human rig i ko dual reporter a549 cells  (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: 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: To characterize the direct E3 ligase activity of TRIM16 and its specific ubiquitin linkage in a cell-free system, recombinant human UbcH5b (HY- P79449 , MedChemExpress) and RIG-I protein (TP317615, OriGene) were employed as the E2 conjugating enzyme and substrate, 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: To characterize the direct E3 ligase activity of TRIM16 and its specific ubiquitin linkage in a cell-free system, recombinant human UbcH5b (HY- P79449 , MedChemExpress) and RIG-I protein (TP317615, OriGene) were employed as the E2 conjugating enzyme and substrate, 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: Identification of the ubiquitination site on RIG-I targeted by FGF8. (a) diagram illustrating the truncated constructs of RIG-I. (b) HEK-293T cells were co-transfected with specified plasmids and exposed to MG132 for 6 hours. Western blot analysis was conducted to assess the ubiquitination of various RIG-I truncation constructs. (C) Western blot analysis identified the ubiquitination site on RIG-I targeted by FGF8, and band intensities were quantified by densitometry to assess the degradation of each mutant. (d) a dual-luciferase assay was conducted in HEK293T cells co-transfected with specified RIG-I mutants and FGF8 to evaluate the impact of FGF8 on IFN-β promoter activity. Error bars indicate the mean ± SEM from three independent experiments. Two-tailed unpaired Student’s t-tests were used. ns (not significant), * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To characterize the direct E3 ligase activity of TRIM16 and its specific ubiquitin linkage in a cell-free system, recombinant human UbcH5b (HY- P79449 , MedChemExpress) and RIG-I protein (TP317615, OriGene) were employed as the E2 conjugating enzyme and substrate, respectively.

Techniques: Ubiquitin Proteomics, Construct, Transfection, Western Blot, Mutagenesis, Luciferase, Activity 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: 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: To characterize the direct E3 ligase activity of TRIM16 and its specific ubiquitin linkage in a cell-free system, recombinant human UbcH5b (HY- P79449 , MedChemExpress) and RIG-I protein (TP317615, OriGene) were employed as the E2 conjugating enzyme and substrate, 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: Theranostics

Article Title: Bispecific Siglec-15/T cell antibody (STAB) activates T cells and suppresses pancreatic ductal adenocarcinoma and non-small cell lung tumors in vivo

doi: 10.7150/thno.103372

Figure Lengend Snippet: Effect of S15 overexpression on survival in PDAC and NSCLC patients and detection of S15 in tumor tissues . (A) S15 is overexpressed in PDAC patients, as shown by data from TCGA. (B-C) S15 is also overexpressed in NSCLC patients, including those with metastatic disease (data from TCGA). (D) Kaplan-Meier survival analysis indicates that pancreatic cancer patients with high S15 expression exhibit significantly lower overall survival (data from TCGA). (E-G) S15 expression was detected in pancreatic cancer patient tumor tissues by immunohistochemistry (IHC) staining. Primary anti-human S15 antibody was used at a 1:500 dilution (0.9 mg/mL), and secondary rabbit anti-human Alexa594 antibody was used at a 1:1000 dilution for detection. (H-J) S15 expression was also detected in NSCLC patient tumor tissues using the same staining protocol with anti-human S15 and rabbit anti-human Alexa594 antibodies.

Article Snippet: Either human CD3ε protein (Novus Biologicals, Cat # NBP2-22752) or human S15 protein (Acro Biosystems, Cat # SG5-H52H3), were coated as antigen onto high binding, half-area, clear 96-well plates (Corning Costar, Cat # 3690) overnight at 4 ̊C.

Techniques: Over Expression, Expressing, Immunohistochemistry, Staining

Journal: Theranostics

Article Title: Bispecific Siglec-15/T cell antibody (STAB) activates T cells and suppresses pancreatic ductal adenocarcinoma and non-small cell lung tumors in vivo

doi: 10.7150/thno.103372

Figure Lengend Snippet: Characterization of STAB in vitro . (A) Schematic representation of the STAB molecule in the IgG-scFv format that binds CD3 and S15. (B) SDS-PAGE gel analysis confirms the expression of the STAB construct, showing correctly sized heavy and light chains. (C) Binding affinity of STAB to S15 compared to the parent S15 mAb, measured by ELISA. (D) Binding affinity of STAB to CD3 compared to the parent CD3 mAb, measured by ELISA. (E-F) Representative flow cytometry histograms demonstrating proliferation of T cells induced by (E) control anti-human IgG and (F) STAB, assessed using the CFSE proliferation assay. PBMCs from different donors were stained with 5 μg CFSE. After staining, PBMCs were cultured with either control IgG or STAB for 3 days, followed by harvesting and preparation of single-cell suspensions, and finally analysis by flow cytometry. (G) Quantification of fraction of CD3⁺ T cells following control IgG or STAB treatment. Data are presented as the mean ± standard error of the mean (SEM). ***p < 0.001.

Article Snippet: Either human CD3ε protein (Novus Biologicals, Cat # NBP2-22752) or human S15 protein (Acro Biosystems, Cat # SG5-H52H3), were coated as antigen onto high binding, half-area, clear 96-well plates (Corning Costar, Cat # 3690) overnight at 4 ̊C.

Techniques: In Vitro, SDS Page, Expressing, Construct, Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Control, Proliferation Assay, Staining, Cell Culture

Journal: Theranostics

Article Title: Bispecific Siglec-15/T cell antibody (STAB) activates T cells and suppresses pancreatic ductal adenocarcinoma and non-small cell lung tumors in vivo

doi: 10.7150/thno.103372

Figure Lengend Snippet: STAB enhances T cell-mediated killing of S15+ human Panc-1 PDAC cells and human H460 NSCLC cells in vitro . PBMCs from different donors were stained with 5 μg CFSE, then cultured with tumor cells in presence of either Control IgG or STAB for 3 days, followed by harvesting and preparation of single-cell suspensions. CD3+CD45+ cell and tumor cell populations were analyzed by flow cytometry (n = 4). (A-B) Representative flow cytometry gating strategy for CD3⁺CD45⁺ T cells in Panc-1 co-culture. (C) Quantification of CD3⁺ T cells and GFP⁺ Panc-1 cells after treatment with Control IgG or STAB. (D-E) Representative flow cytometry gating strategy for GFP⁺ Panc-1 cells. (F) Luminescence-based assay results from Panc-1 co-culture with PBMCs treated with STAB or Control IgG, showing STAB-mediated tumor cell killing. (G-H) Representative flow cytometry gating strategy for CD3⁺CD45+ T cells in H460 co-culture. (I) Quantification of CD3⁺ T cells and mCherry⁺ H460 cells after treatment with Control IgG or STAB. (J-K) Representative flow cytometry gating strategy for mCherry⁺ H460 cells. (L) Luminescence-based assay results from H460 co-culture with PBMCs treated with STAB or Control IgG, showing STAB-mediated tumor cell killing. Percentage of cells in the STAB-treatment group is presented relative to the Control Ab at an effector-to-target (E:T) ratio of 3:1. Statistical differences in cell killing between STAB and control IgG were assessed using two-way ANOVA and are denoted as * (*p < 0.05; **p < 0.01).

Article Snippet: Either human CD3ε protein (Novus Biologicals, Cat # NBP2-22752) or human S15 protein (Acro Biosystems, Cat # SG5-H52H3), were coated as antigen onto high binding, half-area, clear 96-well plates (Corning Costar, Cat # 3690) overnight at 4 ̊C.

Techniques: In Vitro, Staining, Cell Culture, Control, Flow Cytometry, Co-Culture Assay, Luminescence Assay

Journal: Theranostics

Article Title: Bispecific Siglec-15/T cell antibody (STAB) activates T cells and suppresses pancreatic ductal adenocarcinoma and non-small cell lung tumors in vivo

doi: 10.7150/thno.103372

Figure Lengend Snippet: STAB effectively inhibits Panc-1 tumor growth and significantly prolongs survival compared to treatments with anti-S15 or anti-CD3 mAbs. (A&D) Inhibition of Panc-1 tumor growth in NSG mice. Panc-1 cells (1 × 10⁶ in 50 μL PBS) were injected subcutaneously, and tumor size was measured every 2 days using calipers. Panels A and D represent two independent experiments (1st round: n = 5; 2nd round: n = 6-9). (B&E) Kaplan-Meier survival curves for the mice in (A) & (D) . Survival was defined by humane endpoints or tumor size exceeding 1000 mm³. Each group comprised 6-9 mice. (C) Representative images of tumors from the control and STAB treatment groups at the study endpoint. (F) Tumor weights at the study endpoint. (G-J) Quantification of CD3⁺ T cells and activated Ki67⁺CD3⁺ T cells in peripheral blood at 12 and 16 days post-treatment. Blood (60 μL) was collected via submandibular bleeding, stained with CD3, CD45, and Ki67, and analyzed by flow cytometry. Statistical differences between STAB treatment and control anti-human IgG groups were assessed using one-way ANOVA. Results are denoted as * (*p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: Either human CD3ε protein (Novus Biologicals, Cat # NBP2-22752) or human S15 protein (Acro Biosystems, Cat # SG5-H52H3), were coated as antigen onto high binding, half-area, clear 96-well plates (Corning Costar, Cat # 3690) overnight at 4 ̊C.

Techniques: Inhibition, Injection, Control, Staining, Flow Cytometry

Journal: Theranostics

Article Title: Bispecific Siglec-15/T cell antibody (STAB) activates T cells and suppresses pancreatic ductal adenocarcinoma and non-small cell lung tumors in vivo

doi: 10.7150/thno.103372

Figure Lengend Snippet: STAB treatment increases T cell infiltration in tumors and reduces tumor-associated fibroblasts in desmoplastic Panc-1 tumors. Tumors were harvested at the study endpoint from different treatment groups, fixed in 10% formalin overnight, and then stored in 70% ethanol before being embedded in paraffin. (A-D) Tumor sections were stained with anti-CD3 (Alexa 594, red) and anti-S15 (Alexa 488, green) to evaluate T cell infiltration and S15 expression. Negative staining (without primary antibody or without secondary antibody) and control staining were performed. (E-G) Tumor sections were stained with anti-αSMA (Alexa 488, green) and DAPI (blue) to assess tumor-associated fibroblasts. Negative staining and control staining were also performed. (H) Bar graph showing quantification of αSMA staining as an indicator of fibroblast presence.

Article Snippet: Either human CD3ε protein (Novus Biologicals, Cat # NBP2-22752) or human S15 protein (Acro Biosystems, Cat # SG5-H52H3), were coated as antigen onto high binding, half-area, clear 96-well plates (Corning Costar, Cat # 3690) overnight at 4 ̊C.

Techniques: Staining, Expressing, Negative Staining, Control