Journal: Journal of Virology

Article Title: IFIT3 RNA-binding activity promotes influenza A virus infection and translation efficiency

doi: 10.1128/jvi.00286-25

Figure Lengend Snippet: IFIT3 enhances influenza A virus replication. (A) IFIT3 RNA expression measured by RT-qPCR from A549 CRISPRa cells inoculated with non-targeting or IFIT3-targeting TRPPC viruses (MOI = 5). Data are normalized to the non-targeting control. (B) Influenza virus NP protein levels measured 8 hpi in A549 CRISPRa cells inoculated with non-targeting or IFIT3-targeting TRPPC viruses (MOI = 5). Data are normalized to the non-targeting control. (C) Single-cycle replication was measured in A549 CRISPRa cells infected with virus targeting IFIT3 or a non-targeting control (MOI = 0.05). Viral titers were determined 18 hpi. (D) Multicycle replication kinetics in A549 CRISPRa cells inoculated with virus targeting IFIT3 or a non-targeting control (MOI = 0.05). Titers were determined at the indicated time points by plaque assay. Data in panels A–D) are the mean of n = 3 ± SD. Significance was assessed by a two-way Student’s t -test. (E) Viral gene expression during single-cycle infection in WT, IFIT3 -/- , and IFIT2 -/- A549 cells using a WSN-based reporter virus measured 8 hpi. Data are the grand mean ± SEM of two independent biological replicates, each with three technical replicates. Significance was assessed by one-way ANOVA with Dunnett’s multiple comparisons test. * P < 0.5, ** P < 0.01, and **** P < 0.0001.

Article Snippet: Human lung alveolar epithelial A549 cells (CCL-185), Madin–Darby canine kidney (MDCK) cells (CCL-34), human embryonic kidney 293T (HEK293T) cells (CRL-3216), and human embryonic kidney 293 (HEK293) cells (CRL-1573) were acquired from the American Type Culture Collection.

Techniques: Virus, RNA Expression, Quantitative RT-PCR, Control, Infection, Plaque Assay, Gene Expression

Journal: Journal of Virology

Article Title: IFIT3 RNA-binding activity promotes influenza A virus infection and translation efficiency

doi: 10.1128/jvi.00286-25

Figure Lengend Snippet: IFIT3 directly binds RNA. (A) Electrophoretic mobility shift assays were performed using in vitro -transcribed RNAs derived from regions of the indicated viral genes in the presence or absence of recombinant IFIT3. RNAs were detected by Sybr gold staining (top), while proteins were visualized with Coomassie staining (bottom). (B) EMSAs were performed with increasing amounts of recombinant IFIT3 and the indicated RNAs, revealing dose-dependent RNA binding. RNA was detected by Sybr gold staining (top), while protein was visualized with Coomassie staining (bottom). (C) EMSAs were performed by forming complexes between NA1 RNA and increasing amounts of IFIT3. Where indicated, complexes were denatured by boiling before electrophoresis. (D) RNA-immunoprecipitations (RIPs) were performed on infected cell lysates. IFIT2 -/- A549 cells were infected with WSN (MOI = 0.02) for 24 hours, lysed, and lysates were immunoprecipitated with an antibody targeting IFIT3 or an IgG control. IFIT3-specific binding was quantified by RT-qPCR relative to an IgG control. A representative Western blot showing expression and specific capture of IFIT3 is shown below, in the input as well as pulldown with the IFIT3 antibody, but not the IgG antibody.

Article Snippet: Human lung alveolar epithelial A549 cells (CCL-185), Madin–Darby canine kidney (MDCK) cells (CCL-34), human embryonic kidney 293T (HEK293T) cells (CRL-3216), and human embryonic kidney 293 (HEK293) cells (CRL-1573) were acquired from the American Type Culture Collection.

Techniques: Electrophoretic Mobility Shift Assay, In Vitro, Derivative Assay, Recombinant, Staining, RNA Binding Assay, Electrophoresis, Infection, Immunoprecipitation, Control, Binding Assay, Quantitative RT-PCR, Western Blot, Expressing

Journal: Journal of Extracellular Vesicles

Article Title: 1,4‐Dioxane Induces Epithelial‐Mesenchymal Transition and Carcinogenesis in an Nrf2‐Dependent Manner

doi: 10.1002/jev2.70072

Figure Lengend Snippet: Characterisation and function analysis of 1,4‐D‐induced EVs in WT and Nrf2 KO cells. (A) Immunoblots showing the expression of PDCD6IP, CD82, CD9, CANX and ACTB in cell lysates and EV samples derived from WT, 1,4‐D‐transformed WT, Nrf2 KO and 1,4‐D‐transformed Nrf2 KO cells. (B) Representative TEM photographs of EVs from the indicated groups. (C and D) EV concentrations across the groups, with 1,4‐D‐induced changes in EV production in WT and Nrf2 KO cells shown as fold changes. Data are presented as mean ± SD, n = 3, * p < 0.05 vs. control (one‐way ANOVA with Bonferroni's multiple comparisons test). (E and F) Size distribution of EVs in each group, determined and quantified using NTA. Data are presented as mean ± SD, n = 3, * p < 0.05 vs. control (one‐way ANOVA with Bonferroni's multiple comparisons test). (G) Effects of EVs from 1,4‐D‐transformed WT (T‐WT) and 1,4‐D‐transformed Nrf2 KO (T‐Nrf2 KO) cells on A549 cell proliferation. Data are presented as mean ± SD, n = 6, ** p < 0.01 vs. control (one‐way ANOVA with Bonferroni's multiple comparisons test). (H–J) Representative images illustrating changes in migration and invasion capabilities of A549 cells treated with EVs from the indicated groups, with quantification of migrated or invasive cells. Data are presented as mean ± SD, n = 10, ** p < 0.01 vs. control (one‐way ANOVA with Bonferroni's multiple comparisons test).

Article Snippet: The human bronchial epithelial cell line BEAS‐2B and the human non‐small cell lung cancer derived hypotriploid alveolar basal epithelial cells A549, purchased from the American Type Culture Collection (ATCC, Manassas, VA), were maintained in complete medium at 37°C under 5% CO 2 .

Techniques: Western Blot, Expressing, Derivative Assay, Transformation Assay, Control, Migration

Journal: Journal of Extracellular Vesicles

Article Title: 1,4‐Dioxane Induces Epithelial‐Mesenchymal Transition and Carcinogenesis in an Nrf2‐Dependent Manner

doi: 10.1002/jev2.70072

Figure Lengend Snippet: Nrf2 activation induced by 1,4‐D promotes EMT protein loading in EVs. (A) Venn diagram illustrating 359 1,4‐D‐induced, Nrf2‐dependent proteins identified in cell lysate proteomics, significantly enriched in ROS and fatty acid metabolism pathways. (B) Venny diagram illustrating 173 1,4‐D‐induced, Nrf2‐dependent proteins identified in EV proteomics, significantly enriched in the MYC and EMT pathways. (C) Overlapping proteins between the EV and cell proteomes are significantly enriched in ROS, mTORC1 signalling and glycolysis pathways. A heatmap illustrates the abundance of overlapped 1,4‐D‐induced, Nrf2‐dependent proteins in EVs and cell lysates, which are also associated with EMT. Protein abundances are shown as log2 fold changes. (D) Pathway enrichment analysis (Hallmark collection) reveals significant enrichment of the EMT pathway among genes overlapping between the EV proteome and cell transcriptome. (E) Heatmap displaying the protein abundance and gene expression of SFRP1, MMP14, BMP1, SDC4, ITGB3, COL12A1, COL5A2, ITGAV, PVR and FERMT2 in EV samples and cells. These proteins are expressed in a 1,4‐D‐induced, Nrf2‐dependent manner and are significantly enriched in the EMT pathway. Data are shown as log2 fold changes. (F) Representative immunoblots showing the abundance of SDC4, COL12A1, CAPG, NNMT, MMP14, SLUG, NANOG, VIM, TWIST1 and ACTB in A549 recipient cells following 24‐h EV treatment.

Article Snippet: The human bronchial epithelial cell line BEAS‐2B and the human non‐small cell lung cancer derived hypotriploid alveolar basal epithelial cells A549, purchased from the American Type Culture Collection (ATCC, Manassas, VA), were maintained in complete medium at 37°C under 5% CO 2 .

Techniques: Activation Assay, Quantitative Proteomics, Gene Expression, Western Blot

Journal: Journal of Extracellular Vesicles

Article Title: 1,4‐Dioxane Induces Epithelial‐Mesenchymal Transition and Carcinogenesis in an Nrf2‐Dependent Manner

doi: 10.1002/jev2.70072

Figure Lengend Snippet: 1,4‐D‐induced, SDC4‐enriched EVs are significantly internalised by recipient cells. (A) Effects of Nrf2 knockout on the expression of key EV biomarkers based on EV proteomics. Data are shown as log2 fold changes. (B) Representative immunoblots showing the abundance of SDC4 in EVs derived from the indicated groups. (C) Representative immunoblots showing SDC4 abundance in WT cells transformed by 1,4‐D at concentrations ranging from 1.25 to 20 ppm. (D) Representative immunoblots displaying SDC4 expression in EVs derived from WT and Nrf2 KO cells transformed by 1,4‐D at concentrations ranging from 1.25 to 20 ppm. (E) Representative SDC4 staining of A549 cells after 24‐h treatment with PKH67‐labelled EVs derived from WT, 1,4‐D‐transformed WT, Nrf2 KO and 1,4‐D‐transformed Nrf2 KO cell groups. (F) Quantification results of PKH67 and SDC4 intensities, along with Pearson's correlation and Mander's overlap, are shown for the co‐localisation between PKH67‐labelled EVs and SDC4. Data are presented as mean ± SD, n = 10, * p < 0.05, ** p < 0.01 vs. control (one‐way ANOVA with Bonferroni's multiple comparisons test).

Article Snippet: The human bronchial epithelial cell line BEAS‐2B and the human non‐small cell lung cancer derived hypotriploid alveolar basal epithelial cells A549, purchased from the American Type Culture Collection (ATCC, Manassas, VA), were maintained in complete medium at 37°C under 5% CO 2 .

Techniques: Knock-Out, Expressing, Western Blot, Derivative Assay, Transformation Assay, Staining, Control

Journal: Journal of Extracellular Vesicles

Article Title: 1,4‐Dioxane Induces Epithelial‐Mesenchymal Transition and Carcinogenesis in an Nrf2‐Dependent Manner

doi: 10.1002/jev2.70072

Figure Lengend Snippet: Nrf2 modulates the 1,4‐D‐induced EMT process via SDC4 and SDC4‐enriched EVs. (A) ChIP‐seq analysis identifies Nrf2 binding peaks across the SDC4 gene body, with notable enrichment at the transcription start site (TSS), exon 1, and the first intron. Many of these peaks contain conserved antioxidant response element (ARE) featuring the core sequence TGAG/CTC. The predicted ARE sites on the SDC4 gene are marked with green numbers. (B) ChIP‐qPCR analysis showing Nrf2 occupancy at these ARE elements on the SDC4 gene. Data are presented as mean ± SD, n = 3, ** p < 0.01 vs. control (one‐way ANOVA with Bonferroni's multiple comparisons test). (C) Representative immunoblots showing the abundance of SDC4 in 1,4‐D‐transformed WT cells with SDC4 knockdown by siRNA and in 1,4‐D‐transformed Nrf2 KO cells with SDC4 overexpression via the pcDNA3.1 vector. (D) Representative images from wound healing assay illustrating the effects of SDC4 regulation on the migration capabilities of 1,4‐D‐transformed WT and Nrf2 KO cells. (E) Representative immunoblots showing the abundance of SDC4, COL12A1, CAPG, NNMT and ACTB in EV samples derived from 1,4‐D‐transformed WT cells with SDC4 knockdown and from 1,4‐D‐transformed Nrf2 KO cells with SDC4 overexpression. (F) The effects of EVs derived from the indicated groups on A549 cell proliferation were determined by MTT assay. Data are presented as mean ± SD, n = 6, ** p < 0.01 vs. control (one‐way ANOVA with Bonferroni's multiple comparisons test or unpaired Student's t test). (G and H) Representative images illustrating changes in migration and invasion capabilities of A549 cells treated with EVs from the indicated groups, with quantification of migrated or invasive cells. Data are presented as mean ± SD, n = 10, ** p < 0.01 vs. control (one‐way ANOVA with Bonferroni's multiple comparisons test or unpaired Student's t test).

Article Snippet: The human bronchial epithelial cell line BEAS‐2B and the human non‐small cell lung cancer derived hypotriploid alveolar basal epithelial cells A549, purchased from the American Type Culture Collection (ATCC, Manassas, VA), were maintained in complete medium at 37°C under 5% CO 2 .

Techniques: ChIP-sequencing, Binding Assay, Sequencing, ChIP-qPCR, Control, Western Blot, Transformation Assay, Knockdown, Over Expression, Plasmid Preparation, Wound Healing Assay, Migration, Derivative Assay, MTT Assay

Journal: mBio

Article Title: Cell envelope polysaccharide modifications alter the surface properties and interactions of Mycobacterium abscessus with innate immune cells in a morphotype-dependent manner

doi: 10.1128/mbio.00322-25

Figure Lengend Snippet: Effect of patient-derived ubiA mutations on the uptake of smooth and rough M. abscessus by A549 epithelial cells. A549 cells were infected at a multiplicity of infection of 1. The uptake of bacilli at 2 h post-infection is presented as a ratio of bacterial counts (CFU/mL) at 2 h relative to the inoculum for S and R mutants in A549 epithelial cells. Shown are averages ± SDs of triplicate wells for each strain, and the results are representative of three independent assays using different bacterial cultures. A one-way analysis of variance with Dunnett’s multiple comparison test against UbiA WT (R/S) was performed. *** P < 0.001, **** P < 0.0001.

Article Snippet: A549 human lung alveolar type II epithelial cells (ATCC CCL-185) were grown in 24-well plates to ~90% confluence in Dulbecco’s modified Eagle medium (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin.

Techniques: Derivative Assay, Infection, Comparison