human non small cell lung cancer nsclc cell lines a549  (ATCC)

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Broad-spectrum antiviral activity of the synthetic rocaglate zotatifin against Mayaro virus and other viruses

doi: 10.3389/fcimb.2026.1752166

Figure Lengend Snippet: Zotatifin demonstrates dose-dependent inhibition of Mayaro virus (MAYV) replication across multiple cell lines. (A) Chemical structures of rocaglate compounds evaluated in this study. (B) Cell viability assessment in HDFs and HMC3 cells following 24 h treatment with increasing zotatifin concentrations, determined using the MTT assay. (C–H) Antiviral efficacy evaluation across three cell lines. HDFs (C–E) , HMC3 (F–G) , and A549 cells (H) were pretreated with rocaglates for 2 h, infected with MAYV strain AVR0565 (MOI = 1). Following 24 h incubation with compounds, viral progeny was quantified by plaque-forming assay. The dashed horizontal line indicates the limit of detection (10 PFU/ml). Data shown for rocaglamide (blue bars), CR-1-31-B (orange bars), and zotatifin (violet bars). (I) Morphological analysis of MAYV-infected HDFs with zotatifin treatment (50 nM). Cells were fixed at 48 h post-infection with a 4% formaldehyde solution and stained with a 2% crystal violet solution. Scale bar: 100 μm. Values represent mean ± standard deviation from three independent experiments, each performed in triplicate (C–H) o quadruplicate (B) . Statistical analysis was performed using a one-way ANOVA, followed by a Dunnett’s post hoc test comparing treated groups to vehicle control. Significance levels: ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001.

Article Snippet: Human dermal fibroblasts (HDFs, Cat. # PCS-201-012), human immortalized microglial cells (HMC3, Cat. # CRL-3304), human non-small cell lung cancer A549 cells (CCL-185), BSC40 (Cat. # CRL-2761), and Vero-E6 cells (CRL-1586) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Inhibition, Virus, MTT Assay, Infection, Incubation, Staining, Standard Deviation, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Broad-spectrum antiviral activity of the synthetic rocaglate zotatifin against Mayaro virus and other viruses

doi: 10.3389/fcimb.2026.1752166

Figure Lengend Snippet: Zotatifin reduces MAYV E1 and nsP1 protein levels in a dose-dependent manner. HDFs (A) or A549 cells (B) were pretreated with zotatifin at concentrations of 15, 30, and 50 nM for 2 h. Cells were subsequently infected with MAYV strain AVR0565 (MOI = 1) and maintained in the presence of zotatifin throughout the infection period. At 24 h post-infection, viral protein levels were assessed by Western blot analysis using specific antibodies against MAYV E1 and nsP1 proteins. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the loading control. Representative blots from three independent experiments are shown. (C) A549 cells were treated with 50 nM zotatifin or vehicle control and infected with MAYV strain AVR0565. At 24 h post-infection, cells were fixed and processed for immunofluorescence microscopy using antibodies specific for MAYV E1 and nsP1 proteins. Representative immunofluorescence images from at least 10 fields across two independent experiments are presented. Scale bar: 50 μm.

Article Snippet: Human dermal fibroblasts (HDFs, Cat. # PCS-201-012), human immortalized microglial cells (HMC3, Cat. # CRL-3304), human non-small cell lung cancer A549 cells (CCL-185), BSC40 (Cat. # CRL-2761), and Vero-E6 cells (CRL-1586) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Infection, Western Blot, Control, Immunofluorescence, Microscopy

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Broad-spectrum antiviral activity of the synthetic rocaglate zotatifin against Mayaro virus and other viruses

doi: 10.3389/fcimb.2026.1752166

Figure Lengend Snippet: Zotatifin inhibits MAYV replication through stage-specific mechanisms and enhances innate immune responses. (A) Schematic representation of the experimental design for viral cycle analysis, showing the pre-treatment, binding, entry, and post-entry assays protocols. HDFs were treated with zotatifin (50 nM) and infected with MAYV (AVR0565 strain). Viral titers in culture supernatants were quantified by plaque-forming assay for: (B) pre-treatment assay (cells pre-treated with zotatifin before infection), (C) binding assay (zotatifin present during viral adsorption), (D) entry assay (zotatifin added during viral entry phase), and (E) post-entry assay (zotatifin added after viral internalization). (F) Time-of-addition analysis. HDFs were infected with MAYV and zotatifin (50 nM) was added at indicated time points post-infection. Viral titers were determined by plaque-forming assay at 24 h post-infection. The dashed horizontal line indicates the limit of detection (10 PFU/ml). (G–K) Zotatifin upregulates interferon and interferon-stimulated gene expression. A549 and HMC3 cells were treated or untreated with 50 nM zotatifin for 8 h. Relative mRNA expression levels were analyzed by RT-qPCR for: (G) interferon-α ( IFNα ), (H) RIG-I-like receptor ( DDX58 ), (I) myxovirus resistance protein A ( MxA ), (J) interferon-stimulated gene 15 ( ISG15 ), and (K) Toll-like receptor 3 ( TLR3 ). Expression levels were normalized to β-actin using the ΔΔCt method. Data represent mean ± standard deviation from two independent experiments performed in triplicate. Statistical significance was determined by unpaired Student t-test. Significance levels: ns, not significant; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Human dermal fibroblasts (HDFs, Cat. # PCS-201-012), human immortalized microglial cells (HMC3, Cat. # CRL-3304), human non-small cell lung cancer A549 cells (CCL-185), BSC40 (Cat. # CRL-2761), and Vero-E6 cells (CRL-1586) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Binding Assay, Infection, Adsorption, Gene Expression, Expressing, Quantitative RT-PCR, Standard Deviation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Broad-spectrum antiviral activity of the synthetic rocaglate zotatifin against Mayaro virus and other viruses

doi: 10.3389/fcimb.2026.1752166

Figure Lengend Snippet: Zotatifin inhibits the replication of influenza A, vesicular stomatitis, and vaccinia viruses. A549 cells were pretreated with zotatifin, then infected with PR8-GFP (A–D) , rVSV-GFP (E–G) or vaccinia (H, I) viruses at low (0.5) or high (5) multiplicity of infection (MOI). At 24 h post-infection, the intensity of GFP, and the quantity of viral progeny in PR8-GFP and rVSV-GFP infected cells were assessed using flow cytometry, and a plaque-forming assay. Virus titers in cell lysates of vaccinia virus infected cells were determined by plaque assay. The dashed horizontal line indicates the limit of detection (10 PFU/ml). Data represent mean ± standard deviation from two independent experiments performed in triplicate. Statistical analysis was carried out using a one-way ANOVA followed by a Dunnett’s post hoc test or an unpaired Student t-test. Significance levels: ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001.

Article Snippet: Human dermal fibroblasts (HDFs, Cat. # PCS-201-012), human immortalized microglial cells (HMC3, Cat. # CRL-3304), human non-small cell lung cancer A549 cells (CCL-185), BSC40 (Cat. # CRL-2761), and Vero-E6 cells (CRL-1586) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Infection, Flow Cytometry, Virus, Plaque Assay, Standard Deviation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Broad-spectrum antiviral activity of the synthetic rocaglate zotatifin against Mayaro virus and other viruses

doi: 10.3389/fcimb.2026.1752166

Figure Lengend Snippet: Zotatifin downregulates the synthesis of viral proteins in influenza A virus, vesicular stomatitis virus, and vaccinia virus infected cells. A549 cells were pretreated with zotatifin, then infected with PR8-GFP (A, B) , rVSV-GFP (C, D) , or vaccinia (E, F) viruses, as previously indicated. At the indicated hours after infection, levels of influenza A NP, VSV G, or vaccinia E3L proteins were evaluated using Western blot analysis. β-actin or GAPDH proteins were used as a loading controls. Representative images from three independent experiments are shown.

Article Snippet: Human dermal fibroblasts (HDFs, Cat. # PCS-201-012), human immortalized microglial cells (HMC3, Cat. # CRL-3304), human non-small cell lung cancer A549 cells (CCL-185), BSC40 (Cat. # CRL-2761), and Vero-E6 cells (CRL-1586) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Virus, Infection, Western Blot

Journal: Journal of Pharmaceutical Analysis

Article Title: Oblique-incidence reflectivity difference technology identifies the antiviral drug Ribavirin as an inhibitor of lung tumor progression by targeting AMPK signaling

doi: 10.1016/j.jpha.2025.101306

Figure Lengend Snippet: Identification of 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK)-binding small-molecule compounds by high-throughput screening (HTS). (A) Small molecule compounds are printed onto homemade phenyl-isocyanate-functionalized glass slides and immobilized through amino, hydroxyl, and thiol groups as well as other interactions. In oblique-incidence reflectivity difference (OI-RD) scanning of small-molecule microarrays (SMMs), successful binding of the target protein at a specific location with small-molecule compounds increases the molecular layer thickness, which OI-RD detects as white spots. The binding of proteins to small molecule compounds is usually coupled mediated by nucleophilic groups (hydroxyl, amino, and thiol). Interactions such as hydrogen bonding will be further formed thereby mediating protein interactions with small molecule compounds. No spot is observed if binding does not occur. Created with www.BioRender.com . (B) Categories of the compound library. Approximately 4389 compounds, including 1678 Food and Drug Administration (FDA)-approved drugs, 1456 natural products, and 1255 known inhibitors, were printed on the chip. (C) A representative image showing the binding of the recombinant AMPK protein to compounds printed on an HTS chip. (D) A real-time OI-RD assay depicting the association‒dissociation curves of surface-immobilized Ribavirin binding to the recombinant AMPK protein. (E) A surface plasmon resonance (SPR) experiment was used to evaluate the interaction kinetics and binding affinity between Ribavirin and AMPK. (F, G) Quantitative real-time polymerase chain reaction (qRT-PCR) measurement of Ampk transcripts in mouse Lewis lung carcinoma (LLC) cells (F) or human non-small cell lung cancer cells (A549) (G). The data are presented as the relative fold induction of Ampk/actin transcripts. Arrows indicate groups with significantly reduced Ampk transcripts. The data shown are the means ± standard error of the mean (SEM) from n = 3 biological replicates. Statistical significance was determined by two-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. ∗∗ P < 0.01, ∗∗∗ P < 0.001. mRNA: messenger RNA.

Article Snippet: Human embryonic kidney epithelial cells (HEK293T; ATCC CRL-11268), human non-small cell lung cancer cells (A549; ECACC 86012804), human lung adenocarcinoma cells (PC-9; ECACC 90071810), and mouse Lewis lung carcinoma (LLC) cells (ATCC CRL-1642) (FuHeng, Shanghai, China) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA).

Techniques: Binding Assay, High Throughput Screening Assay, Drug discovery, Recombinant, SPR Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Journal of Pharmaceutical Analysis

Article Title: Oblique-incidence reflectivity difference technology identifies the antiviral drug Ribavirin as an inhibitor of lung tumor progression by targeting AMPK signaling

doi: 10.1016/j.jpha.2025.101306

Figure Lengend Snippet: The impact of Ribavirin on inflammatory factors is regulated downstream of the 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway. (A, B) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of interleukin-6 ( Il - 6 ) transcript levels in mouse Lewis lung carcinoma (LLC) cells (A) and human non-small cell lung cancer cells (A549) (B) cells following 8 h of hypoxia treatment. The data are presented as the relative fold induction of Il - 6/actin transcripts. Arrows indicate groups with significantly reduced Il - 6 transcripts. Statistical significance was determined by two-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001. (C, D) qRT-PCR analysis of Tnf α transcript levels in LLC (C) and A549 (D) cells following 8 h of hypoxia treatment. The data are presented as the relative fold induction of Tnf α/actin transcripts. Arrows indicate groups with significantly reduced Tnf α transcripts. Statistical significance was determined by two-way ANOVA, followed by Tukey’s post hoc test. ∗∗ P < 0.01, ∗∗∗ P < 0.001. (E, F) qRT-PCR analysis of C-X-C motif chemokine ligand 10 ( Cxcl10 ) transcript levels in LLC (E) and A549 (F) cells following 8 h of hypoxia treatment. The data are presented as the relative fold induction of Cxcl10/actin transcripts. The arrows indicate groups with significantly reduced Cxcl10 transcript levels. Statistical significance was determined by two-way ANOVA, followed by Tukey’s post hoc test. ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. (G, H) qRT-PCR analysis of C–C motif chemokine ligand 5 ( Ccl5 ) transcript levels in LLC (G) and A549 (H) cells following 8 h of hypoxia treatment. The data are presented as the relative fold induction of Ccl5/actin transcripts. Arrows indicate groups with significantly reduced Ccl5 transcripts. The data shown are the means ± standard error of the mean (SEM) from n = 3 biological replicates. Statistical significance was determined by two-way ANOVA, followed by Tukey’s post hoc test. ∗ P < 0.05. mRNA: messenger RNA.

Article Snippet: Human embryonic kidney epithelial cells (HEK293T; ATCC CRL-11268), human non-small cell lung cancer cells (A549; ECACC 86012804), human lung adenocarcinoma cells (PC-9; ECACC 90071810), and mouse Lewis lung carcinoma (LLC) cells (ATCC CRL-1642) (FuHeng, Shanghai, China) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA).

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Journal of Pharmaceutical Analysis

Article Title: Oblique-incidence reflectivity difference technology identifies the antiviral drug Ribavirin as an inhibitor of lung tumor progression by targeting AMPK signaling

doi: 10.1016/j.jpha.2025.101306

Figure Lengend Snippet: Ribavirin suppresses lung cancer cell growth both in vitro and in vivo . (A, B) Viability of mouse Lewis lung carcinoma (LLC) cells (A) and human non-small cell lung cancer cells (A549) (B) cells treated with various concentrations of Ribavi rin. The data shown are the means ± standard error of the mean (SEM) from n = 3 biological replicates. (C, D) Viability of LLC (C) and A549 (D) cells following treatment with 10 μM Ribavirin or the control for 1–5 days. The data shown are the means ± SEM from n = 3 biological replicates. Statistical significance was determined by two-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. ∗∗ P < 0.01, ∗∗∗ P < 0.001. (E, F) Colony formation of LLC (E) and A549 (F) cells after treatment with 10 μM Ribavirin or the control. The data shown are the means ± SEM from n = 3 biological replicates. Statistical significance was determined by two-way ANOVA, followed by Tukey’s post hoc test. ∗∗ P < 0.01, ∗∗∗ P < 0.001. (G) Tumor anatomy of LLC subcutaneous tumor-bearing mice. (H) Tumor volume of LLC subcutaneous tumor-bearing mice. Statistical significance between datasets was assessed by one-way ANOVA, followed by Tukey’s multiple comparisons post hoc test between all groups. The values are the means ± SEM; n = 5 mice per group, with differences denoted by ∗ P < 0.05. (I) Tumor weights of LLC subcutaneous tumor-bearing mice. Statistical significance between datasets was assessed by one-way ANOVA, followed by Tukey’s multiple comparisons post hoc test between all groups. The values are the means ± SEM; n = 8 mice per group, with differences denoted by ∗∗ P < 0.01. PBS: phosphate buffered saline.

Article Snippet: Human embryonic kidney epithelial cells (HEK293T; ATCC CRL-11268), human non-small cell lung cancer cells (A549; ECACC 86012804), human lung adenocarcinoma cells (PC-9; ECACC 90071810), and mouse Lewis lung carcinoma (LLC) cells (ATCC CRL-1642) (FuHeng, Shanghai, China) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA).

Techniques: In Vitro, In Vivo, Control, Saline