a549 ko cells  (Addgene inc)

Journal: bioRxiv

Article Title: Enhanced lung delivery of an immunostimulatory duplex RNA augments the antitumor activity by reshaping systemic cytokine pharmacodynamics

doi: 10.64898/2026.05.03.722518

Figure Lengend Snippet: (a) Schematic illustrating how BD shapes the in vivo immunostimulatory activity of self-dimerizing RNA-1 delivered by LungLNPs or LiverLNPs. LungLNP enhances delivery of RNA-1 to the lungs (1, pink), whereas conventional LiverLNP delivery directs RNA-1 to the liver (1, blue). In each case, organ-specific accumulation leads to uptake of RNA-1 into tissue resident immune or non-immune cell populations expressing pattern recognition receptors (PRRs) (2, pink/blue), thereby influencing pharmacodynamic responses, cytokine release, immune activation, and tumor suppression. (b) IFN-luciferase reporter assay in A549 IRF3 dual reporter cells showing induction by RNA-1 formulated in LungLNPs vs LiverLNPs, compared with free RNA-1 and empty controls. Data presented as average ± SD, n = 3. (c) Schematic of the in vivo pharmacodynamic (PD) model used to assess plasma cytokines following systemic administration of LungLNP/RNA-1, LiverLNP/RNA-1 formulations and corresponding empty LNPs. Mice were dosed with 2.2 mg/kg of RNA-1. (d-h) Quantification of peak plasma cytokine levels (2h for IFNα, IFNβ, TNFα and 6h for IFNγ, IFNλ), (i–m) Temporal kinetics of plasma cytokines (IFNα, IFNβ, IFNλ, IFNγ, and TNFα) following treatment at 2, 6 and 24 h. Data are represented as mean ± SEM from a representative experiment of three independent experiments with n = 6–7 (d–h) and n = 5–7 (i–m) biologically independent samples. (n) Schematic presentation depicting the knockout models used to study the innate immune pathway activated by LungLNPs/RNA-1 (2.2 mg/kg) in mice. (o) Quantification of IFNα plasma levels in RIG I KO mice (cytoplasmic sensing) compared with wildtype (WT) control. (p) Quantification of IFNα plasma levels in TLR3 and TLR7 KO mice compared with wildtype (WT) control. Data are represented as mean ± SD from a representative experiment of two independent experiments with n = 3-6 (o–p) biologically independent samples. (q) Molecular illustration depicting an Alphafold3 modeling of mouse RIG I and mouse TLR7 engaged with dsRNA-1 or ssRNA-1 respectively. Panels a, c and n were created with BioRender.com. The data were analyzed by ordinary one-way ANOVA with Tukey’s multiple-comparisons test; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: Human NF-κB-SEAP & IRF-Luc Reporter lung carcinoma (A549) cells (A549 RIG I) and human RIG-I-KO Dual Reporter A549 cells (A549 RIG I KO) (InvivoGen) were used to study the in vitro innate immune activity of RNA-1.

Techniques: In Vivo, Activity Assay, Expressing, Activation Assay, Luciferase, Reporter Assay, Clinical Proteomics, Knock-Out, Control

Journal: bioRxiv

Article Title: Enhanced lung delivery of an immunostimulatory duplex RNA augments the antitumor activity by reshaping systemic cytokine pharmacodynamics

doi: 10.64898/2026.05.03.722518

Figure Lengend Snippet: (a) Schematic illustration depicting a cross-section of the human lung cancer chip model, which recapitulates key physiological and pathophysiological features of human lung cancer. The microfluidic chip top channel containing human lung epithelial cells and human A549 adenocarcinoma alveolar basal epithelial cells stably expressing GFP, bottom channel containing human lung microvascular endothelial cells cultured on all four walls of the lower channel. (b) Treatment regimen for the human lung cancer-chip using LungLNPs/RNA-1 (100 and 200 nM), and empty LungLNP control (LungLNPs/Empty, 200 nM) and untreated chips. The first treatment was administered 4 days post-seeding, followed by establishment of the air–liquid interface on the same day. A second dose was administered on day 8. LNPs were delivered by vascular perfusion for 6 h per treatment. (c) A549 tumor growth curves during the treatment regimen, quantified by longitudinal GFP fluorescence imaging and measurement of fluorescence intensity. Data were analyzed using a two-way mixed effects model with time and treatment as fixed effects, followed by Tukey’s multiple-comparisons test. (d) Representative fluorescence images showing A549 tumor cells (green) on day 11 (scale bar = 1000 µm). (e) Quantification of cytokines and chemokines measured 2 h following the second dose. Data were analyzed by one way ANOVA with Tukey’s multiple comparisons test. (f) LNP uptake in the lung cancer-chip following perfusion of fluorescently labeled LungLNPs/RNA-1 Cy (yellow) at 100 and 200 nM. Endothelial cells were stained for VE-cadherin (purple), A549 tumor cells expressing GFP are shown in blue, and nuclei are shown in white. Chips were imaged 4 days post-treatment using confocal microscopy (scale bar = 20 µm). (g) Schematics depicting the mechanistic insight into RIG I-mediated lung cancer immunotherapy in human lung cancer chip demonstrating internalization into endothelial cells and RIG I activation and secretion of cytokines. Uptake into epithelial cells via direct exposure or via transport through gaps in the endothelial barrier. Panels a, b and d were created with BioRender.com. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: Human NF-κB-SEAP & IRF-Luc Reporter lung carcinoma (A549) cells (A549 RIG I) and human RIG-I-KO Dual Reporter A549 cells (A549 RIG I KO) (InvivoGen) were used to study the in vitro innate immune activity of RNA-1.

Techniques: Stable Transfection, Expressing, Cell Culture, Control, Fluorescence, Imaging, Labeling, Staining, Confocal Microscopy, Activation Assay

Journal: Viruses

Article Title: Mitochondrial Antiviral Signaling (MAVS) Protein Modulates the Transition from Acute to Persistent Parainfluenza Virus Infection and Resistance to Complement-Mediated Cell Lysis

doi: 10.3390/v18040416

Figure Lengend Snippet: Loss of MAVS impairs antiviral gene induction independent of STAT1. ( A ) WT or MAVS KO A549 cells were mock-infected or infected with Le-PIV5 at an MOI of 10 and harvested at 24 or 48 hpi. Total RNA was analyzed by quantitative PCR to assess expression of IFN-β, IFIT1, and OAS2. Gene expression levels are expressed relative to the corresponding mock-infected controls. ( B ) Protein lysates from WT or MAVS KO cells that were mock-infected or infected with Le-PIV5 at a MOI of 10 were collected at 24 and 48 hpi and analyzed by Western blotting for STAT1 protein. β-actin was used as a loading control. ** indicates a p value < 0.01, *** indicates a p value < 0.001, and **** indicates a p value < 0.0001.

Article Snippet: A549 KO-MAVS cells were purchased commercially (A549-DualTM KO-MAVS cells, catalog #a549d-komavs; InvivoGen, San Diego, CA, USA) and grown in DMEM 10% HI FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 μg/mL Normocin.

Techniques: Infection, Real-time Polymerase Chain Reaction, Expressing, Gene Expression, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: S6K1 Modulates STAT3 Activation to Promote Resistance to Radiotherapy in Lung Cancer

doi: 10.3390/ijms27041915

Figure Lengend Snippet: S6K1 signaling confers radioresistance to lung cancer cells. ( A ) Cells were radiated at the shown doses, and the surviving fractions were calculated for each cell line as explained in the . Note the dramatic decrease at 4 Gy of the surviving fraction (SF) in the sensitive cells H23 (SF: 0.0001) and H226 (SF: 0.11) compared to the most resistant H661 (SF: 0.4) and A549 cells (SF: 0.52). ( B ) Cells were irradiated at the indicated doses, and cell proliferation was evaluated 4 days after radiation using Alamar blue. Again, H23 and H226 cells showed a lower proliferation rate in our cell models compared to no-irradiated controls, against the most resistant cells, H661 and A549 ( C ) Clonogenic assays showing the colony formation after a 4 Gy dose of radiation. H23 is clearly the most sensitive cell to radiation, followed by H226, H661, and A549. ( D ) Western blot experiments showing higher phosphoactivation of S6 and S6K1 in most radioresistant cells A549 and H661. ( E ) Quantification of Immunoblots using ImageJ software (version 1.54r). The most radioresistance cells H661 and A549 showed an increase in the expression of pS6, the main target of S6K1, with a fold change of 1.7 and 1.8, respectively, compared to the most sensitive H23, used as an internal control. * Denotes a p value < 0.05. *** Denotes a p value < 0.0001. Statistical differences were determined using Tukey’s test as explained in the methods. ( F ) S6K1 expression levels from control patients (non-tumor tissue; n : 104) and lung tumor patients ( n : 986) were downloaded from the Xena TCGA database (University of California).

Article Snippet: For S6K1 re-expression in A549 KO cells, we used the pRK7-HA-S6K1-F5A-E389-R3A plasmid [ ] (Addgene # 8991; RRID: Addgene_8991, Watertown, MA, USA).

Techniques: Irradiation, Western Blot, Software, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: S6K1 Modulates STAT3 Activation to Promote Resistance to Radiotherapy in Lung Cancer

doi: 10.3390/ijms27041915

Figure Lengend Snippet: Inhibition of S6K1 increases radiation sensitivity of lung cancer cells. ( A ) Top: Immunoblot assay showing that the pharmacological inhibition of S6K1 with PF-4708671 (5 μM) for 48 h reduces the phosphorylation of S6, a downstream target of S6K1. Bottom: quantitation of p-S6 using ImageJ. Note a reduction of 53% (H661) and 95% (A549) in the expression of pS6 in cells treated with PF-4708671 compared to controls. Statistical differences were determined using a Student’s t -test. ( B , C ) Colony formation in cells pre-treated with DMSO or PF-4708671 plus radiation. Then, cells were treated with low doses of radiation (2 Gy). PF-4708671 was kept until the end of the experiment. Surviving fraction was calculated for each condition compared to non-treated controls. Data showed that PF-4708671 dramatically sensitized the resistant cells H661 (SF: 0.12) and A549 (SF:0.12) to low doses of radiation. ( D ) S6K1 KO cells and control wild types were seeded as before for clonogenic assays, and the surviving colonies were stained and counted. S6K1 genetic deletion decreases the average colony formation (CF) (CF-KO1: 4; CF-KO2: 8) compared to control (CF: 24) after radiation. Statistical differences were determined using Tukey’s test as explained in the methods. Right panel: S6K1 KO was confirmed by Western blot. * Denotes a p value < 0.05. ** Denotes a p value < 0.001. # Denotes the number.

Article Snippet: For S6K1 re-expression in A549 KO cells, we used the pRK7-HA-S6K1-F5A-E389-R3A plasmid [ ] (Addgene # 8991; RRID: Addgene_8991, Watertown, MA, USA).

Techniques: Inhibition, Western Blot, Phospho-proteomics, Quantitation Assay, Expressing, Control, Staining

Journal: International Journal of Molecular Sciences

Article Title: S6K1 Modulates STAT3 Activation to Promote Resistance to Radiotherapy in Lung Cancer

doi: 10.3390/ijms27041915

Figure Lengend Snippet: STAT3 activation increases after radiation to promote radioresistance modulated by S6K1. ( A ) Compared to non-irradiated controls, STAT3 and STAT3 phosphoactivation increases after a single dose of 10 Gy in H661 and A549 cells at 24 and 48 h. ( B ) A549 S6K1-KO cells, transfected with a plasmid expressing a constitutively active form of S6K1 protein, showed an increase in the phospho-activation of STAT3 before and after radiation. ( C ) PF-4708671 (5 µM) antagonizes the phospho-activation of STAT3 and the expression of c-myc in A549 cells after radiation. Protein expressions were studied by Western Blot. ( D ) Expression analysis showing the downregulation of STAT3 activation (p-Ser727) in S6K1 KO cells. S6K1 deletion decreases the p-STAT3 expression after radiation. ( E ) A549 cells treated with the inhibitor Stattic plus radiation showed the lowest number of colonies (CF: 1.3) compared to radiotherapy (CF: 10) or static (CF: 13) alone and control (DMSO; CF: 43) (* p < 0.05, ** p < 0.005, and **** p < 0.0001). ( F ) Transcriptional activity of STAT3 was measured in the presence of vehicle, PF-4708671 (5 μM), radiation (10 Gy) or the combination by using the Dual-Glo ® Luciferase Assay System. PF-4708671 decreased the STAT3 transcriptional activity compared to control, before and after radiation (* p < 0.05, *** p < 0.0005). Statistical differences were determined using Tukey’s test as explained in the methods.

Article Snippet: For S6K1 re-expression in A549 KO cells, we used the pRK7-HA-S6K1-F5A-E389-R3A plasmid [ ] (Addgene # 8991; RRID: Addgene_8991, Watertown, MA, USA).

Techniques: Activation Assay, Irradiation, Transfection, Plasmid Preparation, Expressing, Western Blot, Control, Activity Assay, Luciferase