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american mink lung epithelial mv1lu ccl 64 cells (ATCC)

Name: american mink lung epithelial mv1lu ccl 64 cells
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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 616 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

Image Search Results

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

C-Jun and EGFR OA activation, necessary for OA-cell migration, are decoupled in Mv1Lu cells. ( a ) The figure shows representative images of cell migration obtained under control conditions compared to those with 5 µM OA and OA plus 2,5 µM EGFRi after 24 h treatment. Scale bar 200 µm. ( b ) Plot represents cell migration as the difference obtained between the quantified areas at time 0 h and time 24 h in each condition. Asterisks indicate statistically significant differences between the selected conditions according to a one-way ANOVA statistical analysis: (*p < 0.05, **p < 0.005, ***p < 0.001 and ****p < 0.0001). ( c ) Total protein extracts from serum-starved sub-confluent Mv1Lu cells treated with 5 µM OA, EGF or DMSO equivalent volume as vehicle control. Different protein phosphorylation was assayed at the indicated times (h) targeting phospho-EGFR (Tyr 1068), phospho-ERK1/2 (Thr 202/Tyr 204), phospho-JNK1/2 (Thr 183/Tyr 185) and phospho-c-Jun (Ser 63). Additionally, total protein expression was assayed: ERK1/2, JNK1/2 and c-Jun. β-Actin was used as a loading control. A representative experiment is shown. ( d ) Plots with intensity values of each protein assayed by Western-blot, by gathering the data of three independent experiments. Asterisks indicate statistically significant differences between the selected conditions according to a one-way ANOVA statistical analysis: (*p < 0.05, **p < 0.005, ***p < 0.001 and ****p < 0.0001).

Journal: Scientific Reports

Article Title: Oleanolic acid stimulation of cell migration involves a biphasic signaling mechanism

doi: 10.1038/s41598-022-17553-w

Figure Lengend Snippet: C-Jun and EGFR OA activation, necessary for OA-cell migration, are decoupled in Mv1Lu cells. ( a ) The figure shows representative images of cell migration obtained under control conditions compared to those with 5 µM OA and OA plus 2,5 µM EGFRi after 24 h treatment. Scale bar 200 µm. ( b ) Plot represents cell migration as the difference obtained between the quantified areas at time 0 h and time 24 h in each condition. Asterisks indicate statistically significant differences between the selected conditions according to a one-way ANOVA statistical analysis: (*p < 0.05, **p < 0.005, ***p < 0.001 and ****p < 0.0001). ( c ) Total protein extracts from serum-starved sub-confluent Mv1Lu cells treated with 5 µM OA, EGF or DMSO equivalent volume as vehicle control. Different protein phosphorylation was assayed at the indicated times (h) targeting phospho-EGFR (Tyr 1068), phospho-ERK1/2 (Thr 202/Tyr 204), phospho-JNK1/2 (Thr 183/Tyr 185) and phospho-c-Jun (Ser 63). Additionally, total protein expression was assayed: ERK1/2, JNK1/2 and c-Jun. β-Actin was used as a loading control. A representative experiment is shown. ( d ) Plots with intensity values of each protein assayed by Western-blot, by gathering the data of three independent experiments. Asterisks indicate statistically significant differences between the selected conditions according to a one-way ANOVA statistical analysis: (*p < 0.05, **p < 0.005, ***p < 0.001 and ****p < 0.0001).

Article Snippet: Mink Lung Epithelial (Mv1Lu) , , cells were grown in Eagle's Minimum Essential Medium (EMEM) (Biowest, Nuaillé, France).

Techniques: Activation Assay, Migration, Control, Phospho-proteomics, Expressing, Western Blot

Specific inhibitors against EGFR, MEK and JNK, upon OA treatment, show the independent OA-activation on c-Jun at the edge of wound-scratched Mv1Lu cells at wound edge. ( a ) Confluent Mv1Lu cells were scratched and allowed to migrate for 6 h. Cells treated with 5 µM OA or DMSO equivalent volume were immunostained with specific antibodies against c-Jun transcription factor. Additionally, cells were treated 30 min before with the following inhibitors: 2.5 µM EGFRi, 50 µM MEKi and 15 µM JNKi. Co-staining with phalloidin and Hoechst-33258 was used to show actin cytoskeleton and nuclei, respectively. Images of p-c-Jun fluorescence were converted into pseudo-color with ImageJ software to show the intensity of c-Jun staining. Color rainbow scale represents fluorescence intensity for phospho-c-Jun. Actin fibers (F-actin): red. Nuclei: blue. Images were obtained with a confocal microscope. This experiment was repeated at least three times. Representative images are shown. Scale bar indicates 10 µm. ( b ) Plot represents the data obtained by p-c-Jun intensity at cell nuclei. In every condition, each point on the plot represents p-c-Jun intensity at the nucleus of one cell, quantified by ImageJ software. With the collected data of p-c-Jun intensity, a one-way ANOVA statistical analysis was performed (*p < 0.05, **p < 0.005, ***p < 0.001 and ****p < 0.0001).

Journal: Scientific Reports

Article Title: Oleanolic acid stimulation of cell migration involves a biphasic signaling mechanism

doi: 10.1038/s41598-022-17553-w

Figure Lengend Snippet: Specific inhibitors against EGFR, MEK and JNK, upon OA treatment, show the independent OA-activation on c-Jun at the edge of wound-scratched Mv1Lu cells at wound edge. ( a ) Confluent Mv1Lu cells were scratched and allowed to migrate for 6 h. Cells treated with 5 µM OA or DMSO equivalent volume were immunostained with specific antibodies against c-Jun transcription factor. Additionally, cells were treated 30 min before with the following inhibitors: 2.5 µM EGFRi, 50 µM MEKi and 15 µM JNKi. Co-staining with phalloidin and Hoechst-33258 was used to show actin cytoskeleton and nuclei, respectively. Images of p-c-Jun fluorescence were converted into pseudo-color with ImageJ software to show the intensity of c-Jun staining. Color rainbow scale represents fluorescence intensity for phospho-c-Jun. Actin fibers (F-actin): red. Nuclei: blue. Images were obtained with a confocal microscope. This experiment was repeated at least three times. Representative images are shown. Scale bar indicates 10 µm. ( b ) Plot represents the data obtained by p-c-Jun intensity at cell nuclei. In every condition, each point on the plot represents p-c-Jun intensity at the nucleus of one cell, quantified by ImageJ software. With the collected data of p-c-Jun intensity, a one-way ANOVA statistical analysis was performed (*p < 0.05, **p < 0.005, ***p < 0.001 and ****p < 0.0001).

Article Snippet: Mink Lung Epithelial (Mv1Lu) , , cells were grown in Eagle's Minimum Essential Medium (EMEM) (Biowest, Nuaillé, France).

Techniques: Activation Assay, Staining, Fluorescence, Software, Microscopy

OA promotes changes in FAs and actin cytoskeleton. Confluent Mv1Lu cells were scratched and allowed to migrate for 6 and 12 h. Cells treated with 5 µM OA or DMSO equivalent volume (vehicle control) were immunostained with specific antibodies against paxillin (green). Co-staining with phalloidin and Hoechst-33258 was used to show actin cytoskeleton (magenta) and nuclei (blue), respectively. This experiment was repeated at least three times. Representative images are shown. Scale bar indicates 10 µm. Quantification of the density of FA as FA number per filopodia area. Quantification of FA size (average size) at the filopodia area. One-way ANOVA statistical analysis was performed (*p < 0.05, **p < 0.005, ***p < 0.001 and ****p < 0.0001).

Journal: Scientific Reports

Article Title: Oleanolic acid stimulation of cell migration involves a biphasic signaling mechanism

doi: 10.1038/s41598-022-17553-w

Figure Lengend Snippet: OA promotes changes in FAs and actin cytoskeleton. Confluent Mv1Lu cells were scratched and allowed to migrate for 6 and 12 h. Cells treated with 5 µM OA or DMSO equivalent volume (vehicle control) were immunostained with specific antibodies against paxillin (green). Co-staining with phalloidin and Hoechst-33258 was used to show actin cytoskeleton (magenta) and nuclei (blue), respectively. This experiment was repeated at least three times. Representative images are shown. Scale bar indicates 10 µm. Quantification of the density of FA as FA number per filopodia area. Quantification of FA size (average size) at the filopodia area. One-way ANOVA statistical analysis was performed (*p < 0.05, **p < 0.005, ***p < 0.001 and ****p < 0.0001).

Article Snippet: Mink Lung Epithelial (Mv1Lu) , , cells were grown in Eagle's Minimum Essential Medium (EMEM) (Biowest, Nuaillé, France).

Techniques: Control, Staining

OA induces FAK phosphorylation and promotes its localization in focal adhesion with paxillin in Mv1Lu cells. ( a ) The activation of FAK was assessed by Western Blot at Tyr 925, at the indicated times in the presence of 5 µM OA, DMSO equivalent volume or EGF. Additionally, total protein expression was assayed: FAK. β-Actin was used as a loading control. Phospho-FAK intensity values in Western-blot were quantified by ImageJ to conduct a one-way ANOVA analysis showed on the plot (*p < 0.05, **p < 0.005, ***p < 0.001 and ****p < 0.0001). ( b ) Confluent Mv1Lu cells were scratched and allowed to migrate for 6 h. Pictures show the immunostaining with specific antibodies against phospho-FAK (Tyr 925) and paxillin. Co-staining with Hoechst-33258 was used to reveal nuclei. P-FAK: green. Paxillin: red. Nuclei: blue. ( c ) Pictures and graphs represent a colocalization analysis performed by Zeiss Efficient Navigation (ZEN) software. Colocalized pixels are highlighted in white at the immunostaining images. Graphs are dot plots representing both p-FAK and paxillin intensities in terms of number of pixels: p-FAK pixels on Y axis and paxillin pixels on X axis. The 3 quadrant (upper right) represents overlapped (common) pixels between both proteins. ( d ) Pictures of each condition (two) were divided in three horizontally distributed sectors. Pearson’s correlation coefficient in each sector of each condition was calculated by the average pixel intensity of p-FAK and paxillin overlapped pixels. The plot represents Pearson’s correlation values for each condition, each dot representing the value obtained in one sector. Asterisks indicate statistically significant differences between conditions according to one-way ANOVA statistical analysis: (*p < 0.05, **p < 0.005, ***p < 0.001 and ****p < 0.0001). This experiment was repeated at least three times. Representative images are shown. Scale bar indicates 10 µm.

Journal: Scientific Reports

Article Title: Oleanolic acid stimulation of cell migration involves a biphasic signaling mechanism

doi: 10.1038/s41598-022-17553-w

Figure Lengend Snippet: OA induces FAK phosphorylation and promotes its localization in focal adhesion with paxillin in Mv1Lu cells. ( a ) The activation of FAK was assessed by Western Blot at Tyr 925, at the indicated times in the presence of 5 µM OA, DMSO equivalent volume or EGF. Additionally, total protein expression was assayed: FAK. β-Actin was used as a loading control. Phospho-FAK intensity values in Western-blot were quantified by ImageJ to conduct a one-way ANOVA analysis showed on the plot (*p < 0.05, **p < 0.005, ***p < 0.001 and ****p < 0.0001). ( b ) Confluent Mv1Lu cells were scratched and allowed to migrate for 6 h. Pictures show the immunostaining with specific antibodies against phospho-FAK (Tyr 925) and paxillin. Co-staining with Hoechst-33258 was used to reveal nuclei. P-FAK: green. Paxillin: red. Nuclei: blue. ( c ) Pictures and graphs represent a colocalization analysis performed by Zeiss Efficient Navigation (ZEN) software. Colocalized pixels are highlighted in white at the immunostaining images. Graphs are dot plots representing both p-FAK and paxillin intensities in terms of number of pixels: p-FAK pixels on Y axis and paxillin pixels on X axis. The 3 quadrant (upper right) represents overlapped (common) pixels between both proteins. ( d ) Pictures of each condition (two) were divided in three horizontally distributed sectors. Pearson’s correlation coefficient in each sector of each condition was calculated by the average pixel intensity of p-FAK and paxillin overlapped pixels. The plot represents Pearson’s correlation values for each condition, each dot representing the value obtained in one sector. Asterisks indicate statistically significant differences between conditions according to one-way ANOVA statistical analysis: (*p < 0.05, **p < 0.005, ***p < 0.001 and ****p < 0.0001). This experiment was repeated at least three times. Representative images are shown. Scale bar indicates 10 µm.

Article Snippet: Mink Lung Epithelial (Mv1Lu) , , cells were grown in Eagle's Minimum Essential Medium (EMEM) (Biowest, Nuaillé, France).

Techniques: Phospho-proteomics, Activation Assay, Western Blot, Expressing, Control, Immunostaining, Staining, Software

EGFR and MEK inhibitors prevent OA-induced FAK localization at FA in Mv1Lu cells. ( a ) Confluent Mv1Lu cells were scratched and allowed to migrate for 6 h. Cells were treated 30 min before scratch and subsequent OA treatment with specific inhibitors 2,5 µM EGFRi, 50 µM MEKi and 15 µM JNKi. Pictures show the immunostaining with specific antibodies against phospho-FAK (Tyr 925) and paxillin. Co-staining with Hoechst-33258 was used to reveal nuclei. ( b ) The plot represents a colocalization analysis performed by Zeiss Efficient Navigation (ZEN) software. Pictures of each condition (two) were divided in three horizontally distributed sectors. Pearson’s correlation coefficient in each sector of each condition was calculated by the average pixel intensity of p-FAK and paxillin overlapped pixels. The plot represents Pearson’s correlation values for each condition, each dot representing the value obtained in one sector. Asterisks indicate statistically significant differences between conditions according to a one-way ANOVA statistical analysis: (*p < 0.05, **p < 0.005, ***p < 0.001 and ****p < 0.0001). This experiment was repeated at least three times. Representative images are shown. Scale bar indicates 10 µm.

Journal: Scientific Reports

Article Title: Oleanolic acid stimulation of cell migration involves a biphasic signaling mechanism

doi: 10.1038/s41598-022-17553-w

Figure Lengend Snippet: EGFR and MEK inhibitors prevent OA-induced FAK localization at FA in Mv1Lu cells. ( a ) Confluent Mv1Lu cells were scratched and allowed to migrate for 6 h. Cells were treated 30 min before scratch and subsequent OA treatment with specific inhibitors 2,5 µM EGFRi, 50 µM MEKi and 15 µM JNKi. Pictures show the immunostaining with specific antibodies against phospho-FAK (Tyr 925) and paxillin. Co-staining with Hoechst-33258 was used to reveal nuclei. ( b ) The plot represents a colocalization analysis performed by Zeiss Efficient Navigation (ZEN) software. Pictures of each condition (two) were divided in three horizontally distributed sectors. Pearson’s correlation coefficient in each sector of each condition was calculated by the average pixel intensity of p-FAK and paxillin overlapped pixels. The plot represents Pearson’s correlation values for each condition, each dot representing the value obtained in one sector. Asterisks indicate statistically significant differences between conditions according to a one-way ANOVA statistical analysis: (*p < 0.05, **p < 0.005, ***p < 0.001 and ****p < 0.0001). This experiment was repeated at least three times. Representative images are shown. Scale bar indicates 10 µm.

Article Snippet: Mink Lung Epithelial (Mv1Lu) , , cells were grown in Eagle's Minimum Essential Medium (EMEM) (Biowest, Nuaillé, France).

Techniques: Immunostaining, Staining, Software

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