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Experiments for panels A-D were performed in the Gerold lab (Hannover, Germany) and for panels E-G in the Olagnier lab (Aarhus, Denmark). A –D Calu-3 cells were pretreated with the compounds (BARD, 0.1 µM; SFN, 10 µM; 4OI, 100 µM) for 24 h, inoculated with SARS-CoV-2/München-1.2/2020/984,p3 (MOI = 0.005) in the presence of the compounds for 4 h, followed by removing the viral inoculum and adding fresh medium containing the respective compounds and controls. Measurements were made 48 h p.i. A , B Viral genome copies in supernatants ( A ) and cell lysates ( B ) (RT-qPCR). C , D . IFIT1 and CXCL10 mRNA in cell lysates (RT-qPCR). Calu-3 cells were pretreated with the indicated compounds for 48 h ( E , F ) or 24 h ( G ), infected with SARS-CoV-2 Wuhan-like early European B.1 lineage (FR-4286) (MOI = 0.01) for 1 h, followed by removal of the inoculum and incubation in fresh medium containing the compounds. Target gene expression and protein levels were measured 24 h p.i. In F infections were additionally performed with the SARS-CoV-2 variants indicated on the x-axis (MOI = 0.01). E Reduction of SARS-CoV-2 spike and nucleocapsid proteins, and <t>XPO1</t> protein expression, but increase in AKR1B10 and NQO1 levels by 4OI (125 µM) (immunoblot with β-actin as internal reference). F Marked reduction of viral genomic RNA of diverse SARS-CoV-2 variants of concern by 4OI (125 µM). G Marked reduction of viral genomic RNA by the compounds (RT-qPCR normalized against TBP mRNA). n = 3, means ± SEM. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.

Xpo1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "NRF2 activators and the inhibitor of nuclear export, selinexor, restrict coronaviruses by targeting a network involving ACE2, TMPRSS2, and XPO1 through an NRF2-independent mechanism"

Article Title: NRF2 activators and the inhibitor of nuclear export, selinexor, restrict coronaviruses by targeting a network involving ACE2, TMPRSS2, and XPO1 through an NRF2-independent mechanism

Journal: Communications Biology

doi: 10.1038/s42003-026-09724-6

Figure Legend Snippet: Experiments for panels A-D were performed in the Gerold lab (Hannover, Germany) and for panels E-G in the Olagnier lab (Aarhus, Denmark). A –D Calu-3 cells were pretreated with the compounds (BARD, 0.1 µM; SFN, 10 µM; 4OI, 100 µM) for 24 h, inoculated with SARS-CoV-2/München-1.2/2020/984,p3 (MOI = 0.005) in the presence of the compounds for 4 h, followed by removing the viral inoculum and adding fresh medium containing the respective compounds and controls. Measurements were made 48 h p.i. A , B Viral genome copies in supernatants ( A ) and cell lysates ( B ) (RT-qPCR). C , D . IFIT1 and CXCL10 mRNA in cell lysates (RT-qPCR). Calu-3 cells were pretreated with the indicated compounds for 48 h ( E , F ) or 24 h ( G ), infected with SARS-CoV-2 Wuhan-like early European B.1 lineage (FR-4286) (MOI = 0.01) for 1 h, followed by removal of the inoculum and incubation in fresh medium containing the compounds. Target gene expression and protein levels were measured 24 h p.i. In F infections were additionally performed with the SARS-CoV-2 variants indicated on the x-axis (MOI = 0.01). E Reduction of SARS-CoV-2 spike and nucleocapsid proteins, and XPO1 protein expression, but increase in AKR1B10 and NQO1 levels by 4OI (125 µM) (immunoblot with β-actin as internal reference). F Marked reduction of viral genomic RNA of diverse SARS-CoV-2 variants of concern by 4OI (125 µM). G Marked reduction of viral genomic RNA by the compounds (RT-qPCR normalized against TBP mRNA). n = 3, means ± SEM. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.

Techniques Used: Quantitative RT-PCR, Infection, Incubation, Targeted Gene Expression, Expressing, Western Blot

A –C Reduction of ACE2 , TMPRSS2 , and XPO1 mRNA. RT-qPCR analysis of RNA from the experiment shown in Fig. . D 4OI and SEL reduce ACE2 and TMPRSS2 protein levels. Uninfected Calu-3 cells were grown in medium containing 4OI (100 µM) or SEL (1 µM), and cellular ACE2 and TMPRSS2 levels were measured by immunoblot after 1–72 h. E 4OI reduces half-life of ACE2. Uninfected Calu-3 cells were grown in medium containing 4OI with or without cycloheximide (CHX, 50 µg/ml), and cellular ACE2 levels were measured by immunoblot after 1–12 h. Densitometry of the blots shown in ( E ); F , bar chart; G , numerical values; untreated cells = 100%. H NEDD4L knock-down attenuates the ACE2-destroying capacity of 4OI at the protein. Efficiency of NEDD4L mRNA knock-down is shown in Supplementary Figure . I . MDM2 knock-down attenuates the ACE2-destroying capacity of 4OI at the protein level. Efficiency of MDM2 mRNA knock-down is shown in Supplementary Figure . J –N Uninfected Calu-3 cells were grown in medium containing 4OI, SEL, proteasome inhibitor (CFZ, 10 nM), or lysosome inhibitors (BAFA1- 100 nM, CQ- 50 µM), and expression of ACE2 was determined by immunoblot. CFZ 6 h ( J ), BAFA1 24 h ( K , L ), and CQ 24 h ( M , N ). O 4OI and SEL reduce XPO1 protein levels. Uninfected Calu-3 cells were grown in medium containing 4OI or SEL, and XPO1 levels were measured by immunoblot after 1–72 h. P , Q NEDD4L and MDM2 knock-down attenuate ACE2 mRNA reduction by 4OI and SEL (RT-qPCR after 24 h). R 4OI and SEL attenuate STAT3 phosphorylation (immunoblot after 24 h). n = 3 ( D , E , H , I , O : n = 2), means ± SEM. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.

Figure Legend Snippet: A –C Reduction of ACE2 , TMPRSS2 , and XPO1 mRNA. RT-qPCR analysis of RNA from the experiment shown in Fig. . D 4OI and SEL reduce ACE2 and TMPRSS2 protein levels. Uninfected Calu-3 cells were grown in medium containing 4OI (100 µM) or SEL (1 µM), and cellular ACE2 and TMPRSS2 levels were measured by immunoblot after 1–72 h. E 4OI reduces half-life of ACE2. Uninfected Calu-3 cells were grown in medium containing 4OI with or without cycloheximide (CHX, 50 µg/ml), and cellular ACE2 levels were measured by immunoblot after 1–12 h. Densitometry of the blots shown in ( E ); F , bar chart; G , numerical values; untreated cells = 100%. H NEDD4L knock-down attenuates the ACE2-destroying capacity of 4OI at the protein. Efficiency of NEDD4L mRNA knock-down is shown in Supplementary Figure . I . MDM2 knock-down attenuates the ACE2-destroying capacity of 4OI at the protein level. Efficiency of MDM2 mRNA knock-down is shown in Supplementary Figure . J –N Uninfected Calu-3 cells were grown in medium containing 4OI, SEL, proteasome inhibitor (CFZ, 10 nM), or lysosome inhibitors (BAFA1- 100 nM, CQ- 50 µM), and expression of ACE2 was determined by immunoblot. CFZ 6 h ( J ), BAFA1 24 h ( K , L ), and CQ 24 h ( M , N ). O 4OI and SEL reduce XPO1 protein levels. Uninfected Calu-3 cells were grown in medium containing 4OI or SEL, and XPO1 levels were measured by immunoblot after 1–72 h. P , Q NEDD4L and MDM2 knock-down attenuate ACE2 mRNA reduction by 4OI and SEL (RT-qPCR after 24 h). R 4OI and SEL attenuate STAT3 phosphorylation (immunoblot after 24 h). n = 3 ( D , E , H , I , O : n = 2), means ± SEM. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.

Techniques Used: Quantitative RT-PCR, Western Blot, Knockdown, Expressing, Phospho-proteomics

A KEGG pathway analysis based on XPO1 cargoes listed in the ValidNESs database . B Efficiency of ABCB1 mRNA knock-down with siRNA (RT-qPCR). C Comparison of half-life reduction of XPO1 mRNA by ActD, 4OI, or co-treatment with both. Numerical values for half-life were obtained by extrapolation and are listed in the table next to the graph. D Same experiment as in C , but using ABCB1 knock-down (siRNA) A549 cells. Representative of two independent experiments, n = 3, means ± SEM. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.

Figure Legend Snippet: A KEGG pathway analysis based on XPO1 cargoes listed in the ValidNESs database . B Efficiency of ABCB1 mRNA knock-down with siRNA (RT-qPCR). C Comparison of half-life reduction of XPO1 mRNA by ActD, 4OI, or co-treatment with both. Numerical values for half-life were obtained by extrapolation and are listed in the table next to the graph. D Same experiment as in C , but using ABCB1 knock-down (siRNA) A549 cells. Representative of two independent experiments, n = 3, means ± SEM. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.

Techniques Used: Knockdown, Quantitative RT-PCR, Comparison

WT and NRF2 −/− human iPSC-derived ECs ( A , B , E – G ) and A549 cells ( C , D ) were infected with the luciferase-labeled strain hCoV-229E-luc (MOI = 0.3) for 4 h and then cultured for 48 h in fresh medium containing the compounds. Luciferase activity, viral M protein RNA levels, host mRNA expression, and mitochondrial ROS levels were measured after 48 h. A , B Luciferase activity and viral M protein RNA. C Luciferase activity and ( D ) viral M protein RNA were measured in WT and NRF2 −/− A549 cells. E XPO1 mRNA. F Mitochondrial ROS (flow cytometry). G HMOX1 mRNA. H , I Effect of HMOX1 knock-down on 229E-luc infectivity and antiviral activity of the compounds in A549 cells. Infections and treatments were carried out as in A – G , except that WT (transfected with scrambled control siRNA) or siRNA-mediated HMOX1 knock-down A549 cells were used. H Luciferase activity. I M protein RNA. n = 3, means ± SEM. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.

Figure Legend Snippet: WT and NRF2 −/− human iPSC-derived ECs ( A , B , E – G ) and A549 cells ( C , D ) were infected with the luciferase-labeled strain hCoV-229E-luc (MOI = 0.3) for 4 h and then cultured for 48 h in fresh medium containing the compounds. Luciferase activity, viral M protein RNA levels, host mRNA expression, and mitochondrial ROS levels were measured after 48 h. A , B Luciferase activity and viral M protein RNA. C Luciferase activity and ( D ) viral M protein RNA were measured in WT and NRF2 −/− A549 cells. E XPO1 mRNA. F Mitochondrial ROS (flow cytometry). G HMOX1 mRNA. H , I Effect of HMOX1 knock-down on 229E-luc infectivity and antiviral activity of the compounds in A549 cells. Infections and treatments were carried out as in A – G , except that WT (transfected with scrambled control siRNA) or siRNA-mediated HMOX1 knock-down A549 cells were used. H Luciferase activity. I M protein RNA. n = 3, means ± SEM. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.

Techniques Used: Derivative Assay, Infection, Luciferase, Labeling, Cell Culture, Activity Assay, Expressing, Flow Cytometry, Knockdown, Transfection, Control

XPO1 mRNA was knocked down in A549 cells by siRNA, and infectivity of 229E-luc and antiviral efficacy of the compounds were assessed in WT (transfected with scrambled control siRNA) and XPO1 KD cells following the same infection protocol as in Fig. . A XPO1 mRNA (RT-qPCR). B M protein RNA expression. C Luciferase activity. D ANPEP mRNA (RT-qPCR). E NFE2L2 mRNA (RT-qPCR). F HMOX1 mRNA (RT-qPCR). n = 3, means ± SEM. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.

Figure Legend Snippet: XPO1 mRNA was knocked down in A549 cells by siRNA, and infectivity of 229E-luc and antiviral efficacy of the compounds were assessed in WT (transfected with scrambled control siRNA) and XPO1 KD cells following the same infection protocol as in Fig. . A XPO1 mRNA (RT-qPCR). B M protein RNA expression. C Luciferase activity. D ANPEP mRNA (RT-qPCR). E NFE2L2 mRNA (RT-qPCR). F HMOX1 mRNA (RT-qPCR). n = 3, means ± SEM. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.

Techniques Used: Infection, Transfection, Control, Quantitative RT-PCR, RNA Expression, Luciferase, Activity Assay

Image Search Results

Involvement of the Ran, CRM1, and 14-3-3 proteins in the nuclear exclusion of FOXO1. ( a ) Effects of FOXO1 mutant with the S248A or/and S311A substitution on its combination with 14-3-3 determined by co-immunoprecipitation in presence or absence of 740-Y-P. The chicken GCs were transfected with the expression constructs of the pcDNA3,1(+)-FOXO1 wild-type, pcDNA3,1(+)-FOXO1 Ser248A mutant, and pcDNA3,1(+)-FOXO1 Ser311A mutant, respectively. ( b ) The protein combining level of 14-3-3 with FOXO1 protein in the cultured GCs. ( c ) Effects of FOXO1 mutant with the S248A or/and S311A substitution on its combination with the CRM1 determined by co-immunoprecipitation in presence or absence of 740-Y-P. ( d ) The combining level of CRM1 with FOXO1 protein in the cells. ( e ) Effects of RANQ69L overexpression on the combination of FOXO1 with 14-3-3 or CRM1 examined by co-immunoprecipitation in presence or absence of Ly294002. The chicken GCs were transfected with the pcDNA3,1(+)-FOXO1 expression construct, and simultaneously with the pcDNA3,1(+)- RANQ69L plasmid. ( f ) The protein combining level of 14-3-3 with FOXO1 in the cells under FOXO1 overexpression with or without Ly294002 treatment and with or without RANQ69L treatment by using Western blotting. ( g ) The protein combining level of CRM1 with FOXO1 under the same conditions as ( f ). ( h ). Effects of the two PKB phosphorylation sites in FOXO1 on its combination with RAN. The ovarian GCs were transfected with the expression constructs, pcDNA3,1(+)-FOXO1 wild-type, pcDNA3,1(+)-FOXO1 Ser248A mutant, and pcDNA3,1(+)-FOXO1 mutant, in presence or absence of 740-Y-P or in presence or absence of Ly294002, respectively, which were determined by the co-immunoprecipitation assay. ( i ) The combination levels of Ran with FOXO1 in cultured GCs transfected by the plasmid of pcDNA3,1(+)-FOXO1 Ser248A mutant with/without 740-Y-P treatment or with/without Ly294002 treatment. ( j ) The combination levels of Ran with FOXO1 in the cultured GCs transfected by the plasmid of pcDNA3,1(+)-FOXO1 Ser311A mutant with/without 740-Y-P treatment or with/without Ly294002 treatment. n = 3. ** p < 0.01, * p < 0.05.

Journal: Cells

Article Title: FSH-Induced Nuclear Exclusion of FOXO1 Mediated by PI3K/Akt Signaling Pathway in Granulosa Cells Is Associated with Follicle Selection and Growth of the Hen Ovary

doi: 10.3390/cells14231864

Figure Lengend Snippet: Involvement of the Ran, CRM1, and 14-3-3 proteins in the nuclear exclusion of FOXO1. ( a ) Effects of FOXO1 mutant with the S248A or/and S311A substitution on its combination with 14-3-3 determined by co-immunoprecipitation in presence or absence of 740-Y-P. The chicken GCs were transfected with the expression constructs of the pcDNA3,1(+)-FOXO1 wild-type, pcDNA3,1(+)-FOXO1 Ser248A mutant, and pcDNA3,1(+)-FOXO1 Ser311A mutant, respectively. ( b ) The protein combining level of 14-3-3 with FOXO1 protein in the cultured GCs. ( c ) Effects of FOXO1 mutant with the S248A or/and S311A substitution on its combination with the CRM1 determined by co-immunoprecipitation in presence or absence of 740-Y-P. ( d ) The combining level of CRM1 with FOXO1 protein in the cells. ( e ) Effects of RANQ69L overexpression on the combination of FOXO1 with 14-3-3 or CRM1 examined by co-immunoprecipitation in presence or absence of Ly294002. The chicken GCs were transfected with the pcDNA3,1(+)-FOXO1 expression construct, and simultaneously with the pcDNA3,1(+)- RANQ69L plasmid. ( f ) The protein combining level of 14-3-3 with FOXO1 in the cells under FOXO1 overexpression with or without Ly294002 treatment and with or without RANQ69L treatment by using Western blotting. ( g ) The protein combining level of CRM1 with FOXO1 under the same conditions as ( f ). ( h ). Effects of the two PKB phosphorylation sites in FOXO1 on its combination with RAN. The ovarian GCs were transfected with the expression constructs, pcDNA3,1(+)-FOXO1 wild-type, pcDNA3,1(+)-FOXO1 Ser248A mutant, and pcDNA3,1(+)-FOXO1 mutant, in presence or absence of 740-Y-P or in presence or absence of Ly294002, respectively, which were determined by the co-immunoprecipitation assay. ( i ) The combination levels of Ran with FOXO1 in cultured GCs transfected by the plasmid of pcDNA3,1(+)-FOXO1 Ser248A mutant with/without 740-Y-P treatment or with/without Ly294002 treatment. ( j ) The combination levels of Ran with FOXO1 in the cultured GCs transfected by the plasmid of pcDNA3,1(+)-FOXO1 Ser311A mutant with/without 740-Y-P treatment or with/without Ly294002 treatment. n = 3. ** p < 0.01, * p < 0.05.

Article Snippet: rabbit anti-CRM1 antibody , 1:1000 , Bioss Antibodies, Beijing, China , bs-3145R.

Techniques: Mutagenesis, Immunoprecipitation, Transfection, Expressing, Construct, Cell Culture, Over Expression, Plasmid Preparation, Western Blot, Phospho-proteomics, Co-Immunoprecipitation Assay

Journal: Communications Biology

doi: 10.1038/s42003-026-09724-6

Figure Lengend Snippet: Experiments for panels A-D were performed in the Gerold lab (Hannover, Germany) and for panels E-G in the Olagnier lab (Aarhus, Denmark). A –D Calu-3 cells were pretreated with the compounds (BARD, 0.1 µM; SFN, 10 µM; 4OI, 100 µM) for 24 h, inoculated with SARS-CoV-2/München-1.2/2020/984,p3 (MOI = 0.005) in the presence of the compounds for 4 h, followed by removing the viral inoculum and adding fresh medium containing the respective compounds and controls. Measurements were made 48 h p.i. A , B Viral genome copies in supernatants ( A ) and cell lysates ( B ) (RT-qPCR). C , D . IFIT1 and CXCL10 mRNA in cell lysates (RT-qPCR). Calu-3 cells were pretreated with the indicated compounds for 48 h ( E , F ) or 24 h ( G ), infected with SARS-CoV-2 Wuhan-like early European B.1 lineage (FR-4286) (MOI = 0.01) for 1 h, followed by removal of the inoculum and incubation in fresh medium containing the compounds. Target gene expression and protein levels were measured 24 h p.i. In F infections were additionally performed with the SARS-CoV-2 variants indicated on the x-axis (MOI = 0.01). E Reduction of SARS-CoV-2 spike and nucleocapsid proteins, and XPO1 protein expression, but increase in AKR1B10 and NQO1 levels by 4OI (125 µM) (immunoblot with β-actin as internal reference). F Marked reduction of viral genomic RNA of diverse SARS-CoV-2 variants of concern by 4OI (125 µM). G Marked reduction of viral genomic RNA by the compounds (RT-qPCR normalized against TBP mRNA). n = 3, means ± SEM. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.

Article Snippet: The following primary antibodies were used: ACE2 (Cell Signaling Technology, 92485S, 1:1000), XPO1 (Cell Signaling Technology, 46249S, 1:1000), TMPRSS2 (Santa Cruz, sc-515727, 1:500), MDM2 (Cell Signaling Technology, 86934S, 1:1000), NEDD4L (ThermoFisher Scientific, 67276-1-IG-20UL, 1:10,000), P-STAT3 (Cell Signaling Technology, 9145S, 1:1000), STAT3 (Cell Signaling Technology, 4904S, 1:1000), and β-actin (Abcam, ab49900, 1:20,000).

Techniques: Quantitative RT-PCR, Infection, Incubation, Targeted Gene Expression, Expressing, Western Blot

Journal: Communications Biology

doi: 10.1038/s42003-026-09724-6

Figure Lengend Snippet: A –C Reduction of ACE2 , TMPRSS2 , and XPO1 mRNA. RT-qPCR analysis of RNA from the experiment shown in Fig. . D 4OI and SEL reduce ACE2 and TMPRSS2 protein levels. Uninfected Calu-3 cells were grown in medium containing 4OI (100 µM) or SEL (1 µM), and cellular ACE2 and TMPRSS2 levels were measured by immunoblot after 1–72 h. E 4OI reduces half-life of ACE2. Uninfected Calu-3 cells were grown in medium containing 4OI with or without cycloheximide (CHX, 50 µg/ml), and cellular ACE2 levels were measured by immunoblot after 1–12 h. Densitometry of the blots shown in ( E ); F , bar chart; G , numerical values; untreated cells = 100%. H NEDD4L knock-down attenuates the ACE2-destroying capacity of 4OI at the protein. Efficiency of NEDD4L mRNA knock-down is shown in Supplementary Figure . I . MDM2 knock-down attenuates the ACE2-destroying capacity of 4OI at the protein level. Efficiency of MDM2 mRNA knock-down is shown in Supplementary Figure . J –N Uninfected Calu-3 cells were grown in medium containing 4OI, SEL, proteasome inhibitor (CFZ, 10 nM), or lysosome inhibitors (BAFA1- 100 nM, CQ- 50 µM), and expression of ACE2 was determined by immunoblot. CFZ 6 h ( J ), BAFA1 24 h ( K , L ), and CQ 24 h ( M , N ). O 4OI and SEL reduce XPO1 protein levels. Uninfected Calu-3 cells were grown in medium containing 4OI or SEL, and XPO1 levels were measured by immunoblot after 1–72 h. P , Q NEDD4L and MDM2 knock-down attenuate ACE2 mRNA reduction by 4OI and SEL (RT-qPCR after 24 h). R 4OI and SEL attenuate STAT3 phosphorylation (immunoblot after 24 h). n = 3 ( D , E , H , I , O : n = 2), means ± SEM. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.

Techniques: Quantitative RT-PCR, Western Blot, Knockdown, Expressing, Phospho-proteomics

Journal: Communications Biology

doi: 10.1038/s42003-026-09724-6

Figure Lengend Snippet: A KEGG pathway analysis based on XPO1 cargoes listed in the ValidNESs database . B Efficiency of ABCB1 mRNA knock-down with siRNA (RT-qPCR). C Comparison of half-life reduction of XPO1 mRNA by ActD, 4OI, or co-treatment with both. Numerical values for half-life were obtained by extrapolation and are listed in the table next to the graph. D Same experiment as in C , but using ABCB1 knock-down (siRNA) A549 cells. Representative of two independent experiments, n = 3, means ± SEM. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.

Techniques: Knockdown, Quantitative RT-PCR, Comparison

Journal: Communications Biology

doi: 10.1038/s42003-026-09724-6

Figure Lengend Snippet: WT and NRF2 −/− human iPSC-derived ECs ( A , B , E – G ) and A549 cells ( C , D ) were infected with the luciferase-labeled strain hCoV-229E-luc (MOI = 0.3) for 4 h and then cultured for 48 h in fresh medium containing the compounds. Luciferase activity, viral M protein RNA levels, host mRNA expression, and mitochondrial ROS levels were measured after 48 h. A , B Luciferase activity and viral M protein RNA. C Luciferase activity and ( D ) viral M protein RNA were measured in WT and NRF2 −/− A549 cells. E XPO1 mRNA. F Mitochondrial ROS (flow cytometry). G HMOX1 mRNA. H , I Effect of HMOX1 knock-down on 229E-luc infectivity and antiviral activity of the compounds in A549 cells. Infections and treatments were carried out as in A – G , except that WT (transfected with scrambled control siRNA) or siRNA-mediated HMOX1 knock-down A549 cells were used. H Luciferase activity. I M protein RNA. n = 3, means ± SEM. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.

Techniques: Derivative Assay, Infection, Luciferase, Labeling, Cell Culture, Activity Assay, Expressing, Flow Cytometry, Knockdown, Transfection, Control

Journal: Communications Biology

doi: 10.1038/s42003-026-09724-6

Figure Lengend Snippet: XPO1 mRNA was knocked down in A549 cells by siRNA, and infectivity of 229E-luc and antiviral efficacy of the compounds were assessed in WT (transfected with scrambled control siRNA) and XPO1 KD cells following the same infection protocol as in Fig. . A XPO1 mRNA (RT-qPCR). B M protein RNA expression. C Luciferase activity. D ANPEP mRNA (RT-qPCR). E NFE2L2 mRNA (RT-qPCR). F HMOX1 mRNA (RT-qPCR). n = 3, means ± SEM. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.

Techniques: Infection, Transfection, Control, Quantitative RT-PCR, RNA Expression, Luciferase, Activity Assay

Leptomycin B (LMB) causes a dose-dependent reduction in Dhori virus (DHOV) titer and viral ribonucleoprotein (vRNP) retention in the nucleus. ( A ) DHOV titer in Huh7 cells (MOI = 0.1) with treatment with the indicated dose of LMB or vehicle control. Virus infectivity titers in supernatants are shown in focus-forming unit (FFU)/mL. Dashed lines indicate the residual DHOV titer following infection and washing with SFM (DHOV inoculum) and DHOV titer from untreated cells (DHOV untreated). ( B ) (left, magenta Y-axis) DHOV titer from LMB-treated cells expressed as a percentage of vehicle control-treated cells. (right, black Y-axis) Cell viability of Huh7 cells treated with the indicated dose of LMB expressed as a percentage of the corresponding dose of vehicle control. ( C ) Localization of DHOV NP and CRM1 in Huh7 cells at 6 hours post-infection (hpi) (MOI = 100) treated with 100 nM of LMB, vehicle control, or untreated, examined by immunofluorescence using anti-CRM1 and custom anti-DHOV NP antibodies. Arrowheads highlight contrast in NP signal localization. Nuclei stained using Hoechst 33342. Images taken using a confocal microscope (ZEISS LSM 980 with Airyscan2) using 10× objective. ( D ) Quantification of the percentage of the NP signal overlapping the nuclei using the Just Another Colocalization (JaCoP) plugin in ImageJ. ( E ) Quantification of the percentage of NP signals overlapping the CRM1 signal using the JaCoP plugin in ImageJ. ns > 0.05, **** P ≤ 0.0001; ordinary one-way ANOVA.

Journal: Journal of Virology

Article Title: Involvement of a tick-borne orthomyxovirus matrix protein in vRNP nuclear export

doi: 10.1128/jvi.01494-25

Figure Lengend Snippet: Leptomycin B (LMB) causes a dose-dependent reduction in Dhori virus (DHOV) titer and viral ribonucleoprotein (vRNP) retention in the nucleus. ( A ) DHOV titer in Huh7 cells (MOI = 0.1) with treatment with the indicated dose of LMB or vehicle control. Virus infectivity titers in supernatants are shown in focus-forming unit (FFU)/mL. Dashed lines indicate the residual DHOV titer following infection and washing with SFM (DHOV inoculum) and DHOV titer from untreated cells (DHOV untreated). ( B ) (left, magenta Y-axis) DHOV titer from LMB-treated cells expressed as a percentage of vehicle control-treated cells. (right, black Y-axis) Cell viability of Huh7 cells treated with the indicated dose of LMB expressed as a percentage of the corresponding dose of vehicle control. ( C ) Localization of DHOV NP and CRM1 in Huh7 cells at 6 hours post-infection (hpi) (MOI = 100) treated with 100 nM of LMB, vehicle control, or untreated, examined by immunofluorescence using anti-CRM1 and custom anti-DHOV NP antibodies. Arrowheads highlight contrast in NP signal localization. Nuclei stained using Hoechst 33342. Images taken using a confocal microscope (ZEISS LSM 980 with Airyscan2) using 10× objective. ( D ) Quantification of the percentage of the NP signal overlapping the nuclei using the Just Another Colocalization (JaCoP) plugin in ImageJ. ( E ) Quantification of the percentage of NP signals overlapping the CRM1 signal using the JaCoP plugin in ImageJ. ns > 0.05, **** P ≤ 0.0001; ordinary one-way ANOVA.

Article Snippet: Rabbit monoclonal antibody against CRM1 was purchased from Cell Signaling Technology (46249S).

Techniques: Virus, Control, Infection, Immunofluorescence, Staining, Microscopy

DHOV M but not NP interacts with CRM1 in a mammalian two-hybrid system. ( A ) Schematic depicting the Promega CheckMate mammalian two-hybrid system using the pBIND (VP16) CRM1 fusion orientation. ( B–E ) CheckMate system luciferase data. HEK-293 cells were co-transfected with 100 ng of the pG5 luc reporter plasmid and combinations of the indicated pACT and pBIND plasmids. After 96 hours post-transfection, cell lysates were harvested, and luciferase assay was performed. Renilla luciferase, expressed from the pBIND vector under a constitutive promoter, was used to normalize firefly luciferase data for transfection efficiency. Interaction between pACT-MyoD and pBIND-ID (Promega) was used as a positive control. ( B–C ) Human CRM1 fused to the VP16 transcriptional activation domain (pBIND) and indicated viral protein fused to the GAL4 DNA-binding domain (pACT). ( D–E ) Orientation comparison demonstrating comparing the luciferase activity of pBIND-CRM1 and pACT-DHOV-M and vice versa. ( B and D ) Raw firefly luciferase data. ( C and E ) Firefly luciferase data normalized to transfection efficiency (Firefly/ Renilla ). Results shown represent at least three independent experiments. Lane 5 repeated in B and C for easier interpretation. ns > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001; t -test. ( A ) Created in BioRender. Swenson, V. (2023) https://BioRender.com/b16s227.

Journal: Journal of Virology

Article Title: Involvement of a tick-borne orthomyxovirus matrix protein in vRNP nuclear export

doi: 10.1128/jvi.01494-25

Figure Lengend Snippet: DHOV M but not NP interacts with CRM1 in a mammalian two-hybrid system. ( A ) Schematic depicting the Promega CheckMate mammalian two-hybrid system using the pBIND (VP16) CRM1 fusion orientation. ( B–E ) CheckMate system luciferase data. HEK-293 cells were co-transfected with 100 ng of the pG5 luc reporter plasmid and combinations of the indicated pACT and pBIND plasmids. After 96 hours post-transfection, cell lysates were harvested, and luciferase assay was performed. Renilla luciferase, expressed from the pBIND vector under a constitutive promoter, was used to normalize firefly luciferase data for transfection efficiency. Interaction between pACT-MyoD and pBIND-ID (Promega) was used as a positive control. ( B–C ) Human CRM1 fused to the VP16 transcriptional activation domain (pBIND) and indicated viral protein fused to the GAL4 DNA-binding domain (pACT). ( D–E ) Orientation comparison demonstrating comparing the luciferase activity of pBIND-CRM1 and pACT-DHOV-M and vice versa. ( B and D ) Raw firefly luciferase data. ( C and E ) Firefly luciferase data normalized to transfection efficiency (Firefly/ Renilla ). Results shown represent at least three independent experiments. Lane 5 repeated in B and C for easier interpretation. ns > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001; t -test. ( A ) Created in BioRender. Swenson, V. (2023) https://BioRender.com/b16s227.

Article Snippet: Rabbit monoclonal antibody against CRM1 was purchased from Cell Signaling Technology (46249S).

Techniques: Luciferase, Transfection, Plasmid Preparation, Positive Control, Activation Assay, Binding Assay, Comparison, Activity Assay

Mutation of NES1 residues within DHOV M reduces interaction with CRM1 in a mammalian two-hybrid system and abrogates the NES phenotype. ( A–D ) CheckMate system luciferase data. HEK-293 cells were co-transfected with 100 ng of the pG5 luc reporter plasmid and combinations of other plasmids as indicated. After 96 hours transfection, cell lysates were harvested, and luciferase assay was performed. Interaction between pACT-CRM1 and pBIND-DHOV-M was used as a positive control. ( A–C ) Data shown as firefly luciferase data normalized to transfection efficiency (Firefly/ Renilla ) and then to vector control (sample/vector). The dashed line indicates the system background of pACT CRM1 alone. ( A ) Alanine point mutants. ( B ) Serine point mutants. ( C ) Combinatorial mutants. ( D ) A–C expressed as percentage of WT DHOV-M signals. Dashed lines indicate the signal of WT M (WT M) and pACT CRM1 alone (CRM1 Only). Results shown represent at least five independent experiments. ( E ) Immunofluorescence images showing localization of the indicated mCherry–DHOV M fusion construct. All mutants shown (All A, All G, All S, All R, 121/2A, and 121/2S) contain the specified mutations within DHOV M 111–128 fused to mCherry. HEK-293 cells were transfected with 0.5 µg of the pCAGGS vector expressing the indicated fusion construct and fixed 24 hours post-transfection. Nuclei were stained using Hoechst 33342. Images taken using a Nikon Eclipse Ts2 microscope with an Excelis MPX-6 camera under 20 × magnification. ns > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001; ( A–C ) t -test.

Journal: Journal of Virology

Article Title: Involvement of a tick-borne orthomyxovirus matrix protein in vRNP nuclear export

doi: 10.1128/jvi.01494-25

Figure Lengend Snippet: Mutation of NES1 residues within DHOV M reduces interaction with CRM1 in a mammalian two-hybrid system and abrogates the NES phenotype. ( A–D ) CheckMate system luciferase data. HEK-293 cells were co-transfected with 100 ng of the pG5 luc reporter plasmid and combinations of other plasmids as indicated. After 96 hours transfection, cell lysates were harvested, and luciferase assay was performed. Interaction between pACT-CRM1 and pBIND-DHOV-M was used as a positive control. ( A–C ) Data shown as firefly luciferase data normalized to transfection efficiency (Firefly/ Renilla ) and then to vector control (sample/vector). The dashed line indicates the system background of pACT CRM1 alone. ( A ) Alanine point mutants. ( B ) Serine point mutants. ( C ) Combinatorial mutants. ( D ) A–C expressed as percentage of WT DHOV-M signals. Dashed lines indicate the signal of WT M (WT M) and pACT CRM1 alone (CRM1 Only). Results shown represent at least five independent experiments. ( E ) Immunofluorescence images showing localization of the indicated mCherry–DHOV M fusion construct. All mutants shown (All A, All G, All S, All R, 121/2A, and 121/2S) contain the specified mutations within DHOV M 111–128 fused to mCherry. HEK-293 cells were transfected with 0.5 µg of the pCAGGS vector expressing the indicated fusion construct and fixed 24 hours post-transfection. Nuclei were stained using Hoechst 33342. Images taken using a Nikon Eclipse Ts2 microscope with an Excelis MPX-6 camera under 20 × magnification. ns > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001; ( A–C ) t -test.

Article Snippet: Rabbit monoclonal antibody against CRM1 was purchased from Cell Signaling Technology (46249S).

Techniques: Mutagenesis, Luciferase, Transfection, Plasmid Preparation, Positive Control, Control, Immunofluorescence, Construct, Expressing, Staining, Microscopy

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