human lung adenocarcinoma alveolar epithelial cell line a549 cells  (Procell Inc)

Journal: bioRxiv

Article Title: Engineered Matrices Reveal Sulfation-Mediated Stress Adaptation and Drug-Specific Modulation of Chemotherapeutic Response

doi: 10.64898/2026.05.21.726894

Figure Lengend Snippet: A) Schematic of alginate modification and fabrication of alginate (Alg) and sulfated alginate (S-Alg) hydrogels with Ca 2+ crosslinking. B) Storage modulus (G’) and loss tangent (G’’/G’) of Alg and S-Alg hydrogels assessed with oscillatory rheology measurements. Three independent replicates were measured with error bars indicating mean ± SD. Statistical analysis was performed using two-tailed t-test, ns not significant. C) Metabolic activity and D) dsDNA quantification of A549 cells encapsulated in Alg and S-Alg hydrogels at days 0, 7,14 and 21. Three independent replicates were measured, and error bars indicate mean ± SD. Statistical analysis was performed using two-tailed t-test, ****p<0.0001. E) Bright-field (upper panel) and phalloidin (red)/DAPI (blue) staining (lower panel) images of A549 cells encapsulated in Alg/S-Alg hydrogels at days 7 and day 21. Scale bar:100 µm. F) Quantification of clump area and G) clump number (>1000 µm 2 ) of cells grown in Alg and S-Alg hydrogels at day 21. Data are shown as mean ± SD of five replicates (the average of 10-30 images per replicate). Statistical analysis was performed using two-tailed t-test, ****p<0.0001, **p<0.01.

Article Snippet: Human lung adenocarcinoma cell line A549 (CCL-185) was purchased from American Tissue Culture Collection (ATCC) and cultured in growth medium DMEM/F12 (Lonza) supplemented with 10% fetal bovine serum (FBS) (Biowest) and 1% penicillin-streptomycin (P/S) (Gibco) with incubation at 37 °C and 5% CO 2 . shRNA-mediated knockdown of PIK3CA in A549 cells was carried out as previously described [ ].

Techniques: Modification, Two Tailed Test, Activity Assay, Staining

Journal: bioRxiv

Article Title: Engineered Matrices Reveal Sulfation-Mediated Stress Adaptation and Drug-Specific Modulation of Chemotherapeutic Response

doi: 10.64898/2026.05.21.726894

Figure Lengend Snippet: A) Dose-response curves and half-inhibitory concentration (IC50) values for A549 cells after 72h treatment with cisplatin, gemcitabine and paclitaxel in 2D monolayer culture. B) Dose-response curves and IC50 values for cisplatin, gemcitabine and paclitaxel for early treatment (day 1) of A549 cells in Alg and S-Alg hydrogels. C) Dose-response curves and IC50 values for cisplatin, gemcitabine and paclitaxel for later treatment (day 16) of A549 cells grown in Alg and S-Alg hydrogels. For all drug treatment set-ups, three independent replicates were measured for each drug concentration and results were normalized to the control group. Dose-response curves were assessed with non-linear regression curve fit analysis.

Article Snippet: Human lung adenocarcinoma cell line A549 (CCL-185) was purchased from American Tissue Culture Collection (ATCC) and cultured in growth medium DMEM/F12 (Lonza) supplemented with 10% fetal bovine serum (FBS) (Biowest) and 1% penicillin-streptomycin (P/S) (Gibco) with incubation at 37 °C and 5% CO 2 . shRNA-mediated knockdown of PIK3CA in A549 cells was carried out as previously described [ ].

Techniques: Concentration Assay, Control

Journal: bioRxiv

Article Title: Engineered Matrices Reveal Sulfation-Mediated Stress Adaptation and Drug-Specific Modulation of Chemotherapeutic Response

doi: 10.64898/2026.05.21.726894

Figure Lengend Snippet: A) Metabolic activity of A549 cells encapsulated in Alg or S-Alg hydrogels for 16 days followed by treatment with cisplatin (10 µM), gemcitabine (5 µM) or paclitaxel (50 nM) for 5 days. Three independent replicates were measured and error bars indicate mean ± SD. Statistical analysis was performed using two-tailed t-test, *p<0.05; **p<0.01; ***p<0.001. B) DCFDA ROS production assessment of A549 cells after treatment with 50 µM TBHP for 4 hours in Alg and S-Alg hydrogels. Three independent replicates were measured, and error bars indicate mean ± SD. Statistical analysis was performed using two-tailed t-test, *p<0.005. C) JC-10 mitochondrial membrane potential assessment of A549 cells after treatment with 50 µM CCCP in Alg and S-Alg hydrogels. Three independent replicates were measured, and error bars indicate mean ± SD. Statistical analysis was performed using two-tailed t-test, *p<0.05. D) Calcein-AM (green) and Propidium iodide (PI) (red) staining of A549 cells encapsulated in Alg or S-Alg hydrogels for 16 days followed by treatment with cisplatin (10 µM), gemcitabine (5 µM) or paclitaxel (50 nM) for 5 days. Scale bar: 70 µm. E) Bright-field microscopy images of cells encapsulated in Alg or S-Alg hydrogels for 16 days followed by treatment with cisplatin (10 µM), gemcitabine (5 µM) or paclitaxel (50 nM) for 5 days. Scale bar: 70 µm.

Article Snippet: Human lung adenocarcinoma cell line A549 (CCL-185) was purchased from American Tissue Culture Collection (ATCC) and cultured in growth medium DMEM/F12 (Lonza) supplemented with 10% fetal bovine serum (FBS) (Biowest) and 1% penicillin-streptomycin (P/S) (Gibco) with incubation at 37 °C and 5% CO 2 . shRNA-mediated knockdown of PIK3CA in A549 cells was carried out as previously described [ ].

Techniques: Activity Assay, Two Tailed Test, Membrane, Staining, Microscopy

Journal: bioRxiv

Article Title: Engineered Matrices Reveal Sulfation-Mediated Stress Adaptation and Drug-Specific Modulation of Chemotherapeutic Response

doi: 10.64898/2026.05.21.726894

Figure Lengend Snippet: A) Representative immunofluorescence staining images for apoptotic marker cleaved caspase-3 expression of A549 cells in Alg and S-Alg hydrogels. Cleaved caspase-3 (red); phalloidin (gray); DAPI (blue). Scale bar: 100 µm. B) Quantification of cleaved caspase-3 fluorescence intensity between Alg and S-Alg hydrogels. Data are shown as mean ± SD of three replicates (the average of 10-30 images per replicates). Statistical analysis was performed using two-tailed t-test, ***p<0.001, *p<0.05. C) Expression level of genes involved in regulation of apoptosis. Gene expression analysis was performed following 3-week culture of A549 cells in Alg and S-Alg hydrogels. Three independent replicates were measured, and error bars indicate mean ± SD. Statistical analysis was performed using two-tailed t-test, **p<0.01; ****p<0.0001.

Article Snippet: Human lung adenocarcinoma cell line A549 (CCL-185) was purchased from American Tissue Culture Collection (ATCC) and cultured in growth medium DMEM/F12 (Lonza) supplemented with 10% fetal bovine serum (FBS) (Biowest) and 1% penicillin-streptomycin (P/S) (Gibco) with incubation at 37 °C and 5% CO 2 . shRNA-mediated knockdown of PIK3CA in A549 cells was carried out as previously described [ ].

Techniques: Immunofluorescence, Staining, Marker, Expressing, Fluorescence, Two Tailed Test, Gene Expression

Journal: bioRxiv

Article Title: Engineered Matrices Reveal Sulfation-Mediated Stress Adaptation and Drug-Specific Modulation of Chemotherapeutic Response

doi: 10.64898/2026.05.21.726894

Figure Lengend Snippet: A) Expression levels of ABCB1 gene in A549 cells grown in Alg and S-Alg hydrogels. Three independent replicates were measured, and error bars indicate mean ± SD. Statistical analysis was performed using two-tailed t-test, *p<0.05 B) Representative immunofluorescence staining images for ABCB1 in Alg and S-Alg hydrogels. ABCB1 (green); Actin (magenta); DAPI (blue). Scale bar: 100 µm. Metabolic activity assessment of A549 cells in S-Alg hydrogels under C) verapamil treatment, D) PIK3CA knockdown, E) concurrent verapamil treatment and PIK3CA -knockdown conditions. Three independent biological replicates were measured, and error bars indicate mean ± SD. Statistical analysis was performed using two-tailed t-test, ns not significant. F) Bright-field microscopy images for A549 cells grown in S-Alg hydrogels under untreated, verapamil-treated, PI3K knockdown and dual inhibition conditions. Scale bar=50 µm. Metabolic activity assessment of A549 cells in S-Alg hydrogels after treatment with cisplatin (10 µM), gemcitabine (5 µM) and paclitaxel (50 nM) under G) verapamil treatment, H) PIK3CA -knockdown, i) concurrent verapamil treatment and PIK3CA knockdown conditions. Three independent replicates were measured; and statistical analysis was performed by using One-way ANOVA, ns not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Article Snippet: Human lung adenocarcinoma cell line A549 (CCL-185) was purchased from American Tissue Culture Collection (ATCC) and cultured in growth medium DMEM/F12 (Lonza) supplemented with 10% fetal bovine serum (FBS) (Biowest) and 1% penicillin-streptomycin (P/S) (Gibco) with incubation at 37 °C and 5% CO 2 . shRNA-mediated knockdown of PIK3CA in A549 cells was carried out as previously described [ ].

Techniques: Expressing, Two Tailed Test, Immunofluorescence, Staining, Activity Assay, Knockdown, Microscopy, Inhibition

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