a549 dual ko rig i  (InvivoGen)

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: Science Advances

Article Title: Effective recognition of double-stranded RNA does not require activation of cellular inflammation

doi: 10.1126/sciadv.ads6498

Figure Lengend Snippet: ( A ) Schematic of dsRNA generation: Complementary RNA strands are transcribed with T7 RNA polymerase, purified by HPLC. In the case of capped transcript generation, the RNA is enzymatically treated to remove uncapped RNA (this step is not shown in the scheme). Last, a pair of complementary strands is heated and slowly cooled down to force duplex formation, i.e., dsRNA. ( B ) Representation of in vitro–transcribed dsRNA with a triphosphate group at the 5′ end. Other 5′ end modifications are shown in fig. S1A. ( C ) Agarose gel analysis of sense and antisense strands (triphosphorylated 5′ ends) and annealed dsRNA. Additional analyses of modified dsRNAs are shown in fig. S1B. ( D ) Heatmap of differentially expressed genes (GO:0051607, “Defense response to virus”) in A549 cells transfected with 5′-modified dsRNAs (ppp-, cap0-, cap1-, and cap2-dsRNA). The full heatmap is shown in fig. S3. ( E ) Top 10 overrepresented GO terms for transfected A549 cells. ( F ) RT-qPCR validation of RNA-seq data: relative gene expression (means ± SD, n = 4). Statistical significance: * P < 0.05 and ** P < 0.01 (one-way ANOVA with Tukey’s multiple comparisons test). Only statistically significant differences are shown.

Article Snippet: A549 (CCL-185) and HeLa (CCL-2) cell lines from American Type Culture Collection, A549 RIG-I KO generated in this study, and A549 MAVS KO, A549OAS3 KO, A549 PKR KO, A549 RNase L KO cell lines provided by S. Weiss ( , ) were maintained at 5% CO 2 and 37°C in Dulbecco’s modified Eagle’ medium (Gibco, 21969035) supplemented with heat-inactivated fetal bovine serum (10%, v/v) (Sigma-Aldrich, F9665), GlutaMAX (1%, v/v) (Gibco, 35050061), and penicillin/streptomycin (1%, v/v) (Gibco, P4333).

Techniques: Purification, In Vitro, Agarose Gel Electrophoresis, Modification, Virus, Transfection, Quantitative RT-PCR, Biomarker Discovery, RNA Sequencing, Gene Expression

Journal: Science Advances

Article Title: Effective recognition of double-stranded RNA does not require activation of cellular inflammation

doi: 10.1126/sciadv.ads6498

Figure Lengend Snippet: ( A ) Schematic of the proteomic approach: A549 cells are transfected with differently capped dsRNAs for 5 hours, UV cross-linked, and lysed, and dsRNA-containing RNP complexes are captured using dsRNA-specific antibodies. The protein composition is analyzed via MS/MS. ( B ) Protein specificity heatmap showing log 2 intensity ratios of identified proteins across experimental conditions. Proteins with a specificity value >4.0 in at least one condition are displayed (full heatmap in fig. S6). ( C ) RT-qPCR analysis of RLR pathway activation in A549 cells transfected with dsRNAs bearing different 5′ end modifications for 5 hours. Data represent the means ± SD ( n = 3). Statistical significance: * P < 0.05 and ** P < 0.01 (one-way ANOVA with Tukey’s multiple comparisons test). Only statistically significant differences are shown. ( D and E ) ppp-ssRNA does not activate RLR or PKR pathways. (D) A549 cells are transfected with ppp-dsRNA, cap1-dsRNA, or ppp-ssRNA for 24 hours (ISG expression) or 7 hours (PKR and eIF2α phosphorylation by Western blotting). The asterisk (*) indicates a nonspecific band. ( F ) RNase L activity assessed by rRNA integrity. rRNA integrity was analyzed in A549 cells 24 hours posttransfection with ppp- or cap1-dsRNA using denaturing agarose gel. ( G ) RLR pathway activation with or without interferon (IFN-α, 200 U/ml) in A549 cells transfected with ppp- or cap1-dsRNA for 24 hours. The asterisk (*) indicates a nonspecific band. ( H ) RNase L activity depends on the dsRNA presence but is independent of the immune state. A549 cells are pretreated with IFN-α (200 U/ml) for 24 hours and transfected with dsRNA for 24 hours, and rRNA integrity is analyzed in denaturing agarose gel.

Article Snippet: A549 (CCL-185) and HeLa (CCL-2) cell lines from American Type Culture Collection, A549 RIG-I KO generated in this study, and A549 MAVS KO, A549OAS3 KO, A549 PKR KO, A549 RNase L KO cell lines provided by S. Weiss ( , ) were maintained at 5% CO 2 and 37°C in Dulbecco’s modified Eagle’ medium (Gibco, 21969035) supplemented with heat-inactivated fetal bovine serum (10%, v/v) (Sigma-Aldrich, F9665), GlutaMAX (1%, v/v) (Gibco, 35050061), and penicillin/streptomycin (1%, v/v) (Gibco, P4333).

Techniques: Transfection, Tandem Mass Spectroscopy, Quantitative RT-PCR, Activation Assay, Expressing, Phospho-proteomics, Western Blot, Activity Assay, Agarose Gel Electrophoresis

Journal: Science Advances

Article Title: Effective recognition of double-stranded RNA does not require activation of cellular inflammation

doi: 10.1126/sciadv.ads6498

Figure Lengend Snippet: ( A ) IFN-β production in A549 cells assessed via luminescence of firefly luciferase under the control of the IFN-β promoter. Cells are transfected with dsRNA for 24 hours, and luciferase activity is measured. Bars represent the means ± SEM from at least three independent biological replicates (each with three technical replicates). Each point represents a technical replicate. Statistical significance: * P < 0.05, ** P < 0.01, and **** P < 0.0001 (one-way ANOVA with Tukey’s multiple comparisons test). Only significant differences are shown. RLU, relative light units. ( B and C ) ISG product expression in A549 (B) or HeLa (C) cells transfected with ppp- or cap1-dsRNA carrying various epitranscriptomic marks for 24 hours, assessed by Western blotting. The asterisk (*) indicates a nonspecific band. ( D ) IFN-β production in A549 RIG-I KO cells assessed via luminescence of firefly luciferase as described in (A). No significant differences are observed (one-way ANOVA with Tukey’s multiple comparisons test). Verification of RIG-I KO in A549 RIG-I KO cells is shown in fig. S9.

Article Snippet: A549 (CCL-185) and HeLa (CCL-2) cell lines from American Type Culture Collection, A549 RIG-I KO generated in this study, and A549 MAVS KO, A549OAS3 KO, A549 PKR KO, A549 RNase L KO cell lines provided by S. Weiss ( , ) were maintained at 5% CO 2 and 37°C in Dulbecco’s modified Eagle’ medium (Gibco, 21969035) supplemented with heat-inactivated fetal bovine serum (10%, v/v) (Sigma-Aldrich, F9665), GlutaMAX (1%, v/v) (Gibco, 35050061), and penicillin/streptomycin (1%, v/v) (Gibco, P4333).

Techniques: Luciferase, Control, Transfection, Activity Assay, Expressing, Western Blot

Journal: Science Advances

Article Title: Effective recognition of double-stranded RNA does not require activation of cellular inflammation

doi: 10.1126/sciadv.ads6498

Figure Lengend Snippet: ( A ) Copurification of endogenous proteins from lysates of IFN-α–treated (200 U/ml) and untreated A549 cells with biotinylated ppp- or cap1-dsRNA. PKR and OAS3 are detected in precipitates by Western blotting. This is a cropped version of fig. S10. The asterisk (*) indicates an unspecific band. ( B ) Activation of the PKR pathway is not affected by the presence of posttranscriptional modifications within dsRNA. A549 or HeLa cells are transfected with ppp- or cap1-dsRNA carrying different epitranscriptomic marks for 7 hours, and the phosphorylation status of PKR and eIF2α is assessed by Western blotting. ( C to E ) The levels of newly synthetized proteins are assessed by puromycin incorporation assay. A549 and PKR KO cells are transfected with ppp- and cap1-dsRNA carrying different posttranscriptional modifications for 24 hours, and then puromycin (4 μg/ml) is added for 30 min. (C) The levels of newly synthetized proteins are assessed by Western blotting with anti-puromycin antibodies. Naphthalene blue staining is performed to control the loading. Verification of PKR KO cells is shown in fig. S11. [(D) and (E)] The levels of newly synthetized proteins are assessed by immunofluorescence with anti-puromycin antibodies. (D) Average intensity of puromycin staining in cells presented in (E) (between 81 and 173 cells are analyzed for each transfection). Bars represent the mean values ± SD from all analyzed cells. Each point represents data from one cell. Statistical significance: ** P < 0.01 and **** P < 0.0001 (one-way ANOVA with Turkey’s multiple comparisons test). Only statistically significant differences were marked on the graph.

Article Snippet: A549 (CCL-185) and HeLa (CCL-2) cell lines from American Type Culture Collection, A549 RIG-I KO generated in this study, and A549 MAVS KO, A549OAS3 KO, A549 PKR KO, A549 RNase L KO cell lines provided by S. Weiss ( , ) were maintained at 5% CO 2 and 37°C in Dulbecco’s modified Eagle’ medium (Gibco, 21969035) supplemented with heat-inactivated fetal bovine serum (10%, v/v) (Sigma-Aldrich, F9665), GlutaMAX (1%, v/v) (Gibco, 35050061), and penicillin/streptomycin (1%, v/v) (Gibco, P4333).

Techniques: Copurification, Western Blot, Activation Assay, Transfection, Phospho-proteomics, Staining, Control, Immunofluorescence

Journal: Science Advances

Article Title: Effective recognition of double-stranded RNA does not require activation of cellular inflammation

doi: 10.1126/sciadv.ads6498

Figure Lengend Snippet: ( A ) RNase L activity assessed by rRNA integrity in A549 (wild type), MAVS KO, OAS3 KO, and RNase L KO cells. Total RNA is isolated 24 hours after transfection with posttranscriptionally modified (m 6 A or Ψ) ppp- or cap1-dsRNA and analyzed on denaturing agarose gel. KO validations for MAVS, OAS3, and RNase L are shown in fig. S11. ( B ) UV-VIS melting profiles of dsRNA bearing m 6 A or containing mismatches (2, 4, 11, and 23%). Fitted curves are shown; raw data are shown in fig. S13. Duplex formation efficiency for mismatched dsRNAs is shown in fig. S14A. ( C ) Comparison of melting temperatures ( T m ) of differently modified dsRNAs; R 2 represents the goodness of fit of absorbance at 260 nm using the dose response-stimulation (four parameters) function. NA, not applicable. ( D ) RNase L activity assessed by rRNA integrity in A549 cells 2 hours after electroporation with ppp-dsRNA bearing m 6 A or Ψ or with ppp-dsRNA containing mismatches (2, 4, 11, and 23%). ( E ) UV-VIS melting profiles of dsRNA with epitranscriptomic marks (m 6 A, m 5 C, and Ψ) and 23% mismatches. The melting profiles of dsRNA_23%_m 5 C and dsRNA_23%_Ψ overlap. Fitted curves are shown; raw data are shown in fig. S13. Duplex formation efficiency for dsRNAs with mismatches and chemical modifications is shown in fig. S14B.

Article Snippet: A549 (CCL-185) and HeLa (CCL-2) cell lines from American Type Culture Collection, A549 RIG-I KO generated in this study, and A549 MAVS KO, A549OAS3 KO, A549 PKR KO, A549 RNase L KO cell lines provided by S. Weiss ( , ) were maintained at 5% CO 2 and 37°C in Dulbecco’s modified Eagle’ medium (Gibco, 21969035) supplemented with heat-inactivated fetal bovine serum (10%, v/v) (Sigma-Aldrich, F9665), GlutaMAX (1%, v/v) (Gibco, 35050061), and penicillin/streptomycin (1%, v/v) (Gibco, P4333).

Techniques: Activity Assay, Isolation, Transfection, Modification, Agarose Gel Electrophoresis, Comparison, Electroporation

Journal: Science Advances

Article Title: Effective recognition of double-stranded RNA does not require activation of cellular inflammation

doi: 10.1126/sciadv.ads6498

Figure Lengend Snippet: ( A ) Immunofluorescence analysis of PKR and Cy5-labeled RNA in A549 cells. Cells are transfected with Cy5-labeled ppp- or cap1-dsRNA (with or without m 6 A) or Cy5-labeled ppp-ssRNA. ( B ) Quantification of (left) Cy5-labeled RNA colocalization with PKR and (right) PKR colocalization with Cy5-labeled RNA. Bars represent all foci counted across 127 to 874 cells per transfection. Yellow indicates RNA-PKR–colocalized foci, while red and green indicate foci with only PKR or RNA signals, respectively.

Article Snippet: A549 (CCL-185) and HeLa (CCL-2) cell lines from American Type Culture Collection, A549 RIG-I KO generated in this study, and A549 MAVS KO, A549OAS3 KO, A549 PKR KO, A549 RNase L KO cell lines provided by S. Weiss ( , ) were maintained at 5% CO 2 and 37°C in Dulbecco’s modified Eagle’ medium (Gibco, 21969035) supplemented with heat-inactivated fetal bovine serum (10%, v/v) (Sigma-Aldrich, F9665), GlutaMAX (1%, v/v) (Gibco, 35050061), and penicillin/streptomycin (1%, v/v) (Gibco, P4333).

Techniques: Immunofluorescence, Labeling, Transfection