total of 10 days in DMEM supplemented with 10% FBS or 10% NCS. The number of NG + HCT-8 cells was counted and averaged from 10 random microscopic fields on D8–D10 ( C ). Significantly more NG + HCT-8 cells were shown from the cells with an FL NG-ns12.9 ds-circDNA on D8–D10 when growing in the DMEM containing 10% FBS when compared with 10% NCS ( D ). ( E ) hCoV-OC43 induced visible and well-defined cytopathic effect (CPE) in both LLC-MK2 and <t>Mv1Lu</t> cells. The monolayer of LLC-MK2 or Mv1Lu cells in ~70% confluence was infected with WT hCoV-OC43 (100 µL supernatant of infected HCT-8 cells). One representative microscopic field is shown for each cell type. ( F ) Mv1Lu cells are more sensitive than LLC-MK2 cells for hCoV-OC43 infection and plaque formation. Plaque assays of LLC-MK2 and Mv1Lu cells were infected with 100 µL of each diluent after serial 10-fold dilutions of WT hCoV-OC43 virus and overlayed with semisolid media (1× DMEM, 0.5% methylcellulose and 10% FBS) for 8 days. The plaques were fixed for 30 min by 3.7% formaldehyde solution and stained with 1% crystal violet.

American Mink Lung Epithelial Mv1lu Ccl 64 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 516 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Discontinuous template switching generates coronavirus subgenomic RNAs from the 3ʹ viral genome end by 5ʹ to 3ʹ transcription"

Article Title: Discontinuous template switching generates coronavirus subgenomic RNAs from the 3ʹ viral genome end by 5ʹ to 3ʹ transcription

Journal: Journal of Virology

doi: 10.1128/jvi.01438-25

Figure Legend Snippet: Optimizing hCoV-OC43 NG-ns12.9 ds-circDNA transfection and co-cultivation for infectious NG virus production. ( A ) The N protein promotes virus production from CPER-derived ds-circDNAs. HEK293T cells were co-transfected with an FL hCoV-OC43 NG-ns12.9 ds-circDNA along with an hCoV-OC43 N expression vector (pCOC42) or a control vector (pFLAG-CMV-5.1) and then co-cultivated with HCT-8 for 8 days. The numbers of NG + HCT-8 cells were counted and averaged from 10 random microscopic fields on days 6, 7, and 8 (D6–D8) post-transfection. ( B ) A representative microscopic field showing the NG + HCT-8 cells co-transfected by an FL hCoV-OC43 NG-ns12.9 ds-circDNA with an hCoV-OC43 N expression vector (pCOC42) or a control vector pFLAG-CMV-5.1 on D6 and D8. ( C, D ) Fetal bovine serum (FBS), but not new calf serum (NCS), is required for efficient virus production in HCT-8 cells. HEK293T cells were co-transfected with an FL hCoV-OC43 NG-ns12.9 ds-circDNA along with an N expression vector pCOC42 and maintained in DMEM supplemented with 2% FBS overnight. The transfected cells were then co-cultivated by the addition of HCT-8 cells with cell passage every 3 days for a total of 10 days in DMEM supplemented with 10% FBS or 10% NCS. The number of NG + HCT-8 cells was counted and averaged from 10 random microscopic fields on D8–D10 ( C ). Significantly more NG + HCT-8 cells were shown from the cells with an FL NG-ns12.9 ds-circDNA on D8–D10 when growing in the DMEM containing 10% FBS when compared with 10% NCS ( D ). ( E ) hCoV-OC43 induced visible and well-defined cytopathic effect (CPE) in both LLC-MK2 and Mv1Lu cells. The monolayer of LLC-MK2 or Mv1Lu cells in ~70% confluence was infected with WT hCoV-OC43 (100 µL supernatant of infected HCT-8 cells). One representative microscopic field is shown for each cell type. ( F ) Mv1Lu cells are more sensitive than LLC-MK2 cells for hCoV-OC43 infection and plaque formation. Plaque assays of LLC-MK2 and Mv1Lu cells were infected with 100 µL of each diluent after serial 10-fold dilutions of WT hCoV-OC43 virus and overlayed with semisolid media (1× DMEM, 0.5% methylcellulose and 10% FBS) for 8 days. The plaques were fixed for 30 min by 3.7% formaldehyde solution and stained with 1% crystal violet.

Techniques Used: Transfection, Virus, Derivative Assay, Expressing, Plasmid Preparation, Control, Infection, Staining

Positional NG insertion into the hCoV-OC43 genome to determine hCoV-OC43 replication and synthesis direction of viral sgRNAs. ( A ) Agarose gel electrophoresis of CPER-derived hCoV-OC43 ds-circDNAs (black arrow) of NG-Δns2 (lane 1) and NG-ns12.9 (lane 2). See for details. ( B and C ) Visualization and quantification of NG + HCT-8 cells after co-cultivation with HEK293T cells transfected with CPER-derived hCoV-OC43 NG-Δns2 or NG-ns12.9 ds-circDNAs. HEK293T cells were transfected with individual FL ds-circDNA products along with an hCoV-OC43 N protein expression vector pCOC42 and then co-cultivated by addition of HCT-8 cells for the indicated days. ( B ) Representative microscopic images on D6 showing NG + HCT-8 cells from the NG-Δns2 or NG-ns12.9 ds-circDNA. ( C ) Numbers of NG + cells quantified from 10 random microscopic fields on D5 and D6, showing more NG + cells (**, P < 0.01, Student’s t -test) for the NG-ns12.9 ds-circDNA than that of the NG-Δns2 ds-circDNA. ( D ) Northern blot analysis of hCoV-OC43 RNA from HCT-8 cells in co-cultivation with HEK293T cells transfected by CPER-derived NG-Δns2 ds-circDNA (lane 1) or NG-ns12.9 ds-circDNA (lane 2) or infected by wild-type (lane 3) viruses. Total RNA extracted from the D6 cells was analyzed by Northern blot using a 32 P-labeled probe antisense to the hCoV-OC43 N ORF. The sgRNAs containing inserted NG are labeled in green. ( E, F ) Virus titration of the HCT-8 cell culture supernatant collected on D8 co-cultivation by using methylcellulose-overlay fluorescent foci ( E ) and plaque ( F ) assays in Mv1Lu cells. ( E ) Representative images of fluorescent foci on D8 of infected Mv1Lu cells (left panel). The Mv1Lu cells were infected by 100 µL of each diluent after serial 10-fold dilutions of the collected HCT-8 culture supernatant collected on D6 described above ( C ). Virus titers are calculated as fluorescent-forming units (FFU/mL) (right bar graph), showing a higher titer of hCoV-OC43 NG-ns12.9 virus (1.2 × 10 6 FFU/mL) than hCoV-OC43 NG-Δns2 virus (4.0 × 10 4 FFU/mL). ( F ) Titration of hCoV-OC43 NG-Δns2 and NG-ns12.9 virus titers by plaque assays using Mv1Lu cells infected by 100 µL of each diluent after serial 10-fold dilutions of the HCT-8 culture supernatant collected on D6 of the cell co-cultivation. The visible virus plaques of hCoV-OC43 NG-Δns2 and hCoV-OC43 NG-ns12.9 on D8 in Mv1Lu cells were visualized by crystal violet staining.

Figure Legend Snippet: Positional NG insertion into the hCoV-OC43 genome to determine hCoV-OC43 replication and synthesis direction of viral sgRNAs. ( A ) Agarose gel electrophoresis of CPER-derived hCoV-OC43 ds-circDNAs (black arrow) of NG-Δns2 (lane 1) and NG-ns12.9 (lane 2). See for details. ( B and C ) Visualization and quantification of NG + HCT-8 cells after co-cultivation with HEK293T cells transfected with CPER-derived hCoV-OC43 NG-Δns2 or NG-ns12.9 ds-circDNAs. HEK293T cells were transfected with individual FL ds-circDNA products along with an hCoV-OC43 N protein expression vector pCOC42 and then co-cultivated by addition of HCT-8 cells for the indicated days. ( B ) Representative microscopic images on D6 showing NG + HCT-8 cells from the NG-Δns2 or NG-ns12.9 ds-circDNA. ( C ) Numbers of NG + cells quantified from 10 random microscopic fields on D5 and D6, showing more NG + cells (**, P < 0.01, Student’s t -test) for the NG-ns12.9 ds-circDNA than that of the NG-Δns2 ds-circDNA. ( D ) Northern blot analysis of hCoV-OC43 RNA from HCT-8 cells in co-cultivation with HEK293T cells transfected by CPER-derived NG-Δns2 ds-circDNA (lane 1) or NG-ns12.9 ds-circDNA (lane 2) or infected by wild-type (lane 3) viruses. Total RNA extracted from the D6 cells was analyzed by Northern blot using a 32 P-labeled probe antisense to the hCoV-OC43 N ORF. The sgRNAs containing inserted NG are labeled in green. ( E, F ) Virus titration of the HCT-8 cell culture supernatant collected on D8 co-cultivation by using methylcellulose-overlay fluorescent foci ( E ) and plaque ( F ) assays in Mv1Lu cells. ( E ) Representative images of fluorescent foci on D8 of infected Mv1Lu cells (left panel). The Mv1Lu cells were infected by 100 µL of each diluent after serial 10-fold dilutions of the collected HCT-8 culture supernatant collected on D6 described above ( C ). Virus titers are calculated as fluorescent-forming units (FFU/mL) (right bar graph), showing a higher titer of hCoV-OC43 NG-ns12.9 virus (1.2 × 10 6 FFU/mL) than hCoV-OC43 NG-Δns2 virus (4.0 × 10 4 FFU/mL). ( F ) Titration of hCoV-OC43 NG-Δns2 and NG-ns12.9 virus titers by plaque assays using Mv1Lu cells infected by 100 µL of each diluent after serial 10-fold dilutions of the HCT-8 culture supernatant collected on D6 of the cell co-cultivation. The visible virus plaques of hCoV-OC43 NG-Δns2 and hCoV-OC43 NG-ns12.9 on D8 in Mv1Lu cells were visualized by crystal violet staining.

Techniques Used: Agarose Gel Electrophoresis, Derivative Assay, Transfection, Expressing, Plasmid Preparation, Northern Blot, Infection, Labeling, Virus, Titration, Cell Culture, Staining

Determination of virus infectivity and replication capacity of hCoV-OC43 NG-Δns2 and NG-ns12.9 virions by de novo virus infection of HCT-8 and Mv1Lu cells. ( A and B ) hCoV-OC43 NG-Δns2 and NG-ns12.9 virions exhibit equal infectivity to HCT-8 cells. HCT-8 cells were infected in parallel with 0.01 MOI of hCoV-OC43 NG-Δns2 or NG-ns12.9 virions titrated, as described in . NG + HCT-8 cells were quantified from five random microscopic fields at days 1, 2, and 3 post infection (D1–D3) of the indicated virus. ( A ) No difference in NG + HCT-8 cells from infection with the NG-Δns2 to NG-ns12.9 virions. ( B ) Selectively shown are one representative fluorescent image each from the HCT-8 cells infected with the NG-Δns2 or NG-ns12.9 virions at D3 post-infection. ( C and D ) hCoV-OC43 NG-Δns2 and NG-ns12.9 virions from the infected HCT-8 cell culture supernatant exhibit equal replication capacity with similar virus titers in Mv1Lu cells. The supernatant from the HCT-8 cells infected by 0.01 MOI of NG-Δns2 or NG-ns12.9 virions was collected on D3 post infection and, after serial 10-fold dilutions, 100 µL of each diluent was used to infect Mv1Lu cells, and titration of viral infectivity was carried out by the methylcellulose-overlay fluorescent foci assay ( C ). Virus titers for NG-Δns2 and NG-ns12.9 virions were calculated as fluorescent-forming units (FFU/mL) shown in a bar graph with NG-Δns2, 1.5 × 10 4 FFU/mL and NG-ns12.9, 2.0 × 10 4 FFU/mL ( D ).

Figure Legend Snippet: Determination of virus infectivity and replication capacity of hCoV-OC43 NG-Δns2 and NG-ns12.9 virions by de novo virus infection of HCT-8 and Mv1Lu cells. ( A and B ) hCoV-OC43 NG-Δns2 and NG-ns12.9 virions exhibit equal infectivity to HCT-8 cells. HCT-8 cells were infected in parallel with 0.01 MOI of hCoV-OC43 NG-Δns2 or NG-ns12.9 virions titrated, as described in . NG + HCT-8 cells were quantified from five random microscopic fields at days 1, 2, and 3 post infection (D1–D3) of the indicated virus. ( A ) No difference in NG + HCT-8 cells from infection with the NG-Δns2 to NG-ns12.9 virions. ( B ) Selectively shown are one representative fluorescent image each from the HCT-8 cells infected with the NG-Δns2 or NG-ns12.9 virions at D3 post-infection. ( C and D ) hCoV-OC43 NG-Δns2 and NG-ns12.9 virions from the infected HCT-8 cell culture supernatant exhibit equal replication capacity with similar virus titers in Mv1Lu cells. The supernatant from the HCT-8 cells infected by 0.01 MOI of NG-Δns2 or NG-ns12.9 virions was collected on D3 post infection and, after serial 10-fold dilutions, 100 µL of each diluent was used to infect Mv1Lu cells, and titration of viral infectivity was carried out by the methylcellulose-overlay fluorescent foci assay ( C ). Virus titers for NG-Δns2 and NG-ns12.9 virions were calculated as fluorescent-forming units (FFU/mL) shown in a bar graph with NG-Δns2, 1.5 × 10 4 FFU/mL and NG-ns12.9, 2.0 × 10 4 FFU/mL ( D ).

Techniques Used: Virus, Infection, Cell Culture, Titration

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Article Title: Restore Veto Property in Low Dose Aspirin/ASA Treated Preeclampsia Placenta Mesenchymal Stem Cells: Insights Into ASA-mediated Clinical Response*
Article Snippet: CCL-64 mink lung epithelial cell was purchased from American Type Culture Collection.

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Journal: Journal of Virology

Article Title: Discontinuous template switching generates coronavirus subgenomic RNAs from the 3ʹ viral genome end by 5ʹ to 3ʹ transcription

doi: 10.1128/jvi.01438-25

Figure Lengend Snippet: Optimizing hCoV-OC43 NG-ns12.9 ds-circDNA transfection and co-cultivation for infectious NG virus production. ( A ) The N protein promotes virus production from CPER-derived ds-circDNAs. HEK293T cells were co-transfected with an FL hCoV-OC43 NG-ns12.9 ds-circDNA along with an hCoV-OC43 N expression vector (pCOC42) or a control vector (pFLAG-CMV-5.1) and then co-cultivated with HCT-8 for 8 days. The numbers of NG + HCT-8 cells were counted and averaged from 10 random microscopic fields on days 6, 7, and 8 (D6–D8) post-transfection. ( B ) A representative microscopic field showing the NG + HCT-8 cells co-transfected by an FL hCoV-OC43 NG-ns12.9 ds-circDNA with an hCoV-OC43 N expression vector (pCOC42) or a control vector pFLAG-CMV-5.1 on D6 and D8. ( C, D ) Fetal bovine serum (FBS), but not new calf serum (NCS), is required for efficient virus production in HCT-8 cells. HEK293T cells were co-transfected with an FL hCoV-OC43 NG-ns12.9 ds-circDNA along with an N expression vector pCOC42 and maintained in DMEM supplemented with 2% FBS overnight. The transfected cells were then co-cultivated by the addition of HCT-8 cells with cell passage every 3 days for a total of 10 days in DMEM supplemented with 10% FBS or 10% NCS. The number of NG + HCT-8 cells was counted and averaged from 10 random microscopic fields on D8–D10 ( C ). Significantly more NG + HCT-8 cells were shown from the cells with an FL NG-ns12.9 ds-circDNA on D8–D10 when growing in the DMEM containing 10% FBS when compared with 10% NCS ( D ). ( E ) hCoV-OC43 induced visible and well-defined cytopathic effect (CPE) in both LLC-MK2 and Mv1Lu cells. The monolayer of LLC-MK2 or Mv1Lu cells in ~70% confluence was infected with WT hCoV-OC43 (100 µL supernatant of infected HCT-8 cells). One representative microscopic field is shown for each cell type. ( F ) Mv1Lu cells are more sensitive than LLC-MK2 cells for hCoV-OC43 infection and plaque formation. Plaque assays of LLC-MK2 and Mv1Lu cells were infected with 100 µL of each diluent after serial 10-fold dilutions of WT hCoV-OC43 virus and overlayed with semisolid media (1× DMEM, 0.5% methylcellulose and 10% FBS) for 8 days. The plaques were fixed for 30 min by 3.7% formaldehyde solution and stained with 1% crystal violet.

Article Snippet: The human embryonic kidney HEK293T (CRL-3216), human colorectal adenocarcinoma HCT-8 (CCL-244), monkey kidney epithelial LLC-MK2 (CCL-7) and American mink lung epithelial Mv1Lu (CCL-64) cells were obtained from ATCC.

Techniques: Transfection, Virus, Derivative Assay, Expressing, Plasmid Preparation, Control, Infection, Staining

Journal: Journal of Virology

Article Title: Discontinuous template switching generates coronavirus subgenomic RNAs from the 3ʹ viral genome end by 5ʹ to 3ʹ transcription

doi: 10.1128/jvi.01438-25

Figure Lengend Snippet: Positional NG insertion into the hCoV-OC43 genome to determine hCoV-OC43 replication and synthesis direction of viral sgRNAs. ( A ) Agarose gel electrophoresis of CPER-derived hCoV-OC43 ds-circDNAs (black arrow) of NG-Δns2 (lane 1) and NG-ns12.9 (lane 2). See for details. ( B and C ) Visualization and quantification of NG + HCT-8 cells after co-cultivation with HEK293T cells transfected with CPER-derived hCoV-OC43 NG-Δns2 or NG-ns12.9 ds-circDNAs. HEK293T cells were transfected with individual FL ds-circDNA products along with an hCoV-OC43 N protein expression vector pCOC42 and then co-cultivated by addition of HCT-8 cells for the indicated days. ( B ) Representative microscopic images on D6 showing NG + HCT-8 cells from the NG-Δns2 or NG-ns12.9 ds-circDNA. ( C ) Numbers of NG + cells quantified from 10 random microscopic fields on D5 and D6, showing more NG + cells (**, P < 0.01, Student’s t -test) for the NG-ns12.9 ds-circDNA than that of the NG-Δns2 ds-circDNA. ( D ) Northern blot analysis of hCoV-OC43 RNA from HCT-8 cells in co-cultivation with HEK293T cells transfected by CPER-derived NG-Δns2 ds-circDNA (lane 1) or NG-ns12.9 ds-circDNA (lane 2) or infected by wild-type (lane 3) viruses. Total RNA extracted from the D6 cells was analyzed by Northern blot using a 32 P-labeled probe antisense to the hCoV-OC43 N ORF. The sgRNAs containing inserted NG are labeled in green. ( E, F ) Virus titration of the HCT-8 cell culture supernatant collected on D8 co-cultivation by using methylcellulose-overlay fluorescent foci ( E ) and plaque ( F ) assays in Mv1Lu cells. ( E ) Representative images of fluorescent foci on D8 of infected Mv1Lu cells (left panel). The Mv1Lu cells were infected by 100 µL of each diluent after serial 10-fold dilutions of the collected HCT-8 culture supernatant collected on D6 described above ( C ). Virus titers are calculated as fluorescent-forming units (FFU/mL) (right bar graph), showing a higher titer of hCoV-OC43 NG-ns12.9 virus (1.2 × 10 6 FFU/mL) than hCoV-OC43 NG-Δns2 virus (4.0 × 10 4 FFU/mL). ( F ) Titration of hCoV-OC43 NG-Δns2 and NG-ns12.9 virus titers by plaque assays using Mv1Lu cells infected by 100 µL of each diluent after serial 10-fold dilutions of the HCT-8 culture supernatant collected on D6 of the cell co-cultivation. The visible virus plaques of hCoV-OC43 NG-Δns2 and hCoV-OC43 NG-ns12.9 on D8 in Mv1Lu cells were visualized by crystal violet staining.

Techniques: Agarose Gel Electrophoresis, Derivative Assay, Transfection, Expressing, Plasmid Preparation, Northern Blot, Infection, Labeling, Virus, Titration, Cell Culture, Staining

Journal: Journal of Virology

Article Title: Discontinuous template switching generates coronavirus subgenomic RNAs from the 3ʹ viral genome end by 5ʹ to 3ʹ transcription

doi: 10.1128/jvi.01438-25

Figure Lengend Snippet: Determination of virus infectivity and replication capacity of hCoV-OC43 NG-Δns2 and NG-ns12.9 virions by de novo virus infection of HCT-8 and Mv1Lu cells. ( A and B ) hCoV-OC43 NG-Δns2 and NG-ns12.9 virions exhibit equal infectivity to HCT-8 cells. HCT-8 cells were infected in parallel with 0.01 MOI of hCoV-OC43 NG-Δns2 or NG-ns12.9 virions titrated, as described in . NG + HCT-8 cells were quantified from five random microscopic fields at days 1, 2, and 3 post infection (D1–D3) of the indicated virus. ( A ) No difference in NG + HCT-8 cells from infection with the NG-Δns2 to NG-ns12.9 virions. ( B ) Selectively shown are one representative fluorescent image each from the HCT-8 cells infected with the NG-Δns2 or NG-ns12.9 virions at D3 post-infection. ( C and D ) hCoV-OC43 NG-Δns2 and NG-ns12.9 virions from the infected HCT-8 cell culture supernatant exhibit equal replication capacity with similar virus titers in Mv1Lu cells. The supernatant from the HCT-8 cells infected by 0.01 MOI of NG-Δns2 or NG-ns12.9 virions was collected on D3 post infection and, after serial 10-fold dilutions, 100 µL of each diluent was used to infect Mv1Lu cells, and titration of viral infectivity was carried out by the methylcellulose-overlay fluorescent foci assay ( C ). Virus titers for NG-Δns2 and NG-ns12.9 virions were calculated as fluorescent-forming units (FFU/mL) shown in a bar graph with NG-Δns2, 1.5 × 10 4 FFU/mL and NG-ns12.9, 2.0 × 10 4 FFU/mL ( D ).

Techniques: Virus, Infection, Cell Culture, Titration

Transiently overexpressed WWOX/TIAF1/p53 triad induces apoptosis, and determination of the kinetics of the triad formation. a L929 cells were electroporated with indicated plasmids. Overexpression of WWOX, TIAF1 and p53 induced apoptosis. Dominant-negative p53 (S46G) suppressed apoptosis mediated by p53 and TIAF1. EP: electroporation with medium only (Mean ± S.D., n = 3, * p < 0.05, student’s t test). b Ras rescue-based yeast two-hybrid analysis revealed the binding of TIAF1 to the N -terminal first WW domain of WWOX, as shown by the growth of yeast at 37 °C [ – ]. WWOX(Y33R) mutant abolished the binding. The SDR domain failed to bind TIAF1. In positive binding controls, MafB self-association and WWOX/p53 binding are shown, and empty pSos versus empty pMyr as negative controls. cDNAs constructed in the pSos are shown in the left, and pMyr in the right. c TNFα, at 100 ng/ml, rapidly upregulated the expression of TIAF1 in 30 min in L929 cells. Co-immunoprecipitation showed the binding of TIAF1 with WWOX. d By co-immunoprecipitation, TGF-β1 (5 ng/ml) had little or no effect on the binding of TIAF1 with pY-WWOX during treatment for 30 min in MCF7 cells. IgH: IgG heavy chain. e , f NCI-H1299 cells were transiently transfected with p53-DsRed, TIAF1-EGFP, and WWOX-ECFP. By 3-protein FRET analysis [ , ], Prima-1 induced p53 activation-dependent binding with WWOX and TIAF1 with time (see the number in red under FRETc columns, e Experiments were designed to let the FRET energy transfer from WWOX-ECFP to TIAF1-EGFP, and finally to p53-DsRed [ , ]. Prima-1 increased the triad complex formation in COS7 cells ( f ). g In Mv1Lu cells, TGF-β1 rapidly increased the triad complex formation in 1 h, followed by reduction ( n = 6; Student’s t test; experiments versus time 0 controls). h Prima-1 increased the binding of the first WW domain of WWOX with TIAF1 and p53 with time. Each bar represents an average of two experiments in ( f ) and ( h )

Journal: Cell Communication and Signaling : CCS

Article Title: A p53/TIAF1/WWOX triad exerts cancer suppression but may cause brain protein aggregation due to p53/WWOX functional antagonism

doi: 10.1186/s12964-019-0382-y

Figure Lengend Snippet: Transiently overexpressed WWOX/TIAF1/p53 triad induces apoptosis, and determination of the kinetics of the triad formation. a L929 cells were electroporated with indicated plasmids. Overexpression of WWOX, TIAF1 and p53 induced apoptosis. Dominant-negative p53 (S46G) suppressed apoptosis mediated by p53 and TIAF1. EP: electroporation with medium only (Mean ± S.D., n = 3, * p < 0.05, student’s t test). b Ras rescue-based yeast two-hybrid analysis revealed the binding of TIAF1 to the N -terminal first WW domain of WWOX, as shown by the growth of yeast at 37 °C [ – ]. WWOX(Y33R) mutant abolished the binding. The SDR domain failed to bind TIAF1. In positive binding controls, MafB self-association and WWOX/p53 binding are shown, and empty pSos versus empty pMyr as negative controls. cDNAs constructed in the pSos are shown in the left, and pMyr in the right. c TNFα, at 100 ng/ml, rapidly upregulated the expression of TIAF1 in 30 min in L929 cells. Co-immunoprecipitation showed the binding of TIAF1 with WWOX. d By co-immunoprecipitation, TGF-β1 (5 ng/ml) had little or no effect on the binding of TIAF1 with pY-WWOX during treatment for 30 min in MCF7 cells. IgH: IgG heavy chain. e , f NCI-H1299 cells were transiently transfected with p53-DsRed, TIAF1-EGFP, and WWOX-ECFP. By 3-protein FRET analysis [ , ], Prima-1 induced p53 activation-dependent binding with WWOX and TIAF1 with time (see the number in red under FRETc columns, e Experiments were designed to let the FRET energy transfer from WWOX-ECFP to TIAF1-EGFP, and finally to p53-DsRed [ , ]. Prima-1 increased the triad complex formation in COS7 cells ( f ). g In Mv1Lu cells, TGF-β1 rapidly increased the triad complex formation in 1 h, followed by reduction ( n = 6; Student’s t test; experiments versus time 0 controls). h Prima-1 increased the binding of the first WW domain of WWOX with TIAF1 and p53 with time. Each bar represents an average of two experiments in ( f ) and ( h )

Article Snippet: Cell lines used in this study included human breast MCF-7 and MDA-MB-231 cancer cells [ ], human prostate DU145 cells [ ], human monocytic U937 cells [ ], human lung p53-deficient NCI-H1299 [ ], murine L929 fibroblasts [ – ], monkey kidney SV40 virus-transformed COS7 fibroblasts [ , ], and primary mink lung epithelial Mv1Lu cells [ , ] (American Type Culture Collection).

Techniques: Over Expression, Dominant Negative Mutation, Electroporation, Binding Assay, Mutagenesis, Construct, Expressing, Immunoprecipitation, Transfection, Activation Assay

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american mink lung epithelial mv1lu ccl 64 cells (ATCC)

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1) Product Images from "Discontinuous template switching generates coronavirus subgenomic RNAs from the 3ʹ viral genome end by 5ʹ to 3ʹ transcription"

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