Journal: Biochemistry and Biophysics Reports

Article Title: ATM inhibition restores IFN-γ sensitivity and induces ferroptosis in NSCLC via DNA damage response

doi: 10.1016/j.bbrep.2026.102568

Figure Lengend Snippet: IFN-γ responsiveness correlates DNA damage and repair responses in NSCLC cell lines (A) Relative cell viability of A549 or PC-9 treated with the indicated concentration of IFN-γ for 24 h are shown. Data are presented as mean ± SD. (B) Reactome analysis of the GSE180942 dataset was performed using differential CRISPR β-scores under IFN-γ treatment (Δβ = PC-9 − A549). Bars indicate the normalized enrichment score (NES) for each pathway indicated. Positive NES denotes pathways whose constituent genes are more essential in A549, whereas negative NES denotes pathways more essential in PC-9 upon IFN-γ treatment.

Article Snippet: The human non-small cell lung cancer cell lines PC-9 (kindly gifted from Dr. Kiura, Okayama University, Japan) and A549 (CCL-185, obtained from American Type Culture Collection) were used.

Techniques: Concentration Assay, CRISPR

Journal: Biochemistry and Biophysics Reports

Article Title: ATM inhibition restores IFN-γ sensitivity and induces ferroptosis in NSCLC via DNA damage response

doi: 10.1016/j.bbrep.2026.102568

Figure Lengend Snippet: Inhibition of ATM restores NSCLC cells to IFN-γ by inducing DNA damage response (A) Cell viability of A549 (left panel) or PC-9 (right panel) treated with IFN-γ (1000 ng/ml) and/or KU-55933 (10 μM) for 24 h are shown. Data are presented as mean ± SD. * p < 0.05. (B) Expression of γH2AX and b-Actin (loading control) in A549 (left panel) or PC-9 (right panel) cells treated with IFN-γ (1000 ng/ml) and/or KU-55933 (10 μM) for 24 h are shown.

Article Snippet: The human non-small cell lung cancer cell lines PC-9 (kindly gifted from Dr. Kiura, Okayama University, Japan) and A549 (CCL-185, obtained from American Type Culture Collection) were used.

Techniques: Inhibition, Expressing, Control

Journal: Biochemistry and Biophysics Reports

Article Title: ATM inhibition restores IFN-γ sensitivity and induces ferroptosis in NSCLC via DNA damage response

doi: 10.1016/j.bbrep.2026.102568

Figure Lengend Snippet: Inhibition of ATM in combination with IFN-γ induce ferroptosis in NSCLCs Cell viability of A549 (A) or PC-9 (B) treated with the indicated combination of IFN-γ (1000 ng/ml), KU-55933 (10 μM), Ferrostatin-1 (5 μM), and Liproxstatin-1 (5 μM) for 24 h are shown. Data are presented as mean ± SD. * p < 0.05.

Article Snippet: The human non-small cell lung cancer cell lines PC-9 (kindly gifted from Dr. Kiura, Okayama University, Japan) and A549 (CCL-185, obtained from American Type Culture Collection) were used.

Techniques: Inhibition

Journal: PLOS Pathogens

Article Title: Collectin-11, a complement pattern recognition molecule, mediates pulmonary SARS-CoV-2 neutralization and protection

doi: 10.1371/journal.ppat.1014216

Figure Lengend Snippet: (A-C) Neutralization of infectious SARS-CoV-2 following incubation with two-fold dilutions of rCL-11 (black) in A549-hACE-2 cells (starting at 40 µg/ml) (A) , Calu-3 cells (starting at 20 µg/ml) (B) , and Huh7.5 cells (starting at 20 µg/ml) (C) using the conventional experimental setup. The mAb clone 61 was used as a neutralization control (orange) and rCL-11 buffer (gray) in corresponding dilutions was used to monitor buffer-mediated effects on the cells. The percentage of protected cells was determined from the number of SARS-CoV-2 infected cells in experimental wells compared to the number of infected cells in virus-only control wells after anti-S protein immunostaining. Data are shown as means ± SD of four (A549-hACE-2 and Calu-3 cells) or six (Huh7.5 cells) replicates.

Article Snippet: The human lung adenocarcinoma A549-hACE-2 cell line (InvivoGen, San Diego, California, USA) (A549 cells expressing human ACE-2) was maintained as described previously [ ].

Techniques: Neutralization, Incubation, Control, Infection, Virus, Immunostaining

Journal: Oncology Letters

Article Title: BUD23 is associated with malignancy and correlates with immune infiltration in NSCLC

doi: 10.3892/ol.2026.15608

Figure Lengend Snippet: BUD23 knockdown suppresses the proliferative and migration of NSCLC cells. (A) RT-qPCR was used to quantify BUD23 mRNA levels in HBE cells and a panel of NSCLC cell lines. Validation of BUD23 knockdown efficiency in (B) A549 and (C) H1299 cells by RT-qPCR. Assessment of cell viability in (D) A549 and (E) H1299 cells via CCK-8 assay and cell migration in (F) A549 and (G) H1299 cells by wound healing assay (magnification, ×10). CCK-8 assays of (H) A549 and (I) H1299 cells 24 h after BUD23 knockdown with or without subsequent co-culture with Jurkat T cells for 48 h in Transwell chambers. (J) Quantification of apoptosis in A549 and H1299 cells via Annexin V/PI flow cytometry. **P<0.01 and ***P<0.001 vs. HBE, NC, Con or as indicated. NSCLC, non-small cell lung cancer; RT-qPCR, reverse transcription-quantitative PCR; HBE, human bronchial epithelial; CCK-8, Cell Counting Kit-8; NC, negative control; Con, control; Si1/2, small interfering RNA targeting BUD23.

Article Snippet: The HBE [full name: HBE4-E6/E7 (Human Bronchial Epithelial Cells; cat. no. CRL-2078)] cell line, A549 lung adenocarcinoma cell line (cat. no. CCL-185), H1299 lung large cell carcinoma cell line (cat. no. CRL-5803), H460 lung large cell carcinoma cell line (cat. no. HTB-177) and Jurkat T cells (cat. no. TIB-152; a childhood T acute lymphoblastic leukemia T-cell line), were purchased from the American Type Culture Collection.

Techniques: Knockdown, Migration, Quantitative RT-PCR, Biomarker Discovery, CCK-8 Assay, Wound Healing Assay, Co-Culture Assay, Flow Cytometry, Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Counting, Negative Control, Control, Small Interfering RNA

Journal: Oncology Letters

Article Title: BUD23 is associated with malignancy and correlates with immune infiltration in NSCLC

doi: 10.3892/ol.2026.15608

Figure Lengend Snippet: BUD23 knockdown significantly downregulates POLR2J expression in non-small cell lung cancer. (A) Venn diagram showing the intersection between LUAD GSEA core enrichment genes (key genes driving Hallmark DNA repair pathway enrichment) and LUSC GSEA core enrichment genes within the Hallmark DNA repair gene set, identifying 49 common genes. (B) Venn diagram showing the intersection between BUD23-correlated genes (correlation index >0.3) derived from the TCGA-LUAD cohort and BUD23-correlated genes (correlation index >0.3) derived from the TCGA-LUSC cohort via multi-gene correlation analysis, yielding 79 overlapping genes. (C) Venn diagram showing the secondary intersection between the 49 overlapping GSEA core enrichment genes (from panel A) and the 79 overlapping BUD23-correlated genes (from panel B), identifying 4 shared common genes: TAF6, POLR2J, RFC2 and VPS37D. (D) Validation of TAF6, POLR2J, RFC2 and VPS37D mRNA expression following BUD23 knockdown in A549 cells via reverse transcription-quantitative PCR. Cell-cycle analysis in (E) A549 and (F) H1299 cells following BUD23 knockdown. ***P<0.001 vs. Con. POLR2J, RNA polymerase II subunit J; LUAD, lung adenocarcinoma; GSEA, gene set enrichment analysis; LUSC, lung squamous cell carcinoma; TCGA, The Cancer Genome Atlas; TAF6, TATA-box binding protein associated factor 6; RFC2, replication factor C subunit 2; VPS37D, vacuolar protein sorting-associated protein 37D; Con, control; Si1, small interfering RNA targeting BUD23.

Article Snippet: The HBE [full name: HBE4-E6/E7 (Human Bronchial Epithelial Cells; cat. no. CRL-2078)] cell line, A549 lung adenocarcinoma cell line (cat. no. CCL-185), H1299 lung large cell carcinoma cell line (cat. no. CRL-5803), H460 lung large cell carcinoma cell line (cat. no. HTB-177) and Jurkat T cells (cat. no. TIB-152; a childhood T acute lymphoblastic leukemia T-cell line), were purchased from the American Type Culture Collection.

Techniques: Knockdown, Expressing, Derivative Assay, Biomarker Discovery, Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Cycle Assay, Binding Assay, Control, Small Interfering RNA

Journal: Frontiers in Oncology

Article Title: Selaginella doederleinii -derived extracellular vesicle-like particles suppress lung cancer with ferroptosis-associated changes and modulation of the FABP4/PPARG/GPX4 axis

doi: 10.3389/fonc.2026.1829211

Figure Lengend Snippet: Internalization of SD-EVLPs and its inhibitory effects on lung cancer cell proliferation (A) Internalization of SD-EVLPs by lung cancer cells; (B, C) CCK-8 assays assessing the effects of SD-EVLPs on lung cancer cell viability; (D–G) EdU assays evaluating the proliferation of A549 and NCI-H1299 cells; (H–I) Colony formation assays assessing the proliferative capacity of A549 and NCI-H1299 cells. Data are presented as the mean ± SEM, n = 3 independent experiments. Compared with the control group, * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Human lung cancer cell lines A549 (Cat. No.: CL-0016) and NCI-H1299 (Cat. No.: CL-0165) were purchased from Wuhan Procell Life Science & Technology Co., Ltd. Four-week-old male BALB/c nude mice (SPF grade) were purchased from Hunan Silaike Jingda Laboratory Animal Co., Ltd. (Changsha, China), with the Animal Production License No.: SCXK (Xiang) 2021-0002.

Techniques: CCK-8 Assay, Control

Journal: Frontiers in Oncology

Article Title: Selaginella doederleinii -derived extracellular vesicle-like particles suppress lung cancer with ferroptosis-associated changes and modulation of the FABP4/PPARG/GPX4 axis

doi: 10.3389/fonc.2026.1829211

Figure Lengend Snippet: SD-EVLPs inhibit migration and invasion of lung cancer cells (A–D) Wound healing assays assessing the effect of SD-EVLPs on the migration of A549 and NCI-H1299 cells; (E, F) Transwell assays evaluating the invasion ability of A549 and NCI-H1299 cells; (G–I) Western blot analysis of E-cadherin and N-cadherin protein expression. Data are presented as the mean ± SEM, n = 3 independent experiments. Compared with the control group, * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Human lung cancer cell lines A549 (Cat. No.: CL-0016) and NCI-H1299 (Cat. No.: CL-0165) were purchased from Wuhan Procell Life Science & Technology Co., Ltd. Four-week-old male BALB/c nude mice (SPF grade) were purchased from Hunan Silaike Jingda Laboratory Animal Co., Ltd. (Changsha, China), with the Animal Production License No.: SCXK (Xiang) 2021-0002.

Techniques: Migration, Western Blot, Expressing, Control

Journal: Frontiers in Oncology

Article Title: Selaginella doederleinii -derived extracellular vesicle-like particles suppress lung cancer with ferroptosis-associated changes and modulation of the FABP4/PPARG/GPX4 axis

doi: 10.3389/fonc.2026.1829211

Figure Lengend Snippet: RT-qPCR and Western blot analysis of ferroptosis-related gene expression in lung cancer cells (A, B) RT-qPCR analysis of GPX4 and SLC7A11 mRNA expression in A549 cells; (C, D) RT-qPCR analysis of GPX4 and SLC7A11 mRNA expression in NCI-H1299 cells; (E–H) Western blot analysis of GPX4 and xCT protein expression in lung cancer cells; (I) TEM assessment of mitochondrial ultrastructure. Data are presented as the mean ± SEM, n = 3 independent experiments. Compared with the control group, * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Human lung cancer cell lines A549 (Cat. No.: CL-0016) and NCI-H1299 (Cat. No.: CL-0165) were purchased from Wuhan Procell Life Science & Technology Co., Ltd. Four-week-old male BALB/c nude mice (SPF grade) were purchased from Hunan Silaike Jingda Laboratory Animal Co., Ltd. (Changsha, China), with the Animal Production License No.: SCXK (Xiang) 2021-0002.

Techniques: Quantitative RT-PCR, Western Blot, Gene Expression, Expressing, Control

Journal: Frontiers in Oncology

Article Title: Selaginella doederleinii -derived extracellular vesicle-like particles suppress lung cancer with ferroptosis-associated changes and modulation of the FABP4/PPARG/GPX4 axis

doi: 10.3389/fonc.2026.1829211

Figure Lengend Snippet: SD-EVLPs modulate the FABP4/PPARG/GPX4-associated pathway in lung cancer cells (A–D) RT-qPCR analysis of FABP4 and PPARG mRNA expression in A549 and NCI-H1299 cells; (E–H) Western blot analysis of FABP4 and PPARG protein expression in A549 and NCI-H1299 cells. Data are presented as the mean ± SEM, n = 3 independent experiments. Compared with the control group, * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Human lung cancer cell lines A549 (Cat. No.: CL-0016) and NCI-H1299 (Cat. No.: CL-0165) were purchased from Wuhan Procell Life Science & Technology Co., Ltd. Four-week-old male BALB/c nude mice (SPF grade) were purchased from Hunan Silaike Jingda Laboratory Animal Co., Ltd. (Changsha, China), with the Animal Production License No.: SCXK (Xiang) 2021-0002.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Control

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