human lung carcinoma a 549 cells  (ATCC)

Journal: Virulence

Article Title: FGF8-mediated TRIM16 regulation promotes K48-linked ubiquitination and degradation of RIG-I to facilitate Influenza a virus immune evasion

doi: 10.1080/21505594.2026.2677346

Figure Lengend Snippet: Analysis of A549 cell transcriptomes post-influenza a virus infection. (a) genes showing differential expression in A549 cells after a 24-hour infection with H1N1 or H13N2. (b) gene ontology (GO) enrichment analysis was conducted on genes commonly upregulated in A549 cells following infection with H1N1 and H13N2. (C) GO enrichment analysis of genes consistently downregulated in A549 cells following infection with H1N1 and H13N2. (d) transcriptomic data validation was conducted via RT-qPCR on selected differentially expressed genes in A549 cells infected with H1N1 or H13N2. Error bars indicate the mean±SEM from three independent experiments. Statistical significance was assessed using two-tailed unpaired Student’s t-tests, with thresholds set at * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Human lung adenocarcinoma cell line A549 (Procell Life Science & Technology Co., Ltd., Wuhan, China; Cat. No. CL-0016), human embryonic kidney cell line HEK293T (Procell Life Science & Technology Co., Ltd., Wuhan, China; Cat. No. CL-0005), and Madin-Darby canine kidney cell line MDCK (Procell Life Science & Technology Co., Ltd., Wuhan, China; Cat. No. CL-0154) were used for virus infection experiments, protein interaction validation experiments, and virus titration assays, respectively.

Techniques: Virus, Infection, Quantitative Proteomics, Biomarker Discovery, Quantitative RT-PCR, Two Tailed Test

Journal: Virulence

Article Title: FGF8-mediated TRIM16 regulation promotes K48-linked ubiquitination and degradation of RIG-I to facilitate Influenza a virus immune evasion

doi: 10.1080/21505594.2026.2677346

Figure Lengend Snippet: FGF8 promoted H13N2 influenza virus replication. (a) FGF8 expression in A549 cells was evaluated 24 hours after H1N1 or H13N2 infection (MOI = 0.5) using Western blot analysis, and band intensities were quantified by densitometry. (b, C) validation of FGF8 knockdown (shFGF8) and overexpression (Flag-FGF8) in A549 cell lines was conducted using RT-qPCR and Western blot. Relative protein levels were quantified by densitometric analysis. (D-F) Following 24 hours of H13N2 infection (MOI = 0.5) in A549 cells transiently transfected with FGF8 expression plasmids, viral RNA levels were measured by RT-qPCR (d), viral titers were determined using TCID50 assay (e), and Western blot analysis was performed to assess the expression of viral proteins NP, PB1, and PB2, with band intensities quantified by densitometry (f). (G-I) A549 cells with silenced FGF8 were infected with H13N2 for 24 hours, viral RNA levels were measured by RT-qPCR (G), viral titers were determined using TCID50 assay (H), and Western blot analysis was performed to assess the expression of viral proteins NP, PB1, and PB2, followed by densitometric analysis (i). (J and K) after 2 hours of H13N2 infection (MOI = 5) in A549 cells with FGF8 knockdown or overexpression, NP mRNA levels were detected using RT-qPCR. Error bars indicate the mean ± SEM from three independent experiments. Statistical analysis was performed using two-tailed unpaired Student’s t-tests, with significance thresholds defined as ns p > 0.05, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Human lung adenocarcinoma cell line A549 (Procell Life Science & Technology Co., Ltd., Wuhan, China; Cat. No. CL-0016), human embryonic kidney cell line HEK293T (Procell Life Science & Technology Co., Ltd., Wuhan, China; Cat. No. CL-0005), and Madin-Darby canine kidney cell line MDCK (Procell Life Science & Technology Co., Ltd., Wuhan, China; Cat. No. CL-0154) were used for virus infection experiments, protein interaction validation experiments, and virus titration assays, respectively.

Techniques: Virus, Expressing, Infection, Western Blot, Biomarker Discovery, Knockdown, Over Expression, Quantitative RT-PCR, Transfection, TCID50 Assay, Two Tailed Test

Journal: Virulence

Article Title: FGF8-mediated TRIM16 regulation promotes K48-linked ubiquitination and degradation of RIG-I to facilitate Influenza a virus immune evasion

doi: 10.1080/21505594.2026.2677346

Figure Lengend Snippet: FGF8 negatively regulated IFN-β induced by H13N2 infection. (a, B) luciferase reporter assays were used to assess the impact of FGF8 overexpression on IFN-β and ISRE promoter activity in A549 cells infected with H13N2 at an MOI of 1. (C-F) FGF8-overexpressing A549 cells were infected with H13N2 at an MOI of 1. At 12 hours post-infection (hpi), IFN-β levels in the cell supernatant were measured using ELISA (C), and IFN-β mRNA levels were evaluated by RT-qPCR (d). At 24 hpi, the mRNA levels of interferon-stimulated genes MX1 (e) and IFIT1 (f) were assessed by RT-qPCR. (G-J) stable FGF8-knockdown A549 cells were infected with H13N2 at an MOI of 1. At 12 hpi, IFN-β levels in the cell supernatant were quantified by ELISA (G), and IFN-β mRNA levels were evaluated using RT-qPCR (H). At 24 hpi, the mRNA levels of MX1 (i) and IFIT1 (J) were assessed by RT-qPCR. (K and L) Western blot analysis evaluated RIG-I, p-TBK1, and p-IRF3 expression in A549 cells with FGF8 overexpression (L) or knockdown (K) at 12 hours after H13N2 infection (MOI = 1). Band intensities were quantified by densitometric analysis. Statistical analysis was performed using two-tailed unpaired Student’s t-tests, with significance levels of * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Human lung adenocarcinoma cell line A549 (Procell Life Science & Technology Co., Ltd., Wuhan, China; Cat. No. CL-0016), human embryonic kidney cell line HEK293T (Procell Life Science & Technology Co., Ltd., Wuhan, China; Cat. No. CL-0005), and Madin-Darby canine kidney cell line MDCK (Procell Life Science & Technology Co., Ltd., Wuhan, China; Cat. No. CL-0154) were used for virus infection experiments, protein interaction validation experiments, and virus titration assays, respectively.

Techniques: Infection, Luciferase, Over Expression, Activity Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Knockdown, Western Blot, Expressing, Two Tailed Test

Journal: Virulence

Article Title: FGF8-mediated TRIM16 regulation promotes K48-linked ubiquitination and degradation of RIG-I to facilitate Influenza a virus immune evasion

doi: 10.1080/21505594.2026.2677346

Figure Lengend Snippet: FGF8 drives ubiquitin – proteasomal degradation of RIG-I. (a) FGF8 inhibits RIG-I-mediated signaling. A luciferase reporter assay was performed to evaluate the effect of FGF8 overexpression on IFN-β promoter activation induced by RIG-I. (B and C) FGF8 does not affect RIG-I transcription. RIG-I mRNA levels were quantified by RT-qPCR in FGF8-overexpressing A549 cells at 0, 6, and 12 hours post-infection with H13N2 (b) or H1N1 (C) at an MOI of 1. (d) dose-dependent reduction of RIG-I protein. A549 cells were transfected with increasing amounts of Flag-FGF8 plasmid for 24 hours, followed by infection with H13N2 (MOI = 1) for 12 hours. RIG-I protein levels were analyzed by Western blot, and band intensities were quantified by densitometry. (e) FGF8 reduces RIG-I stability. FGF8-overexpressing A549 cells were infected with H13N2 (MOI = 1) and treated with cycloheximide (CHX, 50 µg/mL) for the indicated time periods. Protein levels were analyzed by Western blot, and the relative abundance of HA-RIG-I was quantified to assess protein degradation rates. (F and G) proteasome inhibition restores RIG-I levels. A549 cells infected with H13N2 (f) or H1N1 (G) at an MOI of 1 were treated with DMSO, chloroquine (CQ, 50 µM), or MG132 (10 µM) for 6 hours. RIG-I expression was analyzed by Western blot, with relative protein levels quantified by densitometry. (H and I) FGF8 promotes K48-linked ubiquitination of RIG-I. HEK-293T cells were co-transfected with the indicated plasmids and treated with MG132 for 6 hours. (H) Total ubiquitination of RIG-I was assessed by immunoprecipitation with anti-HA antibody followed by immunoblotting (ib) with anti-Myc. (i) K48- or K63-linked ubiquitination was analyzed using specific ubiquitin mutants. Error bars indicate the mean ± SEM from three independent experiments. Statistical analysis was performed using two-tailed unpaired Student’s t-tests. ns (not significant), * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Human lung adenocarcinoma cell line A549 (Procell Life Science & Technology Co., Ltd., Wuhan, China; Cat. No. CL-0016), human embryonic kidney cell line HEK293T (Procell Life Science & Technology Co., Ltd., Wuhan, China; Cat. No. CL-0005), and Madin-Darby canine kidney cell line MDCK (Procell Life Science & Technology Co., Ltd., Wuhan, China; Cat. No. CL-0154) were used for virus infection experiments, protein interaction validation experiments, and virus titration assays, respectively.

Techniques: Ubiquitin Proteomics, Luciferase, Reporter Assay, Over Expression, Activation Assay, Quantitative RT-PCR, Infection, Transfection, Plasmid Preparation, Western Blot, Inhibition, Expressing, Immunoprecipitation, Two Tailed Test

Journal: Virulence

Article Title: FGF8-mediated TRIM16 regulation promotes K48-linked ubiquitination and degradation of RIG-I to facilitate Influenza a virus immune evasion

doi: 10.1080/21505594.2026.2677346

Figure Lengend Snippet: TRIM16 mediated RIG-I degradation and promoted influenza virus replication. (a) Co-immunoprecipitation analysis was performed in cells transfected with Flag-TRIM16 and HA-RIG-I, with or without H13N2 infection (MOI = 1), to verify the interaction. (b) immunofluorescence microscopy showing the localization of TRIM16 (green) and RIG-I (red) in cells infected with H13N2 or mock-infected (NC). Nuclei were stained with DAPI (blue). Note that TRIM16 and RIG-I show diffuse distribution in the NC group but form co-localized puncta (yellow) upon H13N2 infection. Scale bar: 5 μm. (C) in vitro ubiquitination assay to verify the direct E3 ligase activity of TRIM16 using wt and ΔB-Box mutant proteins. (d) in vitro ubiquitination assay to determine the linkage specificity of TRIM16-mediated RIG-I ubiquitination using K48-only and K63-only ubiquitin mutants. (e) bioinformatic analysis using PONDR revealed the presence of intrinsically disordered regions (IDRs) in the FGF8 protein sequence. (f) fluorescence microscopy of A549 cells transfected with EGFP-FGF8 (green). Nuclei were stained with DAPI. Scale bar represents 10 μm. (G) TurboID-based proximity labeling assay was performed in cells expressing FGF8-TurboID. Biotinylated proteins were captured using streptavidin beads, and the pulled-down proteins were analyzed by Western blot to detect the presence of RIG-I and TRIM16. (H and I) validation of TRIM16 knockdown. RT-qPCR (H) and Western blot (i) confirmed the silencing efficiency in A549 cells. (J) control and TRIM16-silenced A549 cells were infected with H1N1 or H13N2 (MOI = 0.5) for 24 hours. Viral protein levels (NP, PB1, PB2) were analyzed by Western blot, and band intensities were quantified by densitometry. (K) RT-qPCR analysis of IFN-β mRNA levels in TRIM16-silenced A549 cells 12 hours post-infection with H13N2 (MOI = 1). (L) Western blot confirmation of TRIM16 overexpression (OE-TRIM16). (M) A549 cells overexpressing TRIM16 were infected with H1N1 or H13N2 (MOI = 0.5) for 24 hours. Viral protein expression was analyzed by Western blot and quantified by densitometry. Error bars indicate the mean ± SEM from three independent experiments. Statistical analysis was performed using two-tailed unpaired Student’s t-tests. ns (not significant), * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Human lung adenocarcinoma cell line A549 (Procell Life Science & Technology Co., Ltd., Wuhan, China; Cat. No. CL-0016), human embryonic kidney cell line HEK293T (Procell Life Science & Technology Co., Ltd., Wuhan, China; Cat. No. CL-0005), and Madin-Darby canine kidney cell line MDCK (Procell Life Science & Technology Co., Ltd., Wuhan, China; Cat. No. CL-0154) were used for virus infection experiments, protein interaction validation experiments, and virus titration assays, respectively.

Techniques: Virus, Immunoprecipitation, Transfection, Infection, Immunofluorescence, Microscopy, Staining, In Vitro, Ubiquitin Proteomics, Activity Assay, Mutagenesis, Sequencing, Fluorescence, Labeling, Expressing, Western Blot, Biomarker Discovery, Knockdown, Quantitative RT-PCR, Control, Over Expression, Two Tailed Test

Journal: Virulence

Article Title: FGF8-mediated TRIM16 regulation promotes K48-linked ubiquitination and degradation of RIG-I to facilitate Influenza a virus immune evasion

doi: 10.1080/21505594.2026.2677346

Figure Lengend Snippet: FGF8 is upregulated by VSV infection and promotes viral replication. (a) upregulation of FGF8 by VSV infection. A549 cells were infected with VSV at an MOI of 0.5 for 24 h. FGF8 mRNA levels were determined by RT-qPCR, and protein expression was analyzed by Western blot, with band intensities quantified by densitometry. (B and C) effect of FGF8 on VSV replication. A549 cells with FGF8 overexpression (b) or knockdown (C) were infected with VSV at an MOI of 0.5 for 24 h. The expression levels of the viral protein VSV-G were determined by Western blot, and relative protein levels were quantified by densitometric analysis. Data are presented as mean ± SEM from three independent experiments. Statistical analysis was performed using two-tailed unpaired Student’s t-tests. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Human lung adenocarcinoma cell line A549 (Procell Life Science & Technology Co., Ltd., Wuhan, China; Cat. No. CL-0016), human embryonic kidney cell line HEK293T (Procell Life Science & Technology Co., Ltd., Wuhan, China; Cat. No. CL-0005), and Madin-Darby canine kidney cell line MDCK (Procell Life Science & Technology Co., Ltd., Wuhan, China; Cat. No. CL-0154) were used for virus infection experiments, protein interaction validation experiments, and virus titration assays, respectively.

Techniques: Infection, Quantitative RT-PCR, Expressing, Western Blot, Over Expression, Knockdown, Two Tailed Test

Journal: iScience

Article Title: A comprehensive multi-omics and functional study of evolutionary adaptive responses to smoke

doi: 10.1016/j.isci.2026.115547

Figure Lengend Snippet: ALDH3A1 and NQO1 as guardians of epithelial survival and barrier function upon smoke exposure (A) A549 epithelial cell death upon 4 h of exposure to 0%–100% cigarette smoke extract in ALDH3A1 and NQO1 CRISPR-Cas9 knockout cells. Epithelial cell barrier function change in response to 0%–20% cigarette smoke extract exposure for 24 h, which was measured in real-time monitoring of electrical resistance and capacitance during and upon the establishment of epithelial monolayers using electric cell-substrate impedance sensing (ECIS) in ALDH3A1- (B) and NQO1-knockout (C) A549 cells. ∗ p value <0.05.

Article Snippet: For in vitro experiments human lung adenocarcinoma A549 cells were used (CCL-185, ATCC).

Techniques: CRISPR, Knock-Out, Electric Cell-substrate Impedance Sensing

Journal: bioRxiv

Article Title: Scalable longitudinal imaging and transcriptomics of cells in dynamic enclosures

doi: 10.64898/2026.05.05.723030

Figure Lengend Snippet: A: classification of CCEs in different phenotypes based on the analysis of longitudinal imaging data. Red: CellTrace™ Far Red, blue: Annexin V, green: EGFR. B: UMAP based on the transcriptomic data from 10,604 CCEs containing A549 cells treated with 10 µM Olmutinib. The colors represent different transcriptomic clusters. C: UMAP based on the transcriptomic data (same as panel B) colored according to the imaging-derived CCE classification in panel A. 2,328 CCEs that could not be accurately classified were excluded from the analysis. D: proportion of CCEs (y axis) belonging to each imaging-based phenotype (indicated by the color) within each gene expression cluster (x axis). E: Upset plot showing overlap of significant GSEA pathway enrichments across three classification strategies. The combination of transcriptomic clustering with imaging classification identified 15 unique pathways not found in either single-modality strategy. F: significant interaction effects (p_adj < 0.05) between RNA clusters and imaging phenotypes on the prediction of drug resistance pathway modules (G2M checkpoint, E2F targets, MYC targets, DNA repair, EMT) (see Methods). The daughter cell resistant phenotype showed 7 out of 14 total significant interactions, indicating that pathway activities are maximally explained by the combination of transcript state and the daughter cell resistant phenotypic classification. G: Confusion matrix for elastic net prediction of imaging phenotypes from gene expression. H: STRING PPI network for top 50 positive coefficient genes (associated with daughter cell resistance). I: STRING PPI network for top 50 negative coefficient genes (inversely associated with daughter cell resistance). J: Selected differentially expressed genes between expression-defined clusters (x axis). The color represents the average expression (scaled per gene) and the size of the circle indicates the percentage of CCEs expressing the gene. Cluster 2 showed strong enrichment for cell division pathways and overexpressed the proliferation marker TOP2A. Cluster 3 exhibited activation of multiple EGFR bypass pathways with overexpression of EPHA7 (64), HGF (65), ERBB2 (66), and AXL(67), all capable of activating MAPK signaling independently of EGFR. Cluster 5 displayed enrichment of p53 targets, including upregulation of quiescence-associated genes such as GADD45A, REDD1, ATF3, SFN, and BTG2.

Article Snippet: Human lung cancer A549 cells were purchased from ATCC (CCL-185), and cultured in DMEM supplemented with 10% FBS and 1% Pen-Strep.

Techniques: Imaging, Derivative Assay, Gene Expression, Expressing, Marker, Activation Assay, Over Expression