Journal: International Journal of Molecular Sciences

Article Title: Engineered Fragments of the PSMA-Specific 5D3 Antibody and Their Functional Characterization

doi: 10.3390/ijms21186672

Figure Lengend Snippet: Characterization of 5D3 antibody fragments expressed in E. coli . ( A ) Purity of recombinant proteins was estimated from SDS-PAGE gel stained by Coomassie Brilliant Blue G-250 (CBB). ( B ) Binding of 5D3 variants on live cells was estimated by flow cytometry using anti-6× His antibody together with an anti-mouse IgG-Alexa Fluor 647 conjugate. All molecules used at saturating concentrations showed specific staining of PSMA-positive LNCaP cells with similar signal intensity. PSMA-negative DU145 cells were used as a control. ( C ) Indirect immunofluorescence microscopy using 5D3 scFv LH. PSMA-overexpressing HEK293T cells and the parent HEK293T cell line (a negative control) were fixed with/without permeabilization by Triton X-100 and incubated with 5D3 scFv LH. The fragment was then detected by anti-6× His Ab combined with anti-mouse Alexa Fluor 488 conjugate (green channel). Cell nuclei were counterstained by DAPI (blue channel). The scale bar represents 25 µm.

Article Snippet: PC-3 and DU145 cells were obtained from the American Type Culture Collection, PC-3 PIP and PC-3 FLU cells were generously provided by Dr. Warren Heston (Cleveland Clinic, Cleveland, OH, USA), and LNCaP and CW22Rv1 cells were kindly provided by Z. Hodny (IMG, Prague, Czech Republic) and R. Lapidus (University of Maryland, Baltimore, MD, USA), respectively.

Techniques: Recombinant, SDS Page, Staining, Binding Assay, Flow Cytometry, Control, Immunofluorescence, Microscopy, Negative Control, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Engineered Fragments of the PSMA-Specific 5D3 Antibody and Their Functional Characterization

doi: 10.3390/ijms21186672

Figure Lengend Snippet: Functional characterization of S2-expressed 5D3 variants. ( A ) Thermal stability was determined using nanoDSF. Melting temperature curves and corresponding numerical values are shown. ( B ) Specificity of 5D3 variants evaluated by indirect immunofluorescence microscopy. PC-3 PIP (PSMA-positive) and parent PC-3 (PSMA-negative) cells were fixed by formaldehyde and permeabilized by Triton X-100 prior to incubation with recombinant 5D3 variants and secondary antibodies (green channel). Cell nuclei were counterstained with DAPI (blue channel). The scale bar represents 25 µm. ( C ) Flow cytometry analysis of PSMA-positive (PC-3 PIP, LNCaP) and negative (PC-3, DU145) cells stained by 100 nM purified 5D3 variants. Each gated population represents approximately 30,000 viable cells. ( D , E ) Determination of binding affinity of 5D3 variants by native ELISA ( D ) and flow cytometry ( E ) using purified PSMA and live LNCaP cells, respectively. PBS and DU145 cells were used as negative controls for ELISA and flow cytometry samples, respectively, and corresponding background signals were subtracted.

Article Snippet: PC-3 and DU145 cells were obtained from the American Type Culture Collection, PC-3 PIP and PC-3 FLU cells were generously provided by Dr. Warren Heston (Cleveland Clinic, Cleveland, OH, USA), and LNCaP and CW22Rv1 cells were kindly provided by Z. Hodny (IMG, Prague, Czech Republic) and R. Lapidus (University of Maryland, Baltimore, MD, USA), respectively.

Techniques: Functional Assay, Nano Differential Scanning Fluorimetry, Immunofluorescence, Microscopy, Incubation, Recombinant, Flow Cytometry, Staining, Purification, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: An Engineered Soluble Single‐Chain TCR Engager for KRAS‐G12V Specific Tumor Immunotherapy

doi: 10.1002/advs.202500181

Figure Lengend Snippet: Specific binding and T cell responsiveness of varied 1–2C TCR engagers. A) Schematics representations of six soluble 1–2C TCR engagers architectures. LL, long linker; SL, short linker. UCHT1, anti‐CD3 scFv. The Vα and Vβ domains of 1–2C TCR are respectively labeled as α and β, whereas the VH and VL domains of UCHT1 antibody are labeled as H and L, accordingly. B) Schematic illustration of the binding assay of various 1–2C TCR engagers to T cells, as detected with the KRAS‐G12/HLA‐A*11:01 pHLA tetramer loaded with KRAS‐G12 wildtype (G12wt) or KRAS‐G12V peptides. Created with Biorender.com. C) Flow cytometry analysis of pHLA tetramers binding to T cells pre‐incubated with 1–2C TCR engager proteins. Tetramers were loaded with KRAS‐G12wt (black line) or KRAS‐G12V mutant peptides (red line). The data presented is representative of three independent experiments. D) Schematic depiction of the the functional evaluation assay, where 1–2C TCR engager s redirect T cells against the KRAS‐G12V antigen presented by K562‐HLA‐A11 cells, inducing IFN‐γ production. Created with Biorender.com. E) IFN‐γ ELIspot analysis of T cells co‐cultured with 1–2C TCR engager proteins (100 nM) and K562‐HLA‐A11 cells loaded with either KRAS‐G12wt or KRAS‐G12V peptides. Data points represent individual T cell responses from three donors and columns display the mean magnitude of IFN‐γ producing spots for the three donors.

Article Snippet: T cells were activated by adding 10 μL T Cell TransAct (Miltenyi Biotec) per 1 × 10 6 cells and cultured in UniEx T‐cell serum‐free medium (ExCell) supplemented with 2.5% Helios UltraGRO‐Advanced (AventaCell), 300 IU mL −1 human IL‐2 (Novoprotein), 10 ng mL −1 human IL‐7 (Novoprotein), 100 μg mL −1 streptomycin and 100 IU mL −1 penicillin (Invitrogen).

Techniques: Binding Assay, Labeling, Flow Cytometry, Incubation, Mutagenesis, Functional Assay, Enzyme-linked Immunospot, Cell Culture

Journal: Advanced Science

Article Title: An Engineered Soluble Single‐Chain TCR Engager for KRAS‐G12V Specific Tumor Immunotherapy

doi: 10.1002/advs.202500181

Figure Lengend Snippet: Binding characteristic, thermal stabilities and antigen sensitivity of 1–2C TCR engagers. A) Characterization of the binding profiles of 1–2C‐LL, 1–2C‐2LL, and 1–2C‐KIH TCR engagers, representing the mono‐, bi‐ and tetra‐valent TCRs, respectively, to the KRAS‐G12V/HLA‐A*11:01 pHLA ligand using BLI assay. The biotinylated KRAS‐G12V/HLA‐A*11:01 was immobilized on streptavidin‐coated probes, and serial dilutions of 1–2C engager proteins were flowed over the immobilized pHLA ligands. B) Thermal stability of 1–2C‐LL, 1–2C‐2LL, and 1–2C‐KIH TCR engagers assessed by NanoDSF, showing thermal transition temperatures. C) Aggregation propensity of 1–2C TCR engagers evaluated using NanoDSF by measuring their scattering temperatures. D) antigen sensitivity of 1–2C TCR engagers. T cells were co‐cultured with 1–2C TCR engager proteins (100 nM) and K562‐HLA‐A11 cells loaded with serial diluted KRAS‐G12wt or KRAS‐G12V peptides. The supernatants from the co‐cultures were analyzed using IFN‐γ ELISA to quantify the secreted IFN‐γ. Data points represent the mean concentration of IFN‐γ from duplicate wells, and the data are representative of three independent experiments.

Article Snippet: T cells were activated by adding 10 μL T Cell TransAct (Miltenyi Biotec) per 1 × 10 6 cells and cultured in UniEx T‐cell serum‐free medium (ExCell) supplemented with 2.5% Helios UltraGRO‐Advanced (AventaCell), 300 IU mL −1 human IL‐2 (Novoprotein), 10 ng mL −1 human IL‐7 (Novoprotein), 100 μg mL −1 streptomycin and 100 IU mL −1 penicillin (Invitrogen).

Techniques: Binding Assay, Nano Differential Scanning Fluorimetry, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Advanced Science

Article Title: An Engineered Soluble Single‐Chain TCR Engager for KRAS‐G12V Specific Tumor Immunotherapy

doi: 10.1002/advs.202500181

Figure Lengend Snippet: T96F‐mutated 1–2C‐2LL TCR engager potentially enhanced T cell reactivity and tumor cytotoxicity. A) T cells were co‐cultured with 1–2C‐2LL TCR engager proteins (100 nM) and K562‐HLA‐A11 cells loaded with serially diluted KRAS‐G12wt or KRAS‐G12V peptides. Secreted IFN‐γ in the supernatants was quantified using ELISA. B) T cells were co‐cultured with serially diluted 1–2C‐2LL TCR engager proteins and K562‐HLA‐A11 cells loaded with KRAS‐G12wt or KRAS‐G12V peptides (10 µg mL −1 ). Secreted IFN‐γ in the supernatants was quantified by ELISA. Data points represent the mean concentration of IFN‐γ from triplicate wells in (A) and (B). The data are representative of three independent experiments. C) Schematic of the flow cytometry‐based cytotoxicity assay, where 1–2C‐2LL TCR engager re‐direct T cell to target tumor cells engineered over‐expressing KRAS‐G12V and HLA‐A*11:01. Tumor cells without engineering served as control. created with BioRender.com. D–G) Cytotoxicity analysis of T cells against K562 cells D), CFPAC‐1 cells E), SW‐620 cells F), and PANC‐1 cells G) engineered to over‐express KRAS‐G12V/HLA‐A*11:01. T cells were co‐cultured with tumor cells and 1–2C‐2LL TCR engager proteins (100 nM). The T cells cytotoxicity was assessed by flow cytometry and the specific lysis of tumor cells was calculated as [(tumor cell events in PBS treatment − tumor cell events in TCR engager treatment)/tumor cell events in PBS treatment]. The data points represent T cell responses from at least three donors, with mean ± SEM values shown as columns. Statistical significance was calculated with an unpaired Student's t test (** p < 0.01, * p < 0.05). H) IFN‐γ Cytokine‐producing profiles induced by 1–2C‐2LL TCR engagers. T cells were co‐cultured with wildtype or T96F‐mutated 1–2C‐2LL engager proteins in the presence of K562‐HLA‐A11 cells loaded with KRAS‐G12wt or KRAS‐G12V peptides, and intracellular cytokine staining (ICS) was performed to detect the secretion of IFN‐γ. The number in the panel represents the percentage of cytokine‐staining positive cells. I and J) Functional profiling of KRAS‐G12V specific CD8 + T cells induced by wild‐type or T96F‐mutated 1–2C‐2LL engagers. Percentages of mono‐functional (single) T cells producing one cytokine (IFN‐γ, TNF‐α, or IL‐2), dual‐functional (double) T cells producing two cytokines, and multi‐functional (triple) T cells producing all three cytokines were determined. The data shown are representative of two independent experiments.

Article Snippet: T cells were activated by adding 10 μL T Cell TransAct (Miltenyi Biotec) per 1 × 10 6 cells and cultured in UniEx T‐cell serum‐free medium (ExCell) supplemented with 2.5% Helios UltraGRO‐Advanced (AventaCell), 300 IU mL −1 human IL‐2 (Novoprotein), 10 ng mL −1 human IL‐7 (Novoprotein), 100 μg mL −1 streptomycin and 100 IU mL −1 penicillin (Invitrogen).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay, Flow Cytometry, Cytotoxicity Assay, Expressing, Control, Lysis, Staining, Functional Assay

Journal: Advanced Science

Article Title: An Engineered Soluble Single‐Chain TCR Engager for KRAS‐G12V Specific Tumor Immunotherapy

doi: 10.1002/advs.202500181

Figure Lengend Snippet: 1–2C‐2LL TCR engagers facilitated immune synapses formation between T cells and target cells. A–C) Representative immunofluorescence images depicting the co‐cultures of T96F‐mutated 1–2C‐2LL TCR engager proteins with A) K562‐HLA‐A11 target cells loaded with KRAS‐G12V peptide, B) T cells, or C) both T cells and K562‐HLA‐A11 target cells loaded with KRAS‐G12V peptide. The co‐cultured cells were stained with fluorescence‐labeled anti‐CD8 antibodies and fluorescence‐labeled anti‐his‐tag antibodies, with cell outlines indicated by white lines. The data are representative of two independent experiments. D and E) Quantification of the percentage of CD8 molecules D) and 1–2C‐2LL TCR engagers E) accumulation at the immunological synapse. Data are represented as means ± SEM, and statistical significance was calculated using ordinary one‐way ANOVA test with Kruskal‐Wallis multi‐comparisons. (**** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05). Data are representative of two independent experiments. F and G) Cell avidity measurements using z‐Movi technology evaluate T cells binding to SW‐620‐HLA‐A11‐luci target cells in the presence of T96F‐mutated 1–2C‐2LL engager proteins. F) The median force required to detach T cells from SW‐620‐HLA‐A11‐luci cells, with each point representing an individual T cell in the microfluidic chips. Differences were analyzed using an unpaired Student's t test (***: p < 0.001). G) The binding frequency of T cells to SW‐620‐HLA‐A11‐luci cells as the applied force increases. T cells and target cells co‐cultured with PBS were enrolled as negative control. The data presented are representative of two independent experiments.

Article Snippet: T cells were activated by adding 10 μL T Cell TransAct (Miltenyi Biotec) per 1 × 10 6 cells and cultured in UniEx T‐cell serum‐free medium (ExCell) supplemented with 2.5% Helios UltraGRO‐Advanced (AventaCell), 300 IU mL −1 human IL‐2 (Novoprotein), 10 ng mL −1 human IL‐7 (Novoprotein), 100 μg mL −1 streptomycin and 100 IU mL −1 penicillin (Invitrogen).

Techniques: Immunofluorescence, Cell Culture, Staining, Fluorescence, Labeling, Binding Assay, Negative Control

Journal: Stem Cell Research & Therapy

Article Title: Inhibition of the RhoGTPase Cdc42 by ML141 enhances hepatocyte differentiation from human adipose-derived mesenchymal stem cells via the Wnt5a/PI3K/miR-122 pathway: impact of the age of the donor

doi: 10.1186/s13287-018-0910-5

Figure Lengend Snippet: Pharmacological targeting of Cdc42 activity by ML141 (ML) induces rifampicin responsiveness and restores cell function. hADSCs derived from young and aged subjects were differentiated and treated with or without ML141 as indicated in Fig. . a–c Secreted albumin, urea production, and low-density lipoprotein (LDL) uptake: supernatants of cultured cells and the lysates were collected at D0/D14/D28 of the differentiation for the quantification of albumin, urea, and LDL uptake as indicated in the Methods. Results are expressed per ng/ml (for the production of albumin and urea) and RFU (fluorescent LDL uptake) and are presented as fold variation relative to young at day D0, and are the mean ± SEM of several measures (5, 6 and 10, respectively). d Cytochrome P450 (CYP3A4) activity at day 28 of the Hep-Dif after induction with rifampicin (20 μM, 24 h). The results are expressed as fold variation relative to basal (without rifampicin), and are the mean ± SEM of three independent experiments performed in duplicate. e hepatocyte-like cells derived from aged ADSCs treated with ML141 exhibit hepatic-specific function of glycogen storage. Cells were evaluated for glycogen storage capacity (pink color) using periodic acid-Schiff (PAS) staining as described in the Methods. Cells were examined microscopically and phase-contrast images were captured. Cultures of HepG2 cells were assessed as positive controls. § * P < 0.05, §§ ** P < 0.01; § rifampicin versus controls or aged versus young and *aged treated with ML141 versus untreated

Article Snippet: The Fluorometric LDL Uptake Assay Kit (#ab204716, Abcam) was used to assess the LDL uptake capacity; 3 × 10 4 hADSCs and hepatocyte-like cells were incubated with fluorescent-labeled LDL overnight at 37 °C and, after incubation, cells were washed and fluorescence measured on the Varioskan™ LUX Multimode Microplate Reader .

Techniques: Activity Assay, Cell Function Assay, Derivative Assay, Cell Culture, Staining

Journal: PLoS ONE

Article Title: Dishevelled Proteins Are Associated with Olfactory Sensory Neuron Presynaptic Terminals

doi: 10.1371/journal.pone.0056561

Figure Lengend Snippet: A, B: Dvl-1 expression in the OB was analyzed in the context of two classical markers: NQO1 (zone I/dorsal OE) and OCAM (zones I-IV/ventral OE). All NQO1-expressing glomeruli showed Dvl-1 expression (A, B, D, F, G, H). A few OCAM-expressing glomeruli located in the dorsal part of the OB, also showed Dvl-1 expression (A, B, and open arrows in E, F, G). In the ventral part of the OB, only OCAM-expressing glomeruli were observed (A, B, C). In the transition NQO1/OCAM zone, those glomeruli expressing both markers, showed expression of Dvl-1 only in the NQO1 positive axons (A, B, and open arrowhead in H). Dvl-1 also revealed a subset of glomeruli negative for both classical markers (A, B, and arrows in G, I). In order to test whether these only Dvl-1 expressing glomeruli are functional, we stained for VGlut-2 (blue), Dvl-1 (green) and OCAM+NQO1 (both in red) (I). These glomeruli expressed VGlut-2 (arrows), consistent with functional activity. Scale bar = 200 µm in A, B; 20 µm C–I. ONL: olfactory nerve layer; GL: glomerular layer; EPL: external plexiform layer; MCL: Mitral cell layer; GCL: granule cell layer.

Article Snippet: The following antibodies were used: mouse anti Dvl-1 (Santa Cruz, CA, sc-8025) at 1/200 with antigen retrieval; rabbit anti Dvl-2 (GeneTex, TX, GTX103878) at 1/250; rabbit anti Dvl-3 (GeneTex, TX, GTX102509) at 1/250; goat anti NQO1 (Abcam, MA, ab2346) at 1/3000; rabbit anti OCAM (gift from Dr. K. Mori) at 1/1000; rabbit anti VGlut2 (Synaptic Systems, Germany, 135403) at 1/2000; rabbit anti Synaptotagmin 1 (Synaptic Systems, Germany, 105002); rabbit anti Synaptophysin (Dako, CA, A0010).

Techniques: Expressing, Functional Assay, Staining, Activity Assay

Journal: PLoS ONE

Article Title: Dishevelled Proteins Are Associated with Olfactory Sensory Neuron Presynaptic Terminals

doi: 10.1371/journal.pone.0056561

Figure Lengend Snippet: The ventral domain was not included in order to increase clarity and emphasize the glomeruli expressing multiple markers. This analysis identified OCAM+ glomeruli (red arrow), OCAM+/Dvl-1+ glomeruli (yellow arrow), Dvl-1+ glomeruli (green arrow), NQO1+/Dvl-1+ glomeruli (turquoise arrow), OCAM+/NQO1+/Dvl-1+ glomeruli (white arrow). The X axis represents the line connecting all the glomeruli, stretched out linearly. We removed most of the ventral and lateral tracing (relative position 0–1500) because all these glomeruli were only OCAM positive.

Article Snippet: The following antibodies were used: mouse anti Dvl-1 (Santa Cruz, CA, sc-8025) at 1/200 with antigen retrieval; rabbit anti Dvl-2 (GeneTex, TX, GTX103878) at 1/250; rabbit anti Dvl-3 (GeneTex, TX, GTX102509) at 1/250; goat anti NQO1 (Abcam, MA, ab2346) at 1/3000; rabbit anti OCAM (gift from Dr. K. Mori) at 1/1000; rabbit anti VGlut2 (Synaptic Systems, Germany, 135403) at 1/2000; rabbit anti Synaptotagmin 1 (Synaptic Systems, Germany, 105002); rabbit anti Synaptophysin (Dako, CA, A0010).

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: NK-A 17E-233I: a novel competitive inhibitor of human dihydroorotate dehydrogenase (DHODH) for cancer therapy

doi: 10.1186/s13046-025-03538-w

Figure Lengend Snippet: NK-A 17E-233I as a Novel Competitive Inhibitor of Human DHODH. A The molecular structure of NK-A 17E-233I is illustrated. B Molecular docking analysis indicates the formation of a complex between NK-A 17E-233I and the human DHODH protein (PDB: 1D3G). C A superposition of NK-A 17E-233I, FMN, and Brequinar when bound to human DHODH is presented, emphasizing the narrow tunnel that leads to the active site. D Graphical representations and a tabular summary illustrate the relative inhibitory effects of Brequinar and NK-A 17E-233I on the activity of DHODH at different concentrations of DHO in an enzymatic assay. The lines represent the fit of the equation V = V max /(1 + ( I /IC 50 ) h ) for all normalized data. The reported IC 50 values are the mean ± SD (n = 4–6), with the average of the fitted slopes ± SD provided in parentheses. Statistical analysis was conducted using an unpaired t-test. E,F Thermal stability assessments were performed via nanoDSF. Δ T m reflects the difference in melting temperatures T m of DHODH in the presence of NK-A 17E-233I ( E ) or Brequinar ( F ). The values of the differences in melting temperatures, Δ T m (°C), are presented as mean ± SD (n = 3). The melting temperatures ( T m ) for DHODH, DHODH R136C, and Δ29 DHODH in the absence of a compound were 55.7 °C, 57.7 °C, and 51.5 °C, respectively

Article Snippet: SW620 cells (0.8 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document} × 10 6 ) were cultured in 60-mm Petri dishes and treated with DMSO, Brequinar (100 μM), or NK-A 17E-233I (25 μM) for 24 h, to quantify the reduced and oxidized glutathione (GSH/GSSG) using a GSH/GSSG Assay Kit (#HY-K0311; MedChemExpress) in accordance with the manufacturer's instructions.

Techniques: Activity Assay, Enzymatic Assay, Nano Differential Scanning Fluorimetry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: NK-A 17E-233I: a novel competitive inhibitor of human dihydroorotate dehydrogenase (DHODH) for cancer therapy

doi: 10.1186/s13046-025-03538-w

Figure Lengend Snippet: NK-A 17E-233I Demonstrates Cytotoxic Effects on Human Colorectal Cancer Cells and Patient-Derived Organoids. A Dose–response curves illustrate the cytotoxic effects of NK-A 17E-233I on human colorectal cancer cell lines and fibroblasts following a 48-h treatment period. Data are presented as mean ± SD (n = 3). B Representative brightfield images obtained using a Dmi8 Leica wide-field microscope depict the treatment effects (Tx, 25 µM) after a 48-h period. Scale bar = 50 µm. C,D The viability of colorectal cancer cells was assessed over time in the absence (DMSO) or presence of treatment (Tx, 25 µM), by the Trypan Blue exclusion assay ( C ) and the ATP assay over a 24-h treatment period ( D ). E A clonogenic assay provides representative images of colorectal cancer colonies following treatment (Tx, 25 µM) over a 14-day period at the specified concentrations. F The proliferation of colorectal cancer cells was assessed after 24 h as described in C , both in the absence (DMSO) and in the presence of Tx (25 µM) or BRQ (100 µM), along with physiological concentrations of uridine. G Similar to D, this portion assesses the outcomes following DMSO or treatment (Tx, 25 µM) and the exposure to non-physiological uridine (200 μM) over a 24-h treatment period. H This figure parallels D , but colorectal cancer cells were cultured in the absence or presence of FBS (10%) concurrently with DMSO or Tx (25 µM). Measurements (in relative light unit, RLU) in D , G, and H were obtained via the CellTiter Glo assay. Data are reported as mean ± SEM (n = 4–6). Statistical analyses were conducted using a one-way ANOVA test with Sidak’s multiple comparisons test. I Representative immunofluorescence images illustrate the morphology of BFCNs and SW620 cells following treatment (Tx, 25 µM) for 24 h, highlighting F-actin (in magenta), MAP2 (neurite marker, in cyan), and nuclei (DAPI, in yellow). Scale bar = 25 μm. J A schematic representation outlines the generation of organoids from colorectal cancer patients. K Representative images depict the morphology of healthy and patient-derived intestinal organoids subjected to increasing concentrations of Tx or DMSO for 96 h. Insets, delineated by dashed boxes in the top panel, emphasize the morphological features of the organoids. Scale bar = 10 µm. Tx and BRQ denote NK-A 17E-233I and Brequinar, respectively

Article Snippet: SW620 cells (0.8 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document} × 10 6 ) were cultured in 60-mm Petri dishes and treated with DMSO, Brequinar (100 μM), or NK-A 17E-233I (25 μM) for 24 h, to quantify the reduced and oxidized glutathione (GSH/GSSG) using a GSH/GSSG Assay Kit (#HY-K0311; MedChemExpress) in accordance with the manufacturer's instructions.

Techniques: Derivative Assay, Microscopy, Trypan Blue Exclusion Assay, ATP Assay, Clonogenic Assay, Cell Culture, Glo Assay, Immunofluorescence, Marker

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: NK-A 17E-233I: a novel competitive inhibitor of human dihydroorotate dehydrogenase (DHODH) for cancer therapy

doi: 10.1186/s13046-025-03538-w

Figure Lengend Snippet: The Impact of NK-A 17E-233I on the Transcriptomic and Metabolomic Profile of SW620 Cells. A The Venn diagram illustrates the overlap and divergence of gene counts obtained from RNASeq following DMSO or treatment (Tx, 25 µM) for 48 h. B Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment scatter plot depicting the 20 most significant KEGG pathways, with the size of each point corresponding to the number of genes associated with each biological pathway. C Left: Schematic representation of the DHODH-POLD2 axis in cancer. Right: CldU tract lengths following treatment (Tx, 25 µM) for 1 h in knockdown cells. The median (n = 3) is indicated by a black line, with each dot representing a single fiber (≥ 200 fibers measured per condition). Statistical differences were determined using the Mann–Whitney test. D Construction of a sample-specific genome-scale metabolic model: A genome-scale metabolic model tailored to the specific sample was developed (see methods section for comprehensive details). E, G, and I Heatmaps depicting the metabolic alterations subsequent to treatment with DMSO, Tx (25 µM), or BRQ (100 µM). F , H, and J represent the functional measurements of glutathione, lactate, and orotate/DHO ratio, respectively. Data are expressed as mean ± SEM (n = 4–5). Statistical differences were assessed using a two-way ANOVA test with Sidak´s multiple comparisons test. Tx and BRQ denote NK-A 17E-233I and Brequinar, respectively

Article Snippet: SW620 cells (0.8 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document} × 10 6 ) were cultured in 60-mm Petri dishes and treated with DMSO, Brequinar (100 μM), or NK-A 17E-233I (25 μM) for 24 h, to quantify the reduced and oxidized glutathione (GSH/GSSG) using a GSH/GSSG Assay Kit (#HY-K0311; MedChemExpress) in accordance with the manufacturer's instructions.

Techniques: Knockdown, MANN-WHITNEY, Functional Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: NK-A 17E-233I: a novel competitive inhibitor of human dihydroorotate dehydrogenase (DHODH) for cancer therapy

doi: 10.1186/s13046-025-03538-w

Figure Lengend Snippet: The Impact of NK-A 17E-233I on Mitochondrial Bioenergetics in Cancer Cells. A,B Representative confocal microscopy images demonstrate the effects of DMSO or Tx (25 µM) administered for 24 h on HCT116 colorectal cancer cells, with a focus on mitochondrial activity assessed using MitoTracker Red (grey) to visualize the mitochondrial membrane potential ( A ) or ROS production ( B ) evaluated with CellDeepRed staining (grey). Nuclei were stained with DAPI (blue). Scale bar = 20 µm. Violin plots depict the density of MitoTracker ( A ) or CellDeepRed ( B ) density per cell, calculated as the intensity of MitoTracker or ROS divided by the area of each cell. N represents independent replicates (2–4) for each condition; n denotes the density of MitoTracker or ROS per cell. In A,B , the data are presented as ‘Superplots,’ indicating the mean of the various replicates (large circles) alongside the distribution of ‘MitoTracker or ROS density per cell analyzed’ (color-coded dots), which is superimposed as a beeswarm plot. The black line represents the mean, and a paired two-tailed t-test was utilized to assess the statistical differences. C A diagram illustrating the oxygen consumption rate (OCR) profile delineates the key phases of mitochondrial respiration. D-F Analysis of OCR in colorectal cancer cells following a 24-h treatment with DMSO, Tx (25 µM) or BRQ (100 µM). D DHODH-mediated respiration; E The measurements of OCR under basal conditions and subsequent to the sequential administration of inhibitors of OXPHOS. F Complex I- and complex II-mediated respiration. Data from D-F are expressed as mean ± SEM (n = 3–5). Statistical differences were determined using a two-way ANOVA test with Sidak’s multiple comparisons test. G Mitochondrial DNA copy number was quantified via qPCR at the specified conditions. Tx and BRQ denote NK-A 17E-233I and Brequinar, respectively

Article Snippet: SW620 cells (0.8 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document} × 10 6 ) were cultured in 60-mm Petri dishes and treated with DMSO, Brequinar (100 μM), or NK-A 17E-233I (25 μM) for 24 h, to quantify the reduced and oxidized glutathione (GSH/GSSG) using a GSH/GSSG Assay Kit (#HY-K0311; MedChemExpress) in accordance with the manufacturer's instructions.

Techniques: Confocal Microscopy, Activity Assay, Membrane, Staining, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: NK-A 17E-233I: a novel competitive inhibitor of human dihydroorotate dehydrogenase (DHODH) for cancer therapy

doi: 10.1186/s13046-025-03538-w

Figure Lengend Snippet: The Impact of NK-A 17E-233I on Fork Progression and Induction of DNA Damage in Colorectal Cancer Cells. Cells were treated with DMSO, Tx (25 µM), or BRQ (100 µM) for a 1-h period. A Top: Schematic representation of the IdU/CldU pulse-labeling protocol. Bottom: Representative images of DNA fibers. B-D CldU tract lengths at the specified conditions (n = 3). Each dot represents one fiber (≥ 200 fibers were measured per condition). E Top: Schematic representation of the IdU/CldU pulse-labeling protocol to evaluate fork restart. Bottom: Representative images of DNA fibers following fork restart in LS174T cells at the specified conditions. Scale bar = 20 μm. F CldU tract lengths in LS174T cells from E. Each dot represents one fiber (> 140 fibers were measured per condition), with the horizontal line representing the median from one experimental replicate (n = 3). G Representative fluorescence images demonstrating the staining of DNA breaks in single-cell neutral gel electrophoresis (neutral comet assay). Scale bar = 25 μm. H Quantification of the DNA comet tail length from one experimental replicate. Each dot represents one cell (> 95 cells were measured per condition) (n = 3). I Representative immunofluorescence images displaying staining of the DNA damage marker protein γ-H2AX (cyan) and the nucleus (DAPI, yellow). Red arrows indicate DNA damage in treated cells. J Violin plots illustrating the fluorescence intensity of the nuclear γ-H2AX (n = 3). Each dot represents one cellular nucleus (n ≥ 74 nuclei were measured per condition). K as in I , but displaying staining of Rad51 (cyan). L as in J , but derived from Rad51 images from K . M Western blot analysis showing CHK1-pS345 and GAPDH (loading control). N Top: Percentage of colonies of LS174T cells after treatment with DMSO, Tx (4 µM), VE-821 (0.5 µM), or the combination of both. Bars represent the mean ± SD (n = 9), shown as dots. Bottom: A representative image of human colorectal cancer colonies conducted at the specified conditions for 14 days. Statistical differences were determined using the Mann–Whitney test in all panels, except in H and N where a one-way ANOVA test with Bonferroni’s multiple comparisons test was employed. Unless otherwise specified, all results are expressed as the median (horizontal black line). Tx and BRQ denote NK-A 17E-233I and Brequinar, respectively

Article Snippet: SW620 cells (0.8 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document} × 10 6 ) were cultured in 60-mm Petri dishes and treated with DMSO, Brequinar (100 μM), or NK-A 17E-233I (25 μM) for 24 h, to quantify the reduced and oxidized glutathione (GSH/GSSG) using a GSH/GSSG Assay Kit (#HY-K0311; MedChemExpress) in accordance with the manufacturer's instructions.

Techniques: Labeling, Fluorescence, Staining, Nucleic Acid Electrophoresis, Neutral Comet Assay, Immunofluorescence, Marker, Derivative Assay, Western Blot, Control, MANN-WHITNEY

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: NK-A 17E-233I: a novel competitive inhibitor of human dihydroorotate dehydrogenase (DHODH) for cancer therapy

doi: 10.1186/s13046-025-03538-w

Figure Lengend Snippet: NK-A 17E-233I Inhibits the Progression of Cell Cycle and Induces Apoptosis in Colorectal Cancer Cells. A The left panel depicts a flow cytometry schematic that illustrates the distribution of DNA content across the G1, S, and G2/M phases of the cell cycle. The right panel features a representative FlowJo histogram displaying the DNA content in a cohort of colorectal cancer cells (n = 3). The red arrow indicates the accumulation of cancer cells in the S phase following a 48-h treatment period with DMSO or Tx (25 µM). B The proportion of cancer cells within each phase of the cell cycle is presented, with data expressed as mean ± SD (n ≥ 6). C Flow cytometry analysis conducted using CytExpert reveals the DNA content of colorectal cancer cells. The upper section demonstrates the population of cells arrested following treatment with DMSO, hydroxyurea (HU, 1 mM), or Tx (25 µM), while the lower section presents the washout results after 12 h, comparing Tx-released cells to those treated with HU. A representative image is provided (n = 3), with yellow sections indicating the cells arrested in the S-phase. D Representative microscopy images from time-lapse experiments display brightfield images (cell morphology in grey) alongside nuclei stained with Hoechst 33,342 (magenta). The arrows indicate the cells approaching division and/or in the process of division, demonstrating that treated cells exhibit a prolonged duration prior to division compared to untreated cells. Scale bar = 25 μm. E Representative dot plots from the Annexin V-FITC assay following FACS analysis of colorectal cancer cells with DMSO or Tx (25 µM). F A representative dot plot from FACS analysis illustrates the activation of caspase-3 in colorectal cancer cells in the presence of DMSO or Tx (25 µM). The black boxes denote live cells, while the red ones indicate dead cells. G-L Representative western blots depict the expression levels of Bax, cytochrome c, caspase-8, and caspase-3 (including both non-cleaved and cleaved protein products), as well as DR5, p53, and KRAS protein levels in untreated versus treated cells (n = 3). α-tubulin and GAPDH served as loading controls. Tx and BRQ denote NK-A 17E-233I and Brequinar, respectively

Article Snippet: SW620 cells (0.8 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document} × 10 6 ) were cultured in 60-mm Petri dishes and treated with DMSO, Brequinar (100 μM), or NK-A 17E-233I (25 μM) for 24 h, to quantify the reduced and oxidized glutathione (GSH/GSSG) using a GSH/GSSG Assay Kit (#HY-K0311; MedChemExpress) in accordance with the manufacturer's instructions.

Techniques: Flow Cytometry, Microscopy, Staining, Activation Assay, Western Blot, Expressing

Journal: medRxiv

Article Title: Aspirin improves both reactivity and durability of type-I interferon signaling to achieve functional cure of chronic hepatitis B

doi: 10.1101/2024.06.14.24308555

Figure Lengend Snippet: (A) A diagram showing that IFN-I-induced IFNAR1 degradation mediated by PARP11 and β-TrCP leads to a shift in IFN-I antiviral efficiency from “Efficiency Display” to “Efficiency Loss”. (B and C) Flow cytometry analysis of cytoplasmic membrane IFNAR1 levels (B) or RT-qPCR analysis of Ifit1 mRNA (C) in PBMCs from chronic hepatitis-B (CHB) patients before and 6 h after Pegylated (Peg)-IFN treatment (180 μg each person). B: before treatment; A: after treatment. (D) IFNAR1 (wild-type,WT; S535A mutants) constructs were stably transfected in Ifnar1 -/- HEK293T cells. RT-qPCR was used to analyze Ifit1 mRNA in these cells treated with IFNα (1,000 IU/ml) for indicated times. (E) ChIP-qPCR analysis of the binding of cellular IRF9 proteins to the ISRE promoters of the representative ISG ( Ifit1 ) gene in HEK293T cells treated with IFNα (1,000 IU/ml) for indicated times. (F) Western blot analysis of IRF9 in HT1080 cells treated with IFNα (1,000 IU/ml) for indicated times. (G) ChIP-qPCR analysis of the binding of IRF9 proteins to the ISRE of Ifit1 in HEK293T cells treated with PTP inhibitor I (10 μM) and (or) IFNα (1,000 IU/ml) as indicated. (H) RT-qPCR analysis of Ifit1 mRNA in HEK293T cells treated with PTP inhibitor I (10 μM) and (or) IFNα (1,000 IU/ml) as indicated. NS, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed unpaired Student’s t -test). Data are shown as means ± SD of three biological replicates (B-E, G, H), or are representative of three independent experiments (F).

Article Snippet: The antibodies in this study are listed here: Anti-IRF9 (Santa Cruz, Cat #sc-365893), Anti-pan-pY (Cell Signalling Technology, Cat #8954), Anti-Flag (Sigma, Cat #F7425), Anti-Tubulin (Proteintech, Cat #66031-1-Ig), Anti-GFP (Santa Cruz, Cat #sc-9996), Anti-p-STAT1(Y701) (Cell Signalling Technology, Cat #9167), Anti-STAT1 (Cell Signalling Technology, Cat #9176), Anti-PTP1B (Proteintech, Cat #11334-1-AP), Anti-β-Actin (Proteintech, Cat #66009), Anti-Myc (Abmart, Cat #M20002), Anti-HA (Abcam, Cat #ab9110), Anti-IFNAR1-PE (Invitrogen, Cat #MA5-23630), Anti-IFNAR1 (Sino Biological, Cat #13222-T20), Anti-β-Trcp (Cell Signalling Technology, Cat #11984),Anti-pan-AcK (Santa Cruz, Cat #sc-32268), Anti-Viperin (Abcam, Cat #107359), Anti-IFIT1 (Santa Cruz, Cat #sc-134948), Anti-HA (H1N1) (Sino Biological, Cat #11684-T56), Anti-p-STAT1 (S727) (Cell Signalling Technology, Cat #8826), Anti-p-STAT2 (Y690) (Cell Signalling Technology, Cat #88410), Anti-VSV-G (Abcam, Cat #ab1874), Anti-PKR (Cell Signalling Technology, Cat #3072S) and Anti-GAPDH (Goodhere Biological Technology, Cat #AB-M-M001).

Techniques: Flow Cytometry, Membrane, Quantitative RT-PCR, Construct, Stable Transfection, Transfection, Binding Assay, Western Blot, Two Tailed Test

Journal: medRxiv

Article Title: Aspirin improves both reactivity and durability of type-I interferon signaling to achieve functional cure of chronic hepatitis B

doi: 10.1101/2024.06.14.24308555

Figure Lengend Snippet: (A) The tyrosine phosphorylation site (Tyr112) of IRF9 was identified by mass spectrometry. (B) All tyrosine residues of IRF9 were shown (upper). Immunoprecipitation (IP)-immunoblotting (IB) was performed to analyze pan-tyrosine phosphorylation (pan-pY) of IRF9 in HEK293T cells transfected with WT or mutated Flag-GFP-tagged IRF9 (F-G-IRF9) (lower). (C and D) ChIP-qPCR analysis of the binding of IRF9 proteins to the ISRE of Ifit1 (C) or RT-qPCR analysis of Ifit1 mRNA levels (D) in IRF9-KO cells stably transfected with F-G-IRF9 (WT, Y112F) and then treated with IFNα (1,000 IU/ml) for 8 h. (E) IP-IB analysis of pY112-IRF9 using a specific anti-pY112-IRF9 antibody in HEK293T cells transfected with F-G-IRF9 (WT or Y112F). (F) Western blot analysis of pY112-IRF9 in HEK293T cells treated with IFNα (1,000 IU/ml) for indicated times. (G) Western blot analysis of pY112-IRF9 in PBMCs from the CHB patients in . B: before Peg-IFN treatment; A: 6 h after Peg-IFN treatment. NS, not significant (p > 0.05), **p < 0.01 (two-tailed unpaired Student’s t -test). Data are shown as means ± SD of three biological replicates (C, D), or are representative of three independent experiments (B, E-G).

Article Snippet: The antibodies in this study are listed here: Anti-IRF9 (Santa Cruz, Cat #sc-365893), Anti-pan-pY (Cell Signalling Technology, Cat #8954), Anti-Flag (Sigma, Cat #F7425), Anti-Tubulin (Proteintech, Cat #66031-1-Ig), Anti-GFP (Santa Cruz, Cat #sc-9996), Anti-p-STAT1(Y701) (Cell Signalling Technology, Cat #9167), Anti-STAT1 (Cell Signalling Technology, Cat #9176), Anti-PTP1B (Proteintech, Cat #11334-1-AP), Anti-β-Actin (Proteintech, Cat #66009), Anti-Myc (Abmart, Cat #M20002), Anti-HA (Abcam, Cat #ab9110), Anti-IFNAR1-PE (Invitrogen, Cat #MA5-23630), Anti-IFNAR1 (Sino Biological, Cat #13222-T20), Anti-β-Trcp (Cell Signalling Technology, Cat #11984),Anti-pan-AcK (Santa Cruz, Cat #sc-32268), Anti-Viperin (Abcam, Cat #107359), Anti-IFIT1 (Santa Cruz, Cat #sc-134948), Anti-HA (H1N1) (Sino Biological, Cat #11684-T56), Anti-p-STAT1 (S727) (Cell Signalling Technology, Cat #8826), Anti-p-STAT2 (Y690) (Cell Signalling Technology, Cat #88410), Anti-VSV-G (Abcam, Cat #ab1874), Anti-PKR (Cell Signalling Technology, Cat #3072S) and Anti-GAPDH (Goodhere Biological Technology, Cat #AB-M-M001).

Techniques: Mass Spectrometry, Immunoprecipitation, Western Blot, Transfection, Binding Assay, Quantitative RT-PCR, Stable Transfection, Two Tailed Test

Journal: medRxiv

Article Title: Aspirin improves both reactivity and durability of type-I interferon signaling to achieve functional cure of chronic hepatitis B

doi: 10.1101/2024.06.14.24308555

Figure Lengend Snippet: (A) A drug library containing 214 clinically approved small molecules from plant sources was employed to screen for the potential small molecules that can upregulate pY112-IRF9 in HEK293T cells with IFNα treatment (1,000 IU/ml, 12 h). (B) Western blot analysis of pY112-IRF9 in HEK293T cells pre-treated with 1×PBS (Ctrl) or Aspirin (100 μg/ml) or Salicylate (100 μg/ml) for 6 h and then treated with or without IFNα (1,000 IU/ml) for 12 h. (C) IP-IB analysis of pan-pY of IRF9 in HEK293T cells transfected with F-G-IRF9 and treated with Salicylate (300 and 600 μg/ml) for 6 h. (D) IP-IB analysis of pan-pY of IRF9 in HEK293T cells transfected with F-G-IRF9 (WT, Y112F) and treated with Salicylate (100 μg/ml) for 6 h. (E and F) Western blot analysis of pY112-IRF9 in HEK293T cells treated with Salicylate (100, 200 and 300 μg/ml) for 6 h (E) or Salicylate (100 μg/ml) for indicated times (F). (G) ChIP-qPCR analysis of the binding of IRF9 to the ISRE of Ifit1 , Isg15 or Viperin genes in HEK293T cells transfected with Myc-IRF9 and treated with Salicylate (300 μg/ml) for 6 h. (H) RT-qPCR analysis of Ifit1 mRNA in IRF9-knockout (KO) cells stably transfected with F-G-IRF9 (WT, Y112F) and treated with Salicylate (300 μg/ml) for 6 h. (I) ChIP-qPCR analysis of the binding of IRF9 to the ISRE of Ifit1 in 2fTGH cells pre-treated with Salicylate (300 μg/ml) for 2 h and then treated with IFNα (1,000 IU/ml) for 6 h. (J) RT-qPCR analysis of Ifit1 and Isg54 mRNA in THP1 cells pre-treated with Salicylate (100 and 300 μg/ml) for 2 h and then treated with IFNα (1,000 IU/ml) for 6 h. (K) RT-qPCR analysis of Ifit1 mRNA in HEK293T cells treated with Salicylate (100 μg/ml) and IFNα (1,000 IU/ml) together for indicated times. (L) HepG2 cells transfected with HBV-1.3 constructs were treated with Salicylate (300 μg/ml) and (or) IFNα (1,000 IU/ml) together for 24 h. RT-qPCR was used to analyze HBV DNA levels in cells. (M) RT-qPCR analysis of Ifit1 mRNA in Irf9 +/+ and Irf9 -/- cells pre-treated with Salicylate (300 μg/ml, 2 h) and then treated with IFNα (1,000 IU/ml) for 6 h. (N) RT-qPCR analysis of Ifit1 mRNA in Irf9 -/- HEK293T cells transfected with F-G-IRF9 and treated with Salicylate (100 μg/ml, 2 h), followed by IFNα treatment (1,000 IU/ml) for 6 h. NS, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed unpaired Student’s t -test). Data are shown as means ± SD of at least three biological replicates (G-N), or are representative of three independent experiments (B-F).

Article Snippet: The antibodies in this study are listed here: Anti-IRF9 (Santa Cruz, Cat #sc-365893), Anti-pan-pY (Cell Signalling Technology, Cat #8954), Anti-Flag (Sigma, Cat #F7425), Anti-Tubulin (Proteintech, Cat #66031-1-Ig), Anti-GFP (Santa Cruz, Cat #sc-9996), Anti-p-STAT1(Y701) (Cell Signalling Technology, Cat #9167), Anti-STAT1 (Cell Signalling Technology, Cat #9176), Anti-PTP1B (Proteintech, Cat #11334-1-AP), Anti-β-Actin (Proteintech, Cat #66009), Anti-Myc (Abmart, Cat #M20002), Anti-HA (Abcam, Cat #ab9110), Anti-IFNAR1-PE (Invitrogen, Cat #MA5-23630), Anti-IFNAR1 (Sino Biological, Cat #13222-T20), Anti-β-Trcp (Cell Signalling Technology, Cat #11984),Anti-pan-AcK (Santa Cruz, Cat #sc-32268), Anti-Viperin (Abcam, Cat #107359), Anti-IFIT1 (Santa Cruz, Cat #sc-134948), Anti-HA (H1N1) (Sino Biological, Cat #11684-T56), Anti-p-STAT1 (S727) (Cell Signalling Technology, Cat #8826), Anti-p-STAT2 (Y690) (Cell Signalling Technology, Cat #88410), Anti-VSV-G (Abcam, Cat #ab1874), Anti-PKR (Cell Signalling Technology, Cat #3072S) and Anti-GAPDH (Goodhere Biological Technology, Cat #AB-M-M001).

Techniques: Western Blot, Transfection, Binding Assay, Quantitative RT-PCR, Knock-Out, Stable Transfection, Construct, Two Tailed Test

Journal: medRxiv

Article Title: Aspirin improves both reactivity and durability of type-I interferon signaling to achieve functional cure of chronic hepatitis B

doi: 10.1101/2024.06.14.24308555

Figure Lengend Snippet: (A and B) Western blot analysis of pY701-STAT1 in HEK293T cells pre-treated with Salicylate (100 μg/ml) (A) or Aspirin (100 μg/ml) (B) for 6 h and then treated with IFNα (1,000 IU/ml) as indicated. (C and D) 2fTGH cells transfected with F-G-IRF9 were pre-treated with phosphatase inhibitors (PPi) Cocktail A+B (1:1,000) (C) or PPi Cocktail A or B (1:1,000) (D) for 2 h. Then cells were treated with or without Salicylate (300 μg/ml) for 6 h. IP-IB was performed to analyze pan-pY of IRF9. (E) F-G-IRF9 proteins were co-immunoprecipitated by anti-Flag antibodies from HEK293T cells transfected with F-G-IRF9 (left). Mass spectrometry was used to analyze the potential IRF9-interacting proteins, among which the tyrosine kinases and phosphatases were shown (right). (F) IP-IB analysis of the interaction between the endogenous IRF9 and PTP1B in HEK293T cells. (G) IP-IB analysis of the interaction between F-G-IRF9 and endogenous PTP1B in HEK293T cells transfected with F-G-IRF9. (H) IP-IB analysis of pan-pY of F-G-IRF9 in HEK293T cells transfected with F-G-IRF9 and increased amounts of Myc-PTP1B. (I) IP-IB analysis of pan-pY of F-G-IRF9 in HEK293T cells transfected with F-G-IRF9 (WT, Y112F), together with or without Myc-PTP1B. Data are representative of three independent experiments (A-D, F-I).

Article Snippet: The antibodies in this study are listed here: Anti-IRF9 (Santa Cruz, Cat #sc-365893), Anti-pan-pY (Cell Signalling Technology, Cat #8954), Anti-Flag (Sigma, Cat #F7425), Anti-Tubulin (Proteintech, Cat #66031-1-Ig), Anti-GFP (Santa Cruz, Cat #sc-9996), Anti-p-STAT1(Y701) (Cell Signalling Technology, Cat #9167), Anti-STAT1 (Cell Signalling Technology, Cat #9176), Anti-PTP1B (Proteintech, Cat #11334-1-AP), Anti-β-Actin (Proteintech, Cat #66009), Anti-Myc (Abmart, Cat #M20002), Anti-HA (Abcam, Cat #ab9110), Anti-IFNAR1-PE (Invitrogen, Cat #MA5-23630), Anti-IFNAR1 (Sino Biological, Cat #13222-T20), Anti-β-Trcp (Cell Signalling Technology, Cat #11984),Anti-pan-AcK (Santa Cruz, Cat #sc-32268), Anti-Viperin (Abcam, Cat #107359), Anti-IFIT1 (Santa Cruz, Cat #sc-134948), Anti-HA (H1N1) (Sino Biological, Cat #11684-T56), Anti-p-STAT1 (S727) (Cell Signalling Technology, Cat #8826), Anti-p-STAT2 (Y690) (Cell Signalling Technology, Cat #88410), Anti-VSV-G (Abcam, Cat #ab1874), Anti-PKR (Cell Signalling Technology, Cat #3072S) and Anti-GAPDH (Goodhere Biological Technology, Cat #AB-M-M001).

Techniques: Western Blot, Transfection, Immunoprecipitation, Mass Spectrometry

Journal: medRxiv

Article Title: Aspirin improves both reactivity and durability of type-I interferon signaling to achieve functional cure of chronic hepatitis B

doi: 10.1101/2024.06.14.24308555

Figure Lengend Snippet: (A) IP-IB analysis of the interaction between IRF9 and PTP1B in HEK293T cells treated with Salicylate (300 μg/ml) for indicated times. (B) IP-IB analysis of the interaction between F-G-IRF9 and PTP1B in HEK293T cells transfected with F-G-IRF9 and treated with Salicylate (300 μg/ml) for 6 h. (C) IP-IB analysis of the IRF9-PTP1B interaction in HEK293T cells pre-treated with Salicylate (300 μg/ml) for 2 h and then treated with IFNα (1,000 IU/ml) for 6 h. (D) IP-IB analysis of pan-pY of IRF9 in HEK293T cells transfected with F-G-IRF9 and Myc-PTP1B, and then treated with or without Salicylate (300 μg/ml) for 6 h. (E) Western blot analysis of pY112-IRF9 in HEK293T cells transfected with Myc-PTP1B, and then treated with or without Salicylate (100 μg/ml) for 6 h. (F) The nanoDSF study of the effect of Salicylate on PTP1B thermal stability. The upper panel shows the fluorescence signal ratio change between 350 nm and 330 nm. The lower panel shows the derivative values. Vertical dashed lines show the melting temperatures. (G) Docking of Salicylate to PTP1B. PTP1B is shown as a cartoon and Salicylates are shown as sticks. The calculated binding energies and the equilibrium dissociation constants (Kd) are shown in the rectangle boxes. (H) Predicted hydrophilic interactions between Salicylate and PTP1B at its active pocket. All atoms are shown as balls and sticks, where carbon atoms are colored black, oxygen atoms are red and nitrogen atoms are blue. Chemical bonds are shown as blue or brown sticks. Predicted hydrogen bonds between Salicylate and PTP1B are shown as dashed lines in angstrom. (I) Predicted hydrophobic interactions between Salicylate and PTP1B at its active pocket. Salicylate is show as sticks and PTP1B residues interacting with Salicylate are shown as thin sticks including hydrogen atoms. Data are representative of three independent experiments (A-E).

Article Snippet: The antibodies in this study are listed here: Anti-IRF9 (Santa Cruz, Cat #sc-365893), Anti-pan-pY (Cell Signalling Technology, Cat #8954), Anti-Flag (Sigma, Cat #F7425), Anti-Tubulin (Proteintech, Cat #66031-1-Ig), Anti-GFP (Santa Cruz, Cat #sc-9996), Anti-p-STAT1(Y701) (Cell Signalling Technology, Cat #9167), Anti-STAT1 (Cell Signalling Technology, Cat #9176), Anti-PTP1B (Proteintech, Cat #11334-1-AP), Anti-β-Actin (Proteintech, Cat #66009), Anti-Myc (Abmart, Cat #M20002), Anti-HA (Abcam, Cat #ab9110), Anti-IFNAR1-PE (Invitrogen, Cat #MA5-23630), Anti-IFNAR1 (Sino Biological, Cat #13222-T20), Anti-β-Trcp (Cell Signalling Technology, Cat #11984),Anti-pan-AcK (Santa Cruz, Cat #sc-32268), Anti-Viperin (Abcam, Cat #107359), Anti-IFIT1 (Santa Cruz, Cat #sc-134948), Anti-HA (H1N1) (Sino Biological, Cat #11684-T56), Anti-p-STAT1 (S727) (Cell Signalling Technology, Cat #8826), Anti-p-STAT2 (Y690) (Cell Signalling Technology, Cat #88410), Anti-VSV-G (Abcam, Cat #ab1874), Anti-PKR (Cell Signalling Technology, Cat #3072S) and Anti-GAPDH (Goodhere Biological Technology, Cat #AB-M-M001).

Techniques: Transfection, Western Blot, Nano Differential Scanning Fluorimetry, Fluorescence, Binding Assay

Journal: medRxiv

Article Title: Aspirin improves both reactivity and durability of type-I interferon signaling to achieve functional cure of chronic hepatitis B

doi: 10.1101/2024.06.14.24308555

Figure Lengend Snippet: (A) Mice (n = 5) were injected intraperitoneally ( i.p. ) with PBS or Aspirin (50 μg per gram body mouse). Two hours after injection, mice were injected with mouse IFNβ (1,000 IU/g, i.p. ) and then mouse tissues were collected for further analysis 6 h after IFNβ injection. (B) RT-qPCR analysis of Ifit1 , Isg15 and Mx1 mRNA in mouse spleen tissues from (A). (C) RT-qPCR analysis of Ifit1 mRNA in mouse heart, liver, lung and kidney tissues from (A). (D and E) Western blot analysis of pY112-IRF9 (D) and IFNAR1 (E) in the livers of mice (n=4) injected with Aspirin and IFNβ as (A). (F) CHB patients were recruited and the IA (IFN-I + Aspirin) therapy was performed. Aspirin (0.2 g, qw) was taken orally before Peg-IFN injection and then Peg-IFN (180 μg, qw) was injected subcutaneously in the abdomen. The PBMCs from CHB patients were collected for analysis before injection and 6 h after injection. B: before injection. A: 6 h after injection. (G) Flow cytometry analysis of IFNAR1 in PBMCs of CHB patients from (F). (H) Western blot analysis of pY112-IRF9 in PBMCs of CHB patients from (F). (I) RT-qPCR analysis of Ifit1 mRNA in PBMCs of CHB patients from (F). NS, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed unpaired Student’s t -test). All graphs show the mean ± SEM for five individual mice (B, C). Data are shown as means ± SD of three biological replicates (G, I), or are representative of three independent experiments (D, E, H).

Article Snippet: The antibodies in this study are listed here: Anti-IRF9 (Santa Cruz, Cat #sc-365893), Anti-pan-pY (Cell Signalling Technology, Cat #8954), Anti-Flag (Sigma, Cat #F7425), Anti-Tubulin (Proteintech, Cat #66031-1-Ig), Anti-GFP (Santa Cruz, Cat #sc-9996), Anti-p-STAT1(Y701) (Cell Signalling Technology, Cat #9167), Anti-STAT1 (Cell Signalling Technology, Cat #9176), Anti-PTP1B (Proteintech, Cat #11334-1-AP), Anti-β-Actin (Proteintech, Cat #66009), Anti-Myc (Abmart, Cat #M20002), Anti-HA (Abcam, Cat #ab9110), Anti-IFNAR1-PE (Invitrogen, Cat #MA5-23630), Anti-IFNAR1 (Sino Biological, Cat #13222-T20), Anti-β-Trcp (Cell Signalling Technology, Cat #11984),Anti-pan-AcK (Santa Cruz, Cat #sc-32268), Anti-Viperin (Abcam, Cat #107359), Anti-IFIT1 (Santa Cruz, Cat #sc-134948), Anti-HA (H1N1) (Sino Biological, Cat #11684-T56), Anti-p-STAT1 (S727) (Cell Signalling Technology, Cat #8826), Anti-p-STAT2 (Y690) (Cell Signalling Technology, Cat #88410), Anti-VSV-G (Abcam, Cat #ab1874), Anti-PKR (Cell Signalling Technology, Cat #3072S) and Anti-GAPDH (Goodhere Biological Technology, Cat #AB-M-M001).

Techniques: Injection, Quantitative RT-PCR, Western Blot, Flow Cytometry, Two Tailed Test

Journal: medRxiv

Article Title: Aspirin improves both reactivity and durability of type-I interferon signaling to achieve functional cure of chronic hepatitis B

doi: 10.1101/2024.06.14.24308555

Figure Lengend Snippet: Aspirin and its metabolite Salicylate maintain IFNAR1 protein stability and IRF9 tyrosine phosphorylation, respectively, thus enhancing both the reactivity and durability of IFN-I signaling, which eventually promotes a functional cure of chronic HBV infection.

Article Snippet: The antibodies in this study are listed here: Anti-IRF9 (Santa Cruz, Cat #sc-365893), Anti-pan-pY (Cell Signalling Technology, Cat #8954), Anti-Flag (Sigma, Cat #F7425), Anti-Tubulin (Proteintech, Cat #66031-1-Ig), Anti-GFP (Santa Cruz, Cat #sc-9996), Anti-p-STAT1(Y701) (Cell Signalling Technology, Cat #9167), Anti-STAT1 (Cell Signalling Technology, Cat #9176), Anti-PTP1B (Proteintech, Cat #11334-1-AP), Anti-β-Actin (Proteintech, Cat #66009), Anti-Myc (Abmart, Cat #M20002), Anti-HA (Abcam, Cat #ab9110), Anti-IFNAR1-PE (Invitrogen, Cat #MA5-23630), Anti-IFNAR1 (Sino Biological, Cat #13222-T20), Anti-β-Trcp (Cell Signalling Technology, Cat #11984),Anti-pan-AcK (Santa Cruz, Cat #sc-32268), Anti-Viperin (Abcam, Cat #107359), Anti-IFIT1 (Santa Cruz, Cat #sc-134948), Anti-HA (H1N1) (Sino Biological, Cat #11684-T56), Anti-p-STAT1 (S727) (Cell Signalling Technology, Cat #8826), Anti-p-STAT2 (Y690) (Cell Signalling Technology, Cat #88410), Anti-VSV-G (Abcam, Cat #ab1874), Anti-PKR (Cell Signalling Technology, Cat #3072S) and Anti-GAPDH (Goodhere Biological Technology, Cat #AB-M-M001).

Techniques: Functional Assay, Infection