jurkat lucia nfat cd16 reporter cell line  (InvivoGen)

Journal: bioRxiv

Article Title: De novo masking domains that gate TNF-family ligand assembly and activity

doi: 10.64898/2026.04.20.719557

Figure Lengend Snippet: To evaluate the generality of the masked TNFL platform, we extended the design and screening workflow to the additional TNFL family members OX40L and 4-1BBL using expanded computational design strategies. Unless otherwise noted, protein concentrations are reported per monomeric protomer. a, Expanded computational strategies for masking-domain design. In the scaffold-guided approach, the masking domains from mTNFα7 and mTNFα8 were used as structural scaffolds to guide backbone generation against the trimerization interfaces of OX40L and 4-1BBL. In the superposition-based approach, artificial complexes were generated by superimposing TNFα and 4-1BBL monomers, followed by partial diffusion to generate new 4-1BBL masking-domain backbones using the TNFα masking domains as templates. b, OX40 binding of selected masked OX40L constructs measured by ELISA before (filled circles) and after (open circles) TEV activation. Data are from a single experiment and are shown as representative results. c, Oligomeric states of selected masked OX40L constructs assessed by analytical SEC before (left) and after (right) TEV activation. The dashed line indicates the elution volume of the trimeric OX40L control. d, Oligomeric states of selected masked 4-1BBL constructs assessed by analytical SEC after TEV activation. The dashed line indicates the elution volume of the trimeric 4-1BBL control. e, Functional activity of masked 4-1BBL (m4-1BBL6) assessed in Jurkat-Lucia h4-1BB reporter cells. Responses to the monomer (filled circles) and SEC-purified homotrimer (open circles) are shown. TEV-treated 4-1BBL_control was included as a positive control. Data are shown as mean ± s.d. from n = 3 technical replicates. f, AlphaFold2-predicted complex structures of representative masked OX40L and masked 4-1BBL constructs, highlighting designs from the scaffold-guided and superposition-based approaches.

Article Snippet: The biological activity of antibody-fused masked 4-1BBL was evaluated using Jurkat-Lucia h4-1BB reporter cells in the presence or absence of HER2-positive SK-BR-3 cells (ATCC).

Techniques: Generated, Diffusion-based Assay, Binding Assay, Construct, Enzyme-linked Immunosorbent Assay, Activation Assay, Control, Functional Assay, Activity Assay, Purification, Positive Control

Journal: bioRxiv

Article Title: De novo masking domains that gate TNF-family ligand assembly and activity

doi: 10.64898/2026.04.20.719557

Figure Lengend Snippet: A single masked TNFα or 4-1BBL module was fused to a one-armed, affinity-attenuated trastuzumab variant to generate an antibody format that undergoes activation-dependent homotrimerization. Homotrimerization was first validated using the masked TNFα construct (b–d), and functional consequences were then evaluated using an analogous masked 4-1BBL fusion in assays with HER2-positive SK-BR-3 cells and Jurkat-Lucia h4-1BB reporter cells (e, f). Protein concentrations are reported per molecular species (monomeric or trimeric antibody, as indicated). Where indicated, trimeric species were re-isolated by SEC before downstream assays. a, Proposed mechanism for delivery of membrane-type TNFLs. Before activation, the construct is predominantly monomeric with reduced apparent target engagement and masked TNFL function. Protease-triggered activation promotes assembly into a trimeric state with increased functional valency and the capacity to drive receptor crosslinking, thereby enhancing signaling. b, Design of antibody formats used to test the concept: OATmab_mTNFα, OATmab, and Tmab. All constructs are based on an affinity-attenuated trastuzumab variant and contain a C-terminal peptide tag on the light chain for site-selective drug conjugation . In OATmab_mTNFα, TNFα (orange) within the masked TNFα module was fused to the C-terminus of one heavy chain. One-armed formats were assembled using knob-into-hole Fc heterodimerization mutations. c, Oligomeric states of OATmab and OATmab_mTNFα assessed by analytical SEC before (black) and after (gray) TEV activation. The arrow indicates the TEV-dependent shift in the main peak consistent with homotrimer formation. d, Binding of monomeric and trimeric antibody species to HER2 (left) and TNFR2 (right) measured by ELISA. Data are shown as mean ± s.d. from n = 3 technical replicates. e, Binding of monomeric and trimeric antibody species to HER2-positive SK-BR-3 cells measured by cell-based ELISA. Data are shown as mean ± s.d. from n = 3 technical replicates. f, Functional activity of monomeric and trimeric antibody species measured in Jurkat-Lucia h4-1BB reporter cells cultured alone (dashed lines) or co-cultured with SK-BR-3 cells (solid lines). Data are shown as mean ± s.d. from n = 3 technical replicates.

Article Snippet: The biological activity of antibody-fused masked 4-1BBL was evaluated using Jurkat-Lucia h4-1BB reporter cells in the presence or absence of HER2-positive SK-BR-3 cells (ATCC).

Techniques: Variant Assay, Activation Assay, Construct, Functional Assay, Isolation, Membrane, Drug discovery, Conjugation Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, In-Cell ELISA, Activity Assay, Cell Culture

Journal: Journal of the Endocrine Society

Article Title: Estrogenic activity in tampon products

doi: 10.1210/jendso/bvag094

Figure Lengend Snippet: Estradiol dose-response and recovery of spiked estradiol from tampon extracts. (A) T47D-luc cells were treated with an 11-point estradiol dilution series starting at 30nM, followed by 10nM, 3.3nM, 1.11nM, 370pM, 123pM, 41pM, 14pM, 4.6pM, 1.5pM, and 0.5pM). For each concentration, 2 μL of steroid dilution was added to 198 μL assay medium, and cells were incubated for 24 hours. Estrogen-responsive luciferase activity was quantified using BrightGlo substrate. Luminescence values represent the mean of 4 technical replicates, and a sigmoidal dose-response curve with EC 50 determination was generated using GraphPad v10. (B) Recovery of estradiol from tampon brands A-D following spiking and extraction. One gram of each tampon from Brands A, B, C, and D were spiked with 2nM estradiol, soaked in a 1:1 water:ethanol solution for 4 hours, extracted, evaporated to dryness, and reconstituted in methanol. Extracts were diluted 1:100 in cell culture medium and then serially diluted 1:2 to generate 4 assay points. T47D-luc cells were incubated with a further 1:10 dilution of each tampon extract for 24 hours and luciferase activity measured using BrightGlo. Each point represents mean ± SEM of 4 technical replicates corrected for the blank control sample (1:1 ethanol:water solution SPE-extracted as per samples). The data for the spiked tampons are plotted alongside estradiol standards run in parallel.

Article Snippet: Estrogen Receptor Luciferase Reporter T47D Stable Cell Line cells (Signosis, Santa Clara, CA, USA: Cat # SL-0002, T47D-luc)) were grown to 90% confluence in a 550 mL cell culture flask with RPMI 164 medium supplemented with 10% (v/v) Hyclone fetal bovine serum (GE Lifesciences) and Geneticin (75 μg mL −1 , G148, Life Technologies).

Techniques: Concentration Assay, Incubation, Luciferase, Activity Assay, Generated, Extraction, Cell Culture, Control

Journal: Journal of the Endocrine Society

Article Title: Estrogenic activity in tampon products

doi: 10.1210/jendso/bvag094

Figure Lengend Snippet: Estrogenic activity detected in extracts from 8 commercially available tampon brands (A-H). T47D-luc cells were exposed to methanolic extracts prepared from 1 g of core tampon material (string removed) from 8 tampon brands sold in New Zealand. Each 1 g sample was soaked in a 1:1 ethanol:water solution for 4 hours, sonicated, filtered, extracted, and then evaporated to dryness, and reconstituted in methanol. The final extract was diluted into assay medium at 1:1000 (1), followed by serial dilutions to generate 1:2000 (2), 1:4000 (3), and 1:8000 (4) exposure concentrations. Cells were incubated with each dilution for 24 hours, and estrogen-responsive luciferase activity was quantified using a luminescence plate reader. Data are presented as the mean ± SEM of 2 independently extracted tampons per brand, each tested as 2 portions of 1 g each, and each dilution tested with 4 technical replicates.

Article Snippet: Estrogen Receptor Luciferase Reporter T47D Stable Cell Line cells (Signosis, Santa Clara, CA, USA: Cat # SL-0002, T47D-luc)) were grown to 90% confluence in a 550 mL cell culture flask with RPMI 164 medium supplemented with 10% (v/v) Hyclone fetal bovine serum (GE Lifesciences) and Geneticin (75 μg mL −1 , G148, Life Technologies).

Techniques: Activity Assay, Sonication, Incubation, Luciferase, Microplate Reader Luminescence Measurement

Journal: Journal of the Endocrine Society

Article Title: Estrogenic activity in tampon products

doi: 10.1210/jendso/bvag094

Figure Lengend Snippet: Independent batches of Tampon Brand D show estrogenic activity. T47D-luc cells were treated with methanolic extracts generated from 1 g of core tampon material (string removed) from 3 independent batches of Brand A and Brand D. For each batch, 3 separate tampons were extracted independently. Each 1 g sample was soaked in 1:1 ethanol:water solution for 4 hours, sonicated, filtered, extracted, and evaporated to dryness, and reconstituted in methanol. Reconstituted extracts were diluted into assay medium at 1:1000 (1), followed by serial 1:2 dilutions to produce 1:2000 (2), 1:4000 (3), and 1:8000 (4) exposure concentrations. Cells were incubated for 24 hours, and estrogen-responsive luciferase activity was quantified by using a luminescence plate reader. Data are shown as mean ± SEM of 3 independently extracted tampons per batch, tested as 2 portions of 1 g each with each dilution tested as 4 technical replicates.

Article Snippet: Estrogen Receptor Luciferase Reporter T47D Stable Cell Line cells (Signosis, Santa Clara, CA, USA: Cat # SL-0002, T47D-luc)) were grown to 90% confluence in a 550 mL cell culture flask with RPMI 164 medium supplemented with 10% (v/v) Hyclone fetal bovine serum (GE Lifesciences) and Geneticin (75 μg mL −1 , G148, Life Technologies).

Techniques: Activity Assay, Generated, Sonication, Incubation, Luciferase, Microplate Reader Luminescence Measurement

Journal: Journal of the Endocrine Society

Article Title: Estrogenic activity in tampon products

doi: 10.1210/jendso/bvag094

Figure Lengend Snippet: Estrogenic activity of internationally sourced tampon brands. T47D-luc cells were treated with methanolic extracts prepared from intact tampons (string removed) from a panel of internationally sourced tampon brands (Brands I-R). For each brand, 2 or 3 independent tampons were extracted separately on independent days. Each tampon was soaked in a 1:1 ethanol:water solution for 4 hours, sonicated, filtered, extracted, evaporated to dryness, and reconstituted in methanol. Reconstituted extracts were diluted into assay medium at 1:1000, and cells were incubated for 24 hours before estrogen-responsive luciferase activity was quantified using a luminescence plate reader. Data are shown as mean ± SEM of 2 or 3 independently extracted tampons per brand with each dilution tested as 4 technical replicates.

Article Snippet: Estrogen Receptor Luciferase Reporter T47D Stable Cell Line cells (Signosis, Santa Clara, CA, USA: Cat # SL-0002, T47D-luc)) were grown to 90% confluence in a 550 mL cell culture flask with RPMI 164 medium supplemented with 10% (v/v) Hyclone fetal bovine serum (GE Lifesciences) and Geneticin (75 μg mL −1 , G148, Life Technologies).

Techniques: Activity Assay, Sonication, Incubation, Luciferase, Microplate Reader Luminescence Measurement

Journal: Journal of the Endocrine Society

Article Title: Estrogenic activity in tampon products

doi: 10.1210/jendso/bvag094

Figure Lengend Snippet: Effect of sonication on estrogenic activity of tampon extracts. T47D-luc cells were exposed to methanolic extracts prepared from 2 independent tampons per brand (Brands A and D, string removed), comparing extraction with and without sonication. Each tampon was soaked in a 1:1 ethanol:water solution for 4 hours, either sonicated or left nonsonicated, then filtered, extracted, evaporated to dryness, and reconstituted in methanol. Extracts were diluted into assay medium at 1:1000 (1), followed by serial 1:2 dilutions to generate 1:2000 (2) and 1:4000 (3) exposure concentrations. Cells were incubated for 24 hours, and estrogen-responsive luciferase activity was quantified. Data represent mean ± SEM of t2wo independently extracted tampons per condition with each dilution tested as 4 technical replicates.

Article Snippet: Estrogen Receptor Luciferase Reporter T47D Stable Cell Line cells (Signosis, Santa Clara, CA, USA: Cat # SL-0002, T47D-luc)) were grown to 90% confluence in a 550 mL cell culture flask with RPMI 164 medium supplemented with 10% (v/v) Hyclone fetal bovine serum (GE Lifesciences) and Geneticin (75 μg mL −1 , G148, Life Technologies).

Techniques: Sonication, Activity Assay, Extraction, Incubation, Luciferase

Journal: Nature Communications

Article Title: A tissue-intrinsic mechanism sensitizes HIV-1 particles for TLR-triggered innate immune responses

doi: 10.1038/s41467-026-72586-3

Figure Lengend Snippet: a Experimental workflow. HIV-1 NL4.3 virions were cultured in suspension (S) or embedded in collagen matrices (DC: Dense collagen; LC: Loose collagen). TZM-bl cells were infected and the relative infectivity determined. b Normalized infectivity of HIV-1 NL4.3 particles. Virions were cultured in S, DC or LC for 16 h prior to relative infectivity determination. Data is normalized to suspension condition (gray dotted line). c Kinetics of ERVI. Culture supernatants were harvested at 0, 4, 8 or 16 h post seeding/embedding, the relative infectivity of virions was determined as in b . d Normalized infectivity of differentially cultured HIV-1 NL4.3 virions. PA: polymerizing agent. e . One representative Western Blot analysis showing the gp120/p24 ratio from HIV-1 NL4.3 virions cultured in S, DC or LC. The gp120/p24 ratios are indicated below the blots. f Quantification of western blot analyses from three independent experiments as in e . g Saturation of ERVI. Increasing amounts of virions were cultured in S, DC or LC prior to infectivity determination. h Normalized infectivity of HIV-1 NL4.3 particles after seeding or embedding in different matrices for 16 h. Data are normalized to suspension condition (gray dotted line). i Conservation of sensitivity to ERVI across lentiviruses. The normalized infectivity of a panel of lentiviruses was assessed after ERVI as in a . Data are normalized to respective suspension condition (gray dotted line). Results represent the mean ± SD of three independent experiments. Symbols indicate data from individual experiments. Significance is indicated by p -values, and was calculated by one-way ANOVA test; Dunnett’s post-test ( b , d , h , i ), or by matched two-way ANOVA test; Tukey’s post-test ( g ). Source data are provided as a file.

Article Snippet: 293T cells (ATCC, CRL-3216) and TZM-bl reporter cells (NIH AIDS Reagent Program, courtesy of Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. (ARP-8129)) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% heat-inactivated fetal calf serum (FCS, Capricorn) and 1% penicillin/streptomycin (Gibco).

Techniques: Cell Culture, Suspension, Infection, Western Blot

Journal: Nature Communications

Article Title: A tissue-intrinsic mechanism sensitizes HIV-1 particles for TLR-triggered innate immune responses

doi: 10.1038/s41467-026-72586-3

Figure Lengend Snippet: a Normalized infectivity of HIV-1 NL4.3 virus containing Vpr.Int.GFP fusion proteins after exposure to LC. b Experimental workflow to assess collagen deposition at the surface of virions. HIV-1 NL4.3 Vpr.Int.GFP virions were collected from S, LC or LCAF647 cultures and analyzed by microscopy. c Representative confocal micrographs of HIV1 NL4.3 Vpr.Int.GFP virions within AlexaFluor 647 stained LC matrices. Yellow arrows depict fluorescent virions. Scale bar: 20 µm. d Representative TIRF-micrographs of HIV-1 NL4.3 Vpr.Int.GFP virions were harvested from the supernatants of fluorescently labeled collagen matrices as in ( c ). Scale bar: 5 µm. e Averaged computational slices of a tomogram showing HIV-1 NL4.3 virions after 16 h of culture in S (left panel), DC (middle panel), or LC (right panel). Scale bar = 50 nm. f Quantification of the diameter of virus particles treated as indicated in d . Violin plot shows individual data points with corresponding median, 25% and 75% quartiles. g Averaged computational slices of a tomogram showing disrupted dense (left panel) or loose (right panel) collagen fibers. Scale bar = 100 nm. h Experimental workflow. HIV-1 NL4.3 virions retrieved from suspension or collagen matrices after 16 h of culture were harvested, and equivalent amounts of RT units were incubated with RM-8 or EF-C infectivity-enhancing peptide, nanofibrils (PNFs) or left untreated (UT) prior to infection of TZM-bl reporter cells. i Impact of infectivity enhancers on the infectivity of suspension or collagen primed virions. Results represent the mean ± SD of three independent experiments. Symbols indicate individual experiments. Significance is indicated by p -values, and was calculated by one-way ANOVA, Dunnett’s post-test ( a ), unpaired t-tests ( d ), or by two-way ANOVA test with Geisser Greenhouse correction; Tukey’s post-test ( i ). Source data are provided as a file.

Article Snippet: 293T cells (ATCC, CRL-3216) and TZM-bl reporter cells (NIH AIDS Reagent Program, courtesy of Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. (ARP-8129)) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% heat-inactivated fetal calf serum (FCS, Capricorn) and 1% penicillin/streptomycin (Gibco).

Techniques: Infection, Virus, Microscopy, Staining, Labeling, Suspension, Incubation

Journal: Nature Communications

Article Title: A tissue-intrinsic mechanism sensitizes HIV-1 particles for TLR-triggered innate immune responses

doi: 10.1038/s41467-026-72586-3

Figure Lengend Snippet: a Experimental workflow. HIV-1 NL4.3 Vpr.mRuby2 virions were cultured in S, DC or LC for 16 h. Equivalent amounts of RT units from culture supernatants or untreated virions were then incubated with TZM-bl cells for 2 h at 4 °C. Cell membranes were then stained with Concanavalin-A AF-488, prior to microscopy processing. b Normalized infectivity of HIV-1 NL4.3 Vpr.mRuby2 virions after 16 h of culture as in a . c Representative spinning disk micrographs of cells incubated with HIV-1 NL4.3 Vpr.mRuby2 virions. Yellow arrows indicate Vpr.mRuby2+ spots detected at the surface of the target cells. Scale bar: 15 µm. d Average binding frequency of HIV-1 NL4.3 Vpr.mRuby2 virions to target cells as shown in c . e Average volume of Vpr.mRuby2 spots detected as shown in c . f Experimental workflow. HIV1 NL4.3 Vpr.BlaM virions were as in a . Equivalent amounts of RT units from culture supernatants or untreated virions were then incubated with TZM-bl cells for 4 h at 37 °C, in the presence or absence of T20 fusion inhibitor. Cells were then loaded with the CCF2-AM and processed by flow cytometry. g Normalized infectivity of HIV-1 NL4.3 Vpr.BlaM virions after16h of culture as in a . h Representative flow cytometry dot plots of CCF2 loaded cells. Gates identify cleaved-CCF2+ cells. See Sup. Figure. for gating strategy. i Quantification of the percentage of CCF2-product positive cells measured by flow cytometry. j Correlation between Relative Infectivity and levels of CCF2-product positive cells after ERVI by linear regression. Results represent the mean ± SD of three independent experiments. Symbols indicate individual experiments. Significance is indicated by p -values, and was calculated by one-way ANOVA test; Dunnets’s post-test ( b , g ), or by one-way ANOVA test; Tukey’s post-test ( d , e , i ). Source data are provided as a file.

Article Snippet: 293T cells (ATCC, CRL-3216) and TZM-bl reporter cells (NIH AIDS Reagent Program, courtesy of Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. (ARP-8129)) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% heat-inactivated fetal calf serum (FCS, Capricorn) and 1% penicillin/streptomycin (Gibco).

Techniques: Cell Culture, Incubation, Staining, Microscopy, Infection, Binding Assay, Flow Cytometry

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: bioRxiv

Article Title: Epithelial NCAPD3 expression protects against stress-induced intestinal injury in mice

doi: 10.64898/2026.04.21.719792

Figure Lengend Snippet: Mice were treated with saline or 9-TB-Dox (25 mg/kg i.p.) daily for 10 days (n=5 mice/group). A. Weight change relative to starting weight. B. Gross evaluation of colonic pathology at endpoint. C. Histopathology of distal colon tissue. D. Intestinal barrier integrity assessed by plasma bioassay on HEKBlue-mTLR5 reporter cell line. E. Systemic inflammation assessed by quantification of SAA at endpoint. Mean ±SEM graphed. Significance determined by unpaired 2-tailed t-test.

Article Snippet: Measurement of intestinal permeability by flagellin bioassay was performed by adding diluted plasma in duplicate to HEKBlue-mTLR5 reporter cells (InVivoGen) plated at 2.5 x10 4 cells/well in 96-well plates for 24 hours.

Techniques: Saline, Histopathology, Clinical Proteomics, Bioassay