Journal: Cell

Article Title: Human MCTS1-dependent translation of JAK2 is essential for IFN-γ immunity to mycobacteria

doi: 10.1016/j.cell.2023.09.024

Figure Lengend Snippet: (A-D) Western blot for JAK2, MCTS1, DENR and vinculin on total lysate from (A) MCTS1 KO THP-1 clones (1 to 5) and wild-type (WT) THP-1 cells, (B) SV40-fibroblasts from P2 and a healthy control stably transduced with the indicated overexpression constructs, (C) HSV-T cells from P2, healthy controls and an IL-12Rβ1-deficient patient and (D) T-cell blasts from P2, P5 and healthy controls (C1, C2 and C3). The data shown are representative of three (A, C, D), or (B) two independent experiments.) (E) Quantification of JAK2 protein levels relative to a loading control from (A-D) (F) Gene set enrichment analysis of RNA-seq analysis of T-cell blasts from heathy controls ( n = 9), MCTS1-deficient ( n = 4), TYK2-deficient ( n = 1), IL-12Rβ1-deficient ( n = 1), IRAK4-deficient ( n = 1) and TYK2 P1104A-homozygous ( n = 2) patients were stimulated with IL-1β (24 h) and with IL-12 and IL-23 (6 h). Dot heatmaps are shown for the 10 gene sets for the healthy controls most strongly affected by stimulation with IL-23 + IL-1β relative to IL-1β alone. (G, I) Western blots of the indicated HEK-Blue cell lines stably transduced with the indicated lentiviral vectors. The data shown are representative of three biological replicates. (H, J) HEK-Blue colorimetric activity of the indicated cell lines stimulated with the indicated cytokines for 20 hours. Data are shown for three biological replicates and statistical significance was assessed in unpaired, two-tailed t -tests, * p <0.05, ** p <0.005. (K) The effect of gandotinib on the response to IL-23 + IL1-β or IL-12 in NK cells and to IFN-γ or IFN-α in monocytes. Summary plot, see – for details. The data shown are the means of six biological replicates performed on NK cells and monocytes from two heathy donors each.

Article Snippet: human TYK2 , Santa Cruz , Cat# sc-5271, RRID:AB_628419.

Techniques: Western Blot, Clone Assay, Control, Stable Transfection, Transduction, Over Expression, Construct, RNA Sequencing, Activity Assay, Two Tailed Test

Journal: Cell

Article Title: Human MCTS1-dependent translation of JAK2 is essential for IFN-γ immunity to mycobacteria

doi: 10.1016/j.cell.2023.09.024

Figure Lengend Snippet: Key resources table

Article Snippet: human TYK2 , Santa Cruz , Cat# sc-5271, RRID:AB_628419.

Techniques: Virus, Clinical Proteomics, Recombinant, Staining, Selection, Reverse Transcription, Polymerase Chain Reaction, Plasmid Preparation, Reporter Assay, Sequencing, Mutagenesis, Variant Assay, Cloning, Knock-Out, Software, Next-Generation Sequencing

Journal:

Article Title: Blocking of Interferon-Induced Jak-Stat Signaling by Japanese Encephalitis Virus NS5 through a Protein Tyrosine Phosphatase-Mediated Mechanism

doi: 10.1128/JVI.02714-05

Figure Lengend Snippet: Reduction of IFN-α-stimulated Stat1 and Tyk2 tyrosine phosphorylation in Vero cells infected with a recombinant Sindbis virus expressing JEV NS5. (A) Schematic representation of a recombinant SIN expressing JEV NS5 protein. The construct, including a respiratory syncytial virus (RSV) promoter-driving expression cassette, was able to transcribe and translate the SIN replicase complex from nonstructural genes (nsP1, nsP2, nsP3, and nsP4). The replicase produced then activates the subgenomic promoter (SP) to drive the expression of SIN structural genes and JEV NS5. (B) Vero cells were mock infected or infected with wt-SIN, recombinant NS5-SIN, or JEV (MOI = 10) for 6 h. The cells were then stimulated with IFN-αA/D (1,000 U/ml) for 30 min or left unstimulated. The cell lysates were harvested for Western blotting with anti-phospho-Stat1 (Tyr701), anti-Stat1, anti-Flag, and anti-JEV NS3 antibodies, as indicated on the right sides of the gels. (C) Vero cells were prepared as described for panel B except that cells were stimulated with IFN-αA/D for 15 min before harvesting for Western blotting with anti-phospho-Tyk2 (Tyr1054/1055), anti-Tyk2, anti-Flag, and anti-JEV NS3 antibodies as indicated.

Article Snippet: Rabbit polyclonal anti-Stat1 (#9172), anti-phospho-Stat1 (Tyr701) (#9171), and anti-phospho-Tyk2 (Tyr1054/1055) (#9321) antibodies were purchased from Cell Signaling Technology.

Techniques: Phospho-proteomics, Infection, Recombinant, Virus, Expressing, Construct, Produced, Western Blot

Journal:

Article Title: Blocking of Interferon-Induced Jak-Stat Signaling by Japanese Encephalitis Virus NS5 through a Protein Tyrosine Phosphatase-Mediated Mechanism

doi: 10.1128/JVI.02714-05

Figure Lengend Snippet: Treatment with sodium orthovanadate reverses the blocking effect of IFN-stimulated Stat1 and Tyk2 phosphorylation in JEV- and NS5-SIN-infected cells. Vero cells pretreated with or without sodium orthovanadate (25 μM) for 16 h were mock infected or infected with wt-SIN, NS5-SIN, or JEV (MOI = 10) in the presence or absence of sodium orthovanadate (25 μM) for 6 h as indicated above the lanes. The cells were then stimulated with IFN-αA/D (1,000 U/ml) for 30 min (or 15 min for Tyk2) or left unstimulated before the cell lysates were harvested for Western blot analyses using anti-phospho-Stat1 (Tyr701), anti-Stat1, anti-phospho-Tyk2 (Tyr1054/1055), anti-Tyk2, anti-Flag, and anti-JEV NS3 antibodies as shown on the right sides of the gels.

Article Snippet: Rabbit polyclonal anti-Stat1 (#9172), anti-phospho-Stat1 (Tyr701) (#9171), and anti-phospho-Tyk2 (Tyr1054/1055) (#9321) antibodies were purchased from Cell Signaling Technology.

Techniques: Blocking Assay, Phospho-proteomics, Infection, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Cirsiliol targets tyrosine kinase 2 to inhibit esophageal squamous cell carcinoma growth in vitro and in vivo

doi: 10.1186/s13046-021-01903-z

Figure Lengend Snippet: TYK2 expression is higher in ESCC and is negatively associated with patient survival. a Representative IHC staining images of ESCC tissue array using specific antibody for TYK2 in adjacent tissues and paired cancer tissues. Scale bar: 50 μm. b Analysis of TYK2 protein levels in ESCC tissue array according to staining results. TYK2 expression was valued as positivity. c Protein levels of TYK2 in ESCC tissue array based on clinical stages. d Survival rates of cancer patients with high or low protein levels of TYK2 in ESCC tumor microarray. Kaplan-Meier method was used to generate the survival curve. P < 0.05 was considered to be statistically significant. e The mRNA levels of TYK2 in different cancers based on the TCGA database. f The mRNA levels of TYK2 in ESCA (TCGA database) based on tumor histology. g The mRNA levels of TYK2 in ESCA (TCGA database) based on clinical stages. Due to some of the clinical stage information missing, the cases number in ( g ) is not the same with ( f ). Statistical analysis was performed using Student’s unpaired t -test in ( b , c , e , and f ); ANOVA in ( g ). Asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001) indicate a significant change. TPM, transcription per million

Article Snippet: Active TYK2 (Cat#T21-11G) and inactive STAT3 protein (Cat#S54-54BH) were bought from Signal Chem company (Richmond, BC, Canada).

Techniques: Expressing, Immunohistochemistry, Staining, Microarray

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Cirsiliol targets tyrosine kinase 2 to inhibit esophageal squamous cell carcinoma growth in vitro and in vivo

doi: 10.1186/s13046-021-01903-z

Figure Lengend Snippet: Cohort characteristics of esophageal cancer patients

Article Snippet: Active TYK2 (Cat#T21-11G) and inactive STAT3 protein (Cat#S54-54BH) were bought from Signal Chem company (Richmond, BC, Canada).

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Cirsiliol targets tyrosine kinase 2 to inhibit esophageal squamous cell carcinoma growth in vitro and in vivo

doi: 10.1186/s13046-021-01903-z

Figure Lengend Snippet: TYK2 knockdown suppresses ESCC growth. a Upper panel: TYK2 protein level in the normal esophagus and ESCC cell lines; lower panel: TYK2 knockdown results in KYSE140 and KYSE450 cells. b Left panel: KYSE140 cell viability; right panel: KYSE450 cell viability. c Left panel: the representative colony images of ESCC cell lines. Scale bar: 200 μm. Middle panel: Statistical analysis of the clone ratio in KYSE140 cells. Right panel: Statistical analysis of the clone ratio in KYSE450 cells. d The tumor pictures of the cell-derived tumor xenograft (CDX) mouse model. e The tumor growth status after transplanted TYK2 knockdown cells in the CDX mouse model. f The analysis of tumor weight of the CDX mouse. Student’s unpaired t -test was used in ( b , c , e , and f ). Asterisks (* p < 0.05, *** p < 0.001) indicate a significant change

Article Snippet: Active TYK2 (Cat#T21-11G) and inactive STAT3 protein (Cat#S54-54BH) were bought from Signal Chem company (Richmond, BC, Canada).

Techniques: Knockdown, Derivative Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Cirsiliol targets tyrosine kinase 2 to inhibit esophageal squamous cell carcinoma growth in vitro and in vivo

doi: 10.1186/s13046-021-01903-z

Figure Lengend Snippet: Overexpressed TYK2 increased ESCC growth. a Cell viability of KYSE30, KYSE410 and KYSE510 after transfection with TYK2. b The representative colony images and Statistical analysis of ESCC cell lines. Scale bar: 200 μm. c The change of downstream signaling after TYK2 knockdown in KYSE140 and KYSE450 cells. d The change of downstream signaling after TYK2 overexpressed in KYSE30, KYSE410 and KYSE510 cells. Student’s unpaired t -test was used in ( a and b ). Asterisks (* p < 0.05, ** p < 0.01) indicate a significant change

Article Snippet: Active TYK2 (Cat#T21-11G) and inactive STAT3 protein (Cat#S54-54BH) were bought from Signal Chem company (Richmond, BC, Canada).

Techniques: Transfection, Knockdown

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Cirsiliol targets tyrosine kinase 2 to inhibit esophageal squamous cell carcinoma growth in vitro and in vivo

doi: 10.1186/s13046-021-01903-z

Figure Lengend Snippet: Cirsiliol binds with TYK2 and inhibits the kinase activity. a Computational docking model between cirsiliol and TYK2. b The detailed binding site of cirsiliol on TYK2. c Upper panel: the binding ability of cirsiliol on endogenic TYK2 in vitro , obtained via pull down assay. Down panel: the binding ability of cirsiliol to recombinant TYK2 protein. d The change of affinity response intensity with the passage of time. -60 to 0 s was set as the time before inject cirsiliol solution; 0–120 s was set as contact time between cirsiliol and TYK2; 120–180 s was set as dissociation time. e The variation of response intensity with the increase of cirsiliol concentration. f Upper panel: cirsiliol binding ability with mutant TYK2. Down panel: the protein level of TYK2 in 293 T cell line. 1A (V981A), 1’A (P982A), 2A (double mutant), WT: wild type. g Kinase assay performed with cirsiliol and TYK2. h p-STAT3 inhibition analyzed by ImageJ in three independent assays ( n = 3, * p < 0.05, ** p < 0.01). Student’s unpaired t -test in ( h ). KD, dissociation constant

Article Snippet: Active TYK2 (Cat#T21-11G) and inactive STAT3 protein (Cat#S54-54BH) were bought from Signal Chem company (Richmond, BC, Canada).

Techniques: Activity Assay, Binding Assay, In Vitro, Pull Down Assay, Recombinant, Concentration Assay, Mutagenesis, Kinase Assay, Inhibition

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Cirsiliol targets tyrosine kinase 2 to inhibit esophageal squamous cell carcinoma growth in vitro and in vivo

doi: 10.1186/s13046-021-01903-z

Figure Lengend Snippet: Cirsiliol inhibits ESCC cell proliferation and the inhibiting effect is attenuated when TYK2 knockdown. a MTT assay showed that cirsiliol suppresses KYSE140 (left panel) and KYSE450 (right panel) cell viability. b Colony forming assay. Upper panel: the representative clone pictures of KYSE450 and KYSE140, scale bar: 200 µm; lower panel: Statistical analysis of the colony ratios of both cell lines after cirsiliol treatment. c Cell cycle arrest at the G2/M phase after cirsiliol treatment. Left panel: KYSE140 cells; right panel: KYSE450 cells. d The proliferation inhibiting ability of cirsiliol change after TYK2 knockdown (three independent groups were set up in each cell line and the data was analyzed between vehicle and treatment in each group separately). Upper panel: KYSE140 cells; lower panel: KYSE450 cells. Scale bar: 200 µm. e Representative colony pictures after TYK2 silencing with or without cirsiliol treatment. f Analysis of the colony number. Cell colonies were analyzed using Image-Pro Plus v6.0. g Cell viability after rescue TYK2 protein level in KYSE140 and KYSE450 TYK2 knockdown cell lines. h The change of TYK2 protein level and phosphorylation STAT3 after rescue TYK2 in KYSE140 and KYSE450. Student’s unpaired t -test was used in ( a , b , c , f , g ) and ANOVA in ( d ). Asterisks (** p < 0.01, *** p < 0.001) indicate a significant change

Article Snippet: Active TYK2 (Cat#T21-11G) and inactive STAT3 protein (Cat#S54-54BH) were bought from Signal Chem company (Richmond, BC, Canada).

Techniques: Knockdown, MTT Assay, Phospho-proteomics

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Cirsiliol targets tyrosine kinase 2 to inhibit esophageal squamous cell carcinoma growth in vitro and in vivo

doi: 10.1186/s13046-021-01903-z

Figure Lengend Snippet: Cirsiliol inhibits ESCC through TYK2-STAT3 signaling pathways. a Immunofluorescence staining of KYSE140 and KYSE450: cells were treated for 24 h, and then stained for p-STAT3 (Tyr705) and STAT3 (100× magnification). b The analysis of p-STAT3 fluorescence intensity in KYSE140 and KYSE450 cells. Analyzed by student’s unpaired t -test (* p < 0.05, ** p < 0.01). c The change of STAT3 dimer formation after treated by cirsiliol in KYSE140 and KYSE450 cells. d The Nucleus localization variation of STAT3 after treated with 20 μM cirsiliol in KYSE140 and KYSE450 cells. e The effects of cirsiliol on the TYK2-related signal pathway in KYSE140 and KYSE450 cells. IOD, Integrated Optical Density

Article Snippet: Active TYK2 (Cat#T21-11G) and inactive STAT3 protein (Cat#S54-54BH) were bought from Signal Chem company (Richmond, BC, Canada).

Techniques: Protein-Protein interactions, Immunofluorescence, Staining, Fluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Cirsiliol targets tyrosine kinase 2 to inhibit esophageal squamous cell carcinoma growth in vitro and in vivo

doi: 10.1186/s13046-021-01903-z

Figure Lengend Snippet: Cirsiliol inhibits ESCC patient-derived xenograft (PDX) tumor growth in vivo. a The information of two PDX cases. b The protein levels of TYK2 in different PDX cases. c The change of average tumor volume in different group of LEG73 and LEG104 cases after cirsiliol treatment (* p < 0.05) and tumor images of different groups after sacrifice. d Tumor weight analysis in different groups of LEG73 and LEG104 cases after cirsiliol treatment and the tumor growth inhibition of cirsiliol compared with the average tumor weight of the vehicle group (* p < 0.05, ** p < 0.01). e Upper panel: Representative IHC images of LEG73 and LEG104 tumor tissue slices (100 × magnifications), tumor tissues were stained with p-STAT3 and anti-Ki67; lower panel: Statistical analysis of IHC positive staining of p-STAT3 and Ki67 in both LEG73 and LEG104 cases (* p < 0.05, ** p < 0.01, *** p < 0.001). f Schematic diagram of the signaling pathway of esophageal cancer proliferation inhibited by cirsiliol. Data for each treatment group were compared to the control group and statistical significance was analyzed by student’s unpaired t -test

Article Snippet: Active TYK2 (Cat#T21-11G) and inactive STAT3 protein (Cat#S54-54BH) were bought from Signal Chem company (Richmond, BC, Canada).

Techniques: Derivative Assay, In Vivo, Inhibition, Staining, Control

Journal: iScience

Article Title: Schlafen 11 expression in human acute leukemia cells with gain-of-function mutations in the interferon-JAK signaling pathway

doi: 10.1016/j.isci.2021.103173

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-phospho (Y1054/1055)-TYK2 (D7T8A) , Cell signaling Technology , Cat# 68790S; RRID: AB_2799752.

Techniques: Recombinant, Reverse Transcription, SYBR Green Assay, Protease Inhibitor, Knock-Out, Software, Real-time Polymerase Chain Reaction