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

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

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

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

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.

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, Western Blot, Knockdown, Expressing, Phospho-proteomics

Journal: Communications Biology

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

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.

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: Knockdown, Quantitative RT-PCR, Comparison

Journal: Communications Biology

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

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.

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

Journal: Communications Biology

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

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.

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