Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: VEGF-induced vascular permeability is reduced upon CRISPR/Cas9-mediated knockout of Stat3 in zebrafish. (A) VEGF-inducible zebrafish were crossed to Stat3 +/− (heterozygous) zebrafish to generate VEGF-inducible; Stat3 +/− double transgenic fish, which were intercrossed to generate VEGF-inducible; Stat3 −/− (KO) zebrafish. (B) CRISPR/Cas9-generated Stat3 KO zebrafish (bottom) display no overt vascular defects relative to wild-type (WT) zebrafish (top). The vascular system of 3 days post-fertilization (dpf) zebrafish was visualized by microangiography with 2000 kDa FITC-dextran. Representative images of at least three zebrafish per group are shown. Scale bars: 100 μm. (C) Microangiography using 70 kDa Texas Red-dextran permeabilizing tracer (red) and 2000 kDa FITC-dextran intersegmental vessel marker (green) was performed on 3 dpf Stat3 +/+ (negative controls without VEGF induction; left) , VEGF-induced, Stat3 +/+ (middle) and VEGF-induced, Stat3 −/− (right) zebrafish. Representative images shown were obtained using a Zeiss Apotome 2 microscope with a Fluar 5×/0.25 NA lens at room temperature (RT). Scale bars: 50 μm. (D) Quantitative analysis of vascular permeability upon VEGF stimulation in WT Stat3 +/+ ( n =30) and KO Stat3 −/− ( n =9) zebrafish. Mean±s.e.m., unpaired, two-tailed Student's t -test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Permeability, CRISPR, Knock-Out, Transgenic Assay, Generated, Marker, Microscopy, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: Endothelial cell-specific STAT3 knockout mice exhibit decreased VEGF-induced permeability. (A) Images of footpads from WT and endothelial cell-specific STAT3 knockout (STAT3 ECKO ) mice following tail vein injection with 1% Evans Blue dye and human recombinant VEGF-165 protein (2.5 µg/ml; left footpads) or PBS vehicle (right footpads) being injected into the root of the footpad. (B,C) Quantitation of Evans Blue leakage in Tie2-Cre negative; STAT3 flox/flox (WT) and Tie2-Cre positive; STAT3 flox/flox (STAT3 ECKO ) mice. n =7 mice in WT group and n =6 mice in STAT3 ECKO group. Each mouse was injected with PBS on the right anterior and posterior footpads and VEGF on the left anterior and posterior footpads. Multiple biological replicates were performed and depicted findings are representative. Mean±s.e.m., one-way ANOVA followed by Bonferroni test. A.U., arbitrary units.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Knock-Out, Permeability, Injection, Recombinant, Quantitation Assay

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: Pharmacological inhibition of STAT3 stabilizes endothelial barrier integrity following VEGF stimulation in human endothelial cells. (A) Serum-starved human umbilical vein endothelial cells (HUVECs) were pretreated with DMSO (vehicle control) for 1 h, 30 µM AQ for 4 h, or 10 µM PYR for 1 h prior to VEGF (25 ng/ml) stimulation for 0, 2 or 5 min. Lysates were immunoblotted. Densitometry was performed, and the values below the rows of bands represent the ratio of phosphorylated protein to respective total protein. (B) Human VEGF-165 recombinant protein (VEGF; 25 ng/ml) stimulation of HUVECs promotes ZO-1 (green) disorganization at endothelial cell junctions (yellow arrows; left column; DMSO vehicle control pretreatment for 1 h prior to VEGF stimulation). ZO-1 organization is maintained upon pretreatment with 30 μM AQ for 4 h (magenta arrows; middle column) or 10 μM PYR for 1 h (magenta arrows; right column) prior to VEGF stimulation. Nuclei were stained with DAPI (blue). (C) Serum-starved human pulmonary artery endothelial cells (HPAECs) were pretreated with 10 µM PYR for 1 h prior to VEGF (25 ng/ml) stimulation for 0, 5 or 30 min. VEGF stimulation promotes disorganization of ZO-1 (green) at endothelial cell junctions (yellow arrows). ZO-1 organization is maintained when HPAECs were pretreated with PYR (magenta arrows). Nuclei were stained with DAPI (blue). (D) VEGF (25 ng/ml) stimulation of human lung microvascular endothelial cells (HMVEC-Ls) promotes ZO-1 (green) disorganization at endothelial cell junctions (yellow arrows). ZO-1 organization is maintained upon pretreatment with 20 μM PYR for 6 h prior to VEGF stimulation (magenta arrows). Nuclei were stained with DAPI (blue). At least two biological replicates were performed for each experiment depicted in A-D. Scale bars: 20 µm.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Inhibition, Recombinant, Staining

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: Suppression of STAT3 activity by pyrimethamine (PYR) inhibits VEGF-induced vascular permeability in zebrafish and mice. (A) Microangiography using 70 kDa Texas Red-dextran permeabilizing tracer (red) and 2000 kDa FITC-dextran intersegmental vessel marker (green) was performed on 3 dpf zebrafish without induced VEGF pretreated with DMSO ( n =6) or 25 μM PYR ( n =5) or 3 dpf zebrafish with induced VEGF pretreated with DMSO ( n =4) or 25 μM PYR ( n =9) for 3 days. Representative images shown were obtained using a Zeiss Apotome 2 microscope with a Fluar 5×/0.25 NA lens at RT. Scale bars: 50 μm. (B) The quantitative analysis of vascular permeability without VEGF stimulation or upon VEGF stimulation in zebrafish pretreated with DMSO or PYR. Mean±s.e.m., one-way ANOVA followed by Bonferroni test. (C) Representative images of footpads from mice treated with vehicle or PYR following tail vein injection with 1% Evans Blue and footpad injection of VEGF (2.5 μg/ml) or PBS vehicle. (D) Quantitation of Evans Blue dye leakage in C57BL/6 WT mice treated with vehicle or PYR. n =9 mice in the vehicle group and n =7 mice in the PYR group. Each mouse was injected with PBS in the right posterior footpad and VEGF in the left posterior footpad. Multiple biological replicates were performed and depicted findings are representative. Mean±s.e.m., one-way ANOVA followed by Bonferroni test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Activity Assay, Permeability, Marker, Microscopy, Injection, Quantitation Assay

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: JAK2 phosphorylates STAT3 to transduce VEGF/VEGFR-2 signaling and promote vascular permeability. (A) To perform a STAT3 GST pull-down of VEGFR-2 and JAK2, lysates of HUVECs stimulated with serum for 30 min were used as prey. GST fusion protein STAT3 expressed in 293F cells was used as bait. GST alone served as a negative control. Binding experiments were analyzed by SDS-PAGE and visualized by immunoblotting. GST-STAT3 and GST were each detected using an anti-GST antibody. Three biological replicates were performed and depicted findings are representative. (B) JAK2 phosphorylates STAT3 in vitro . In vitro kinase assays were performed using purified human STAT3 protein and kinase active JAK2 protein. The results shown here are representative of two independent experiments. (C) Representative images of footpads from C57BL/6 WT mice treated with vehicle or JAK2 inhibitor AG490. Following tail vein injection with 1% Evans Blue dye, human VEGF-165 protein (2.5 μg/ml) or PBS vehicle was injected into the root of the footpad. After 30 min, the mice were euthanized and the footpads were excised. (D) Quantitation of Evans Blue dye leakage in C57BL/6 mice treated with vehicle or AG490. n =4 mice per group. Each mouse was injected with PBS in the right posterior footpad and VEGF in the left posterior footpad. Two biological replicates were performed and depicted findings are representative. Mean±s.e.m., one-way ANOVA followed by Bonferroni test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Transduction, Permeability, Negative Control, Binding Assay, SDS Page, Western Blot, In Vitro, Purification, Injection, Quantitation Assay

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: STAT3 transcriptionally activates ICAM-1, a cell adhesion molecule that promotes vascular permeability. (A) Top: the pGL3-ICAM1-WT plasmid containing the human ICAM-1 promoter with a STAT3 binding site located at −115 to −107 bp. Bottom: the pGL3-ICAM1-SDM plasmid with a site-directed mutation (SDM) in the STAT3 binding site as indicated. (B) Dual luciferase assays were performed in HUVECs that were transfected with pGL3-ICAM1-WT or pGL3-ICAM1-SDM and empty vector or constitutively active STAT3. Firefly and Renilla luminescence was measured and plotted as a ratio. Mean±s.e.m., one-way ANOVA followed by Bonferroni test. n =9 technical replicates. Depicted findings are representative of three independent experiments. (C) HUVECs that had been stably transduced with lentivirus encoding STAT3-specific shRNA or control shRNA were stimulated with human VEGF-165 protein (25 ng/ml) and the lysates were immunoblotted for ICAM1, p-STAT3 (Y705) and total STAT3. Depicted data are representative of three biological replicates. (D) RNA was harvested from VEGF; Stat3 +/+ or VEGF; Stat3 −/− 3 dpf embryos for quantitative PCR. stat3 transcripts are reduced in VEGF; Stat3 −/− ( n =5) compared to VEGF; Stat3 +/+ zebrafish ( n =7). Mean±s.e.m., unpaired, two-tailed Student's t -test. (E) The expression of icam-1 was assessed by real-time quantitative PCR using RNA derived from each zebrafish embryo in the absence of VEGF induction (Stat3 +/+ , n =3; Stat3 −/− , n =2) or 8 h following VEGF induction (Stat3 +/+ , n =4; Stat3 −/− , n =3) in the heat-inducible VEGF; Stat3 mutant zebrafish. Mean±s.e.m., one-way ANOVA followed by Bonferroni test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Permeability, Plasmid Preparation, Binding Assay, Mutagenesis, Luciferase, Transfection, Stable Transfection, Transduction, shRNA, Real-time Polymerase Chain Reaction, Two Tailed Test, Expressing, Derivative Assay

Journal: PLoS ONE

Article Title: Acyloxy Nitroso Compounds Inhibit LIF Signaling in Endothelial Cells and Cardiac Myocytes: Evidence That STAT3 Signaling Is Redox-Sensitive

doi: 10.1371/journal.pone.0043313

Figure Lengend Snippet: HMEC-1 were pretreated for 1 h with vehicle (0.04% v/v DMSO), (A) 100 µM NCP, or (C) 100 µM NCP. Afterwards, cells were dosed for various times with 2 ng/mL LIF. Western immunoblots of cell lysates were probed for STAT3 Y705 phosphorylation and STAT3 as a loading control. (B and D) Results were quantified and expressed as the ratio of phosphorylated STAT3 to total STAT3. **P<0.01 and ***P<0.001 vs. same time point control (n = 4); 2-way ANOVA and Bonferroni post-test.

Article Snippet: Fluorescein-5-maleimide was from Pierce Biotechnology (Rockford, lL USA) and recombinant human STAT3 was from OriGene Technologies (Rockville, MD).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Acyloxy Nitroso Compounds Inhibit LIF Signaling in Endothelial Cells and Cardiac Myocytes: Evidence That STAT3 Signaling Is Redox-Sensitive

doi: 10.1371/journal.pone.0043313

Figure Lengend Snippet: Neonatal rat ventricular myocytes (A & B) were pretreated for 1 h with 100 µM NCP (lanes 5–8) or vehicle (0.04% v/v DMSO; lanes 1–4). Cells were then dosed with 2 ng/mL LIF for various times. Western immunoblots of cell lysates were probed for STAT3 Y705 phosphorylation and STAT3. (A) Representative immunoblot of 4 independent experiments. (B) Compiled data analysis. Adult mouse cardiac myocytes (C & D) were pretreated for 1 h with 500 µM NCP (+) or vehicle (−). Cells were then dosed with 2 ng/mL LIF for 0, 5, or 15 min. (C) Representative immunoblot of 3 independent experiments. (D) Compiled data analysis. **P<0.01 or ***P<0.001 vs. same time point control; 2-way ANOVA and Bonferroni post-test (n = 3).

Article Snippet: Fluorescein-5-maleimide was from Pierce Biotechnology (Rockford, lL USA) and recombinant human STAT3 was from OriGene Technologies (Rockville, MD).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Acyloxy Nitroso Compounds Inhibit LIF Signaling in Endothelial Cells and Cardiac Myocytes: Evidence That STAT3 Signaling Is Redox-Sensitive

doi: 10.1371/journal.pone.0043313

Figure Lengend Snippet: HMEC-1 were pretreated (A) for 1 h with various doses (0−100 µM) of NCA or NCP and the same amount of vehicle (0.04% v/v DMSO) or (B) for 30 min with 0−500 µM Angeli’s salt and the same amount of vehicle (50 µM NaOH). Cells were treated for 15 min with 2 ng/mL LIF. Western immunoblots of cell lysates were probed for STAT3 Y705 phosphorylation and STAT3. Results represent 2 independent experiments for both NCA and NCP and a single experiment for Angeli’s salt.

Article Snippet: Fluorescein-5-maleimide was from Pierce Biotechnology (Rockford, lL USA) and recombinant human STAT3 was from OriGene Technologies (Rockville, MD).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Acyloxy Nitroso Compounds Inhibit LIF Signaling in Endothelial Cells and Cardiac Myocytes: Evidence That STAT3 Signaling Is Redox-Sensitive

doi: 10.1371/journal.pone.0043313

Figure Lengend Snippet: (A) NCA and NCP block thiolate labeling. Recombinant human STAT3 was treated with vehicle (DMSO), NCA (100 µM) or NCP (100 µM) for 1 h at room temperature and then labeled for 2 h with fluorescein-5-maleimide. Equal amounts of protein were separated by SDS-PAGE and fluorescence in the gel detected (upper panel). To ensure equal loading, Western analysis was done on each fluorescein-labeled sample. Separated proteins on nitrocellulose membranes were probed with a STAT3 antibody and imunoreactive bands quantified using the Li-COR Odyssey infrared imaging system (lower panel). Results shown are representative of 3 independent experiments. (B & C) Oxidation of STAT3 is associated with sulfenic acid formation. Purified recombinant STAT3 was immunoprecipitated and pretreated with 10 mM DTT and then treated with nothing or the oxidant o -IBZ (2.5 mM) for 1 hr at 4°C. Immunoprecipitates were processed as described under “ " to determine sulfenic acid formation (STAT3-SOH). (B) Representative blot. (C) Levels of cysteine-sulfenic acid and STAT3 were quantified by the Li-COR Odyssey Detection System. Treatment with o -IBZ resulted in a significant increase in relative sulfenic acid content. **P<0.01 vs. control, n = 3; paired Student’s t-test.

Article Snippet: Fluorescein-5-maleimide was from Pierce Biotechnology (Rockford, lL USA) and recombinant human STAT3 was from OriGene Technologies (Rockville, MD).

Techniques: Blocking Assay, Labeling, Recombinant, SDS Page, Fluorescence, Western Blot, Imaging, Purification, Immunoprecipitation

Journal: PLoS ONE

Article Title: Acyloxy Nitroso Compounds Inhibit LIF Signaling in Endothelial Cells and Cardiac Myocytes: Evidence That STAT3 Signaling Is Redox-Sensitive

doi: 10.1371/journal.pone.0043313

Figure Lengend Snippet: HL-1 cells were treated for 30 min with vehicle (control), 500 µM NCP, 1 mM diamide, or 500 µM NCP and 1 mM diamide together. Cell extracts were prepared. (A) Equal protein amounts of cleared extracts were added to non-reducing Laemmli’s SDS-sample buffer and subjected to SDS-PAGE. Blots were probed for total STAT3 and glutathionylated protein using a rabbit and mouse antibody, respectively. Immunoreactive bands were detected using Li-COR Odyssey system and secondary antibodies that produced a red (anti-rabbit) or green (anti-mouse) signal. The overlay of the red and green signals produced an orange color. Relative levels of glutathionylated STAT3 were quantified. **P<0.01, 1-way ANOVA and Dunnett’s multiple comparison test (n = 3). (B) Cells were treated as in panel A. Cell extracts were added to non-reducing Laemmli’s SDS-sample buffer and subjected to SDS-PAGE. Blots were probed for total STAT3, which showed two bands consistent with STAT3 monomers and dimers. The intensity of the higher (dimer) band relative to the lower (monomer) band for each lane was quantified. *P<0.05 and **P<0.01, 1-way ANOVA and Newman–Keuls post-test (n = 3).

Article Snippet: Fluorescein-5-maleimide was from Pierce Biotechnology (Rockford, lL USA) and recombinant human STAT3 was from OriGene Technologies (Rockville, MD).

Techniques: SDS Page, Produced

Journal: PLoS ONE

Article Title: Acyloxy Nitroso Compounds Inhibit LIF Signaling in Endothelial Cells and Cardiac Myocytes: Evidence That STAT3 Signaling Is Redox-Sensitive

doi: 10.1371/journal.pone.0043313

Figure Lengend Snippet: (A & B) Aliquots of a cleared mouse heart homogenate were incubated for 30 min with vehicle, 500 µM NCP, 1 mM diamide, or 500 µM NCP+1 mM diamide. Samples were processed for SDS-PAGE and Western blot analysis in nonreducing or reducing sample buffer. (A) Membranes were probed for STAT3 using the Li-COR Odyssey detection system. (B) Intensity of the STAT3 band in the nonreduced sample was normalized to the intensity of the band after reduction. ***P<0.001 vs. Control, 1-way ANOVA and Newman–Keuls post-test (n = 3 mouse hearts). (C) Ratio of nonreduced to reduced STAT3 in wild type (WT) and failing (Gaq) mouse hearts. STAT3 levels in mouse myocardial tissue from WT (FVB/N) and heart failure mice (Gaq overexpressing) (n = 3) were determined via immunoblot analysis under nonreducing or reducing (3.75% β-mercaptoethanol (β-ME)) conditions. Protein loads were normalized using the direct blue 71 stained membranes (DB71). *P<0.05 (Student t-test).

Article Snippet: Fluorescein-5-maleimide was from Pierce Biotechnology (Rockford, lL USA) and recombinant human STAT3 was from OriGene Technologies (Rockville, MD).

Techniques: Incubation, SDS Page, Western Blot, Staining

Journal: Advanced Science

Article Title: Targeting C21orf58 is a Novel Treatment Strategy of Hepatocellular Carcinoma by Disrupting the Formation of JAK2/C21orf58/STAT3 Complex

doi: 10.1002/advs.202306623

Figure Lengend Snippet: C21orf58 accelerated cell cycle of HCC cells and increased the expression of phosphorylated STAT3. A,B) The effects of C21orf58 overexpression and knockdown on cell cycle distribution of HCC cells. C,D) The gene set enrichment analysis (GSEA) plot of IL6‐JAK‐STAT3 signaling pathway based on the RNA seq data from control and C21orf58 knockdown HCC cells (shC21orf58‐1 and shC21orf58‐2, n = 3 per group). NES, normalized enrichment score. E,F) STAT3 and p‐STAT3(Y705) protein levels in C21orf58 overexpression and knockdown HCC cells. G) The expression levels of C21orf58 and p‐STAT3(Y705) proteins in paired clinical HCC tissues ( n = 12). The positive correlation between C21orf58 and p‐STAT3(Y705) expression was assessed by linear regression. All * P <0.05, ** P <0.01, *** P <0.001. Scr: Scramble.

Article Snippet: Recombinant human STAT3 GST (N‐Term) protein was purchased from Novus Biologicals (H00006774‐P01).

Techniques: Expressing, Over Expression, Knockdown, RNA Sequencing, Control

Journal: Advanced Science

Article Title: Targeting C21orf58 is a Novel Treatment Strategy of Hepatocellular Carcinoma by Disrupting the Formation of JAK2/C21orf58/STAT3 Complex

doi: 10.1002/advs.202306623

Figure Lengend Snippet: C21orf58 simultaneously interacted with JAK2 and STAT3 to form a ternary complex. A) The exogenous interaction between C21orf58 and STAT3 in HepG2 cells. B) Truncations of STAT3 were constructed as shown in graphic, NTD: N‐terminal domain; CCD: coiled‐coil domain; DBD: DNA‐binding domain; LD: linker domain; SH2: SH2 domain; TAD: transactivation domain. C) The interaction between C21orf58 and NTD domain of STAT3 was validated by immunoprecipitation. D) The exogenous interaction between C21orf58 and JAK2 in HepG2 cells. E) Truncations of JAK2 were constructed as shown in graphic. F) Interaction domains between JAK2 and C21orf58 was detected by immunoprecipitation, SH2 domain was the binding region of C21orf58 on JAK2. G) Co‐immunoprecipitation showed that C21orf58 simultaneously interacted with JAK2 and STAT3 in HepG2 cells. H) In vitro pull‐down assay was performed to conform that C21orf58 formed a ternary complex with JAK2 and STAT3 in HCC cells by direct interaction.

Article Snippet: Recombinant human STAT3 GST (N‐Term) protein was purchased from Novus Biologicals (H00006774‐P01).

Techniques: Construct, Binding Assay, Immunoprecipitation, In Vitro, Pull Down Assay

Journal: Advanced Science

Article Title: Targeting C21orf58 is a Novel Treatment Strategy of Hepatocellular Carcinoma by Disrupting the Formation of JAK2/C21orf58/STAT3 Complex

doi: 10.1002/advs.202306623

Figure Lengend Snippet: C21orf58 facilitated the activity of wildtype and constitutively mutated STAT3 by forming ternary complex. A) C21orf58 overexpression promoted the interaction of JAK2 on STAT3. B) Attenuated C21orf58 expression decreased the interaction of JAK2 on STAT3. C) In vitro kinase activity assay was performed to verify that C21orf58 promoted the phosphorylation of STAT3 by JAK2. D) After kinase activity assay, proteins were examined by western blot and detected that C21orf58 improved the phosphorylation of STAT3 by JAK2. E) C21orf58 improved the interaction between JAK2 and constitutively activated mutants of STAT3. F) Reduction of C21orf58 expression remarkably declined the phosphorylation of constitutively mutated STAT3. G,H) Downregulation of C21orf58 effectively reduced the interaction of JAK2 on constitutively activated mutants of STAT3.

Article Snippet: Recombinant human STAT3 GST (N‐Term) protein was purchased from Novus Biologicals (H00006774‐P01).

Techniques: Activity Assay, Over Expression, Expressing, In Vitro, Kinase Assay, Phospho-proteomics, Western Blot

Journal: Advanced Science

Article Title: Targeting C21orf58 is a Novel Treatment Strategy of Hepatocellular Carcinoma by Disrupting the Formation of JAK2/C21orf58/STAT3 Complex

doi: 10.1002/advs.202306623

Figure Lengend Snippet: C21orf58 promoted sorafenib resistance of HCC cells. A) C21orf58 elevated the IC50 value of HepG2 cells. B,C) The clone formation of C21orf58 overexpressed and knockdown HCC cells treated with sorafenib at different concentrations. D) Construction of sorafenib‐resistant Huh7 cells, which were not vulnerable to sorafenib compared with their parental cells. IC50 was the 50% inhibiting concentration. E) The expression of C21orf58 and p‐STAT3(Y705) were increased in sorafenib‐resistant and Huh7 cells. F) Inhibition of C21orf58 expression using siRNA was effectively to repress the cell growth of HCC cells with sorafenib resistance. G,H) The growth curve, volume and weight of tumors derived from sorafenib‐resistant Huh7 cells were suppressed by siC21orf58, tumors treated with siC21orf58 (5 nmol) twice a week. I) After treating with siC21orf58 and negative control siRNA respectively, the expression of p‐STAT3(Y705), STAT3 and C21orf58 proteins in sorafenib‐resistant Huh7‐derived tumors were detected by western blot. siRNA processing condition: tumors were treated with siC21orf58 (5 nmol) or negative control siRNA twice a week. J) Immunohistochemistry was performed to investigate the expression of C21orf58 and Ki‐67 proteins. Scale bar = 100 µm. All *** P <0.001. siNC: negative control siRNA.

Article Snippet: Recombinant human STAT3 GST (N‐Term) protein was purchased from Novus Biologicals (H00006774‐P01).

Techniques: Knockdown, Concentration Assay, Expressing, Inhibition, Derivative Assay, Negative Control, Western Blot, Immunohistochemistry

Journal: Advanced Science

Article Title: Targeting C21orf58 is a Novel Treatment Strategy of Hepatocellular Carcinoma by Disrupting the Formation of JAK2/C21orf58/STAT3 Complex

doi: 10.1002/advs.202306623

Figure Lengend Snippet: Alminoprofen, a ligand of C21orf58, displayed a promising potential in HCC therapy. A) A 2D hydrogen bond (green dash line) bound the alminoprofen to the amino acid residues of C21orf58. B) 3D model of C21orf58's optimal binding mechanism in the protein pocket (alminoprofen depicted as colored sticks). C) Amino acid residues of C21orf58 interacting with the alminoprofen in 3D (color sticks). D) The inhibitory effect of alminoprofen on cell viability of HepG2 and Huh7 cells by CCK8 assay. E) The effect of alminoprofen on expression of p‐STAT3 and p‐JAK2 proteins in HepG2 and Huh7 cells was examined by western blot. F) Alminoprofen showed a block on ATP consumption mediated by C21orf58 via kinase activity assay in vitro. G) After kinase reaction, the level of p‐STAT3 was investigated by western blot. H) The growth curve of Huh7‐derived tumors treated with alminoprofen (50 mg kg −1 , n = 5) or vehicle ( n = 5). I,J) The picture and weight statistics of tumors treated with alminoprofen (50 mg kg −1 ) or vehicle, P = 0.0022. K) The effect of alminoprofen on the levels of p‐STAT3, p‐JAK2 and C21orf58 proteins were examined by western blot in tumors treated with alminoprofen (50 mg kg −1 ) or vehicle. All ** P < 0.01, *** P < 0.001, ns: not significant.

Article Snippet: Recombinant human STAT3 GST (N‐Term) protein was purchased from Novus Biologicals (H00006774‐P01).

Techniques: Binding Assay, CCK-8 Assay, Expressing, Western Blot, Blocking Assay, Kinase Assay, In Vitro, Derivative Assay

Journal: Advanced Science

Article Title: Targeting C21orf58 is a Novel Treatment Strategy of Hepatocellular Carcinoma by Disrupting the Formation of JAK2/C21orf58/STAT3 Complex

doi: 10.1002/advs.202306623

Figure Lengend Snippet: Schematic representation of the molecular mechanism that C21orf58 played oncogenic adaptor role on promoting cell growth and sorafenib resistance by activating STAT3 cascades in HCC cells with wild‐type STAT3 or constitutively mutated STAT3.

Article Snippet: Recombinant human STAT3 GST (N‐Term) protein was purchased from Novus Biologicals (H00006774‐P01).

Techniques:

Journal: Cells, tissues, organs

Article Title: The role of epithelial Stat3 in amelogenesis during mouse incisor renewal

doi: 10.1159/000486745

Figure Lengend Snippet: (A) Illustration of the adult mouse hemi-mandible showing the incisor and molars, as well as the mineralized dentin and enamel in the incisor. (A’) The proximal region of the incisor denoting the labial and lingual cervical loop (laCL and liCL, respectively, highlighted by dashed, red lines). (A’’) Magnified illustration of the laCL indicating the outer enamel epithelium (OEE), inner enamel epithelium (IEE), transit amplifying (TA) region, and stellate reticulum (SR). (B–I) Immunofluorescence staining for STAT3 in wildtype mouse teeth (sagittal views) at E14.5, E16.5, P1, and 6 weeks showed the presence of STAT3 in developing incisors and molars, and adult incisors. The epithelial component (i.e., cells that ultimately generate enamel) of developing teeth are outlined (dotted red line) with the exception of 6-week old molar where the entire tooth is outlined (I). (J,K’) H&E histological staining of control (Stat3fl/fl) and mutant (Krt14Cre;Stat3fl/fl) adult mice mandibular incisors showed little or no enamel matrix in mutants compared to controls.

Article Snippet: Primary antibodies used were as follows: anti-rabbit STAT3 (1:200; Cat#7197, ProSci, used to detect STAT3 expression in wildtype specimens), anti-rabbit STAT3 (Phospho-Stat3 (Tyr705) (D3A7), Cat#9145S, Cell signaling, used to detect STAT3 expression in Stat3 fl/fl and K rt14 Cre/+ ; Stat3 fl/fl mice), anti-rabbit AMELX (amelogenin; 1:200; Cat#ab54507, Abcam), anti-rabbit AMBN (ameloblastin; 1:200; Cat#ab116347, Abcam), anti-kallikrein-4 or KLK4 (1:200; Abcam, Cat#ab71234, Abcam), enamelin or ENAM (1:200; Cat#sc-33107, Santa Cruz), and KI67 (1:200, Cat#RM-9106, Thermo Scientific).

Techniques: Immunofluorescence, Staining, Control, Mutagenesis

Journal: Cell Reports

Article Title: CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci

doi: 10.1016/j.celrep.2020.108545

Figure Lengend Snippet: IL-6 Signaling Landscape in Primary Human T Cells (A and B) STAT1 and STAT3 phosphorylation in response to various doses (A) and exposure time (B) of IL-6 stimulation in resting and activated primary human CD4 + and CD8 + T cells. Error bars show mean ± SEM from three individual biological replicas. (C and D) Phospho-FLOW analysis of IL-6 signaling pathways in resting (C) and activated primary human CD4 + and CD8 + T cells treated with HyIL-6 or anti-CD3/CD28 (TCR) + IL-2. ns, cells without any stimulation. Heatmaps show fold change in the level of phosphorylation or protein expression of the different proteins. See also and . (E and F) Effect of JAK inhibition (2 μM tofacitinib) on the phosphorylation of STAT1 (E) and STAT3 (F) Tyr701 and Ser727 in resting and activated primary human CD4 + and CD8 + T cells. Error bars show mean ± SEM from three individual biological replicas.

Article Snippet: ATP was purchased from Sigma (Cat# A2383-10G), human recombinant CDKs were purchased from Thermo (CDK7/CyclinH/MNAT1 Cat# PV3868, CDK8/CyclinC Cat# PV4402 and CDK9/CyclinK Cat# PV4335) and human recombinant STAT3 was purchased from NovusBiologicals (Cat# H0000677-P01-10 μg).

Techniques: Expressing, Inhibition

Journal: Cell Reports

Article Title: CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci

doi: 10.1016/j.celrep.2020.108545

Figure Lengend Snippet: STAT1 and STAT3 HyIL-6-Induced Ser727 Phosphorylation Is CDK8/9 Mediated (A and B) Spider plots showing pTyr701 STAT1 (A) or pTyr705 STAT3 (B) (blue line) and pSer727 STAT1 (A) or pSer727 STAT3 (B) (red line) MFI normalized to HyIL-6-treated cells in the presence of different inhibitors in human primary CD4 + Th-1 cells. (C) Effect of different mTOR inhibitors on the STAT1 (top panel) and STAT3 (bottom panel) Ser727 phosphorylation induced by HyIL-6 in human primary CD4 + T cells. (D) Effect of ATM inhibitor (KU53933) and DNA-PK inhibitor (KU57788) on the STAT1 (top panel) and STAT3 (bottom panel) Ser727 phosphorylation induced by HyIL-6 in human primary CD4 + T cells. (E) Effect of different CDK inhibitors on the STAT3 Tyr705 (top panel) and STAT3 Ser727 (bottom panel) phosphorylation induced by HyIL-6 in human primary CD4 + T cells. For all experiments, quantitative data were calculated from three individual biological replicates. Error bars show mean ± SEM.

Article Snippet: ATP was purchased from Sigma (Cat# A2383-10G), human recombinant CDKs were purchased from Thermo (CDK7/CyclinH/MNAT1 Cat# PV3868, CDK8/CyclinC Cat# PV4402 and CDK9/CyclinK Cat# PV4335) and human recombinant STAT3 was purchased from NovusBiologicals (Cat# H0000677-P01-10 μg).

Techniques:

Journal: Cell Reports

Article Title: CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci

doi: 10.1016/j.celrep.2020.108545

Figure Lengend Snippet: PLA Analysis of the Interaction of STAT3 and CDK8/9 Induced upon HyIL-6 Stimulation in Human Primary CD4 + Th-1 Cells (A and B) Kinetics of the STAT3/CDK8 (A) or STAT3/CDK9 (B) interaction induced by 20 nM HyIL-6 in human primary CD4 + Th-1 cells. Scale bars, 20 μm. Statistical significance was calculated by one-way ANOVA. (C and D) STAT3/CDK8 (C) or STAT3/CDK9 (D) interactions were analyzed by PLA upon 20 nM HyIL-6 stimulation in the absence or presence of 2 μM MSC2530818 or 2 μM flavopiridol or upon treatment with the inhibitor only. Scale bars, 20 μm. Statistical significance was calculated by unpaired t test. White arrows in A to D indicate examples of cells where interaction signal was detected. Cumulative plots from n = 15 pictures alongside show the percentage of positive cells. Error bars show mean ± SEM. The p values were calculated based on non-parametric two-tailed Wilcoxon rank-sum test against the control group (first bar on the left). (E) STAT3/CDK9 interaction analyzed by PLA upon 20 nM HyIL-6 stimulation in STAT3 KnD Hut78 cells reconstituted with STAT3 WT-GFP (top panel) or STAT3 S727A-GFP (bottom panels). White arrows indicate examples of cells expressing the recombinant protein and where the STAT3/CDK9 interaction was detected by PLA. Scale bars, 20 μm. Graphs alongside show the nuclear GFP MFI normalized to unstimulated cells (top graph) or the nuclear STAT3/CDK9 PLA MFI in GFP-positive cells normalized to unstimulated cells (bottom graph). Quantitative data generated from n = 15 pictures. Error bars show mean ± SEM.

Article Snippet: ATP was purchased from Sigma (Cat# A2383-10G), human recombinant CDKs were purchased from Thermo (CDK7/CyclinH/MNAT1 Cat# PV3868, CDK8/CyclinC Cat# PV4402 and CDK9/CyclinK Cat# PV4335) and human recombinant STAT3 was purchased from NovusBiologicals (Cat# H0000677-P01-10 μg).

Techniques: Two Tailed Test, Control, Expressing, Recombinant, Generated

Journal: Cell Reports

Article Title: CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci

doi: 10.1016/j.celrep.2020.108545

Figure Lengend Snippet: Transcriptional Program Elicited by Interplay between HyIL-6 and CDK8 in Human Primary CD4 + Th-1 Cells (A) Number of differentially expressed genes (DEGs; fold chang,e >1.5; p < 0.05) between unstimulated versus HyIL-6-, mesenchymal stem cell (MSC)-, or HyIL-6+MSC-stimulated Th-1 cells in three biological replicates. (B) Scatterplot showing mean gene expression values (n = 3) before (x axis) and after indicated stimulation (y axis). Upregulated (red) and downregulated (blue) genes are highlighted. (C) Representative gene expression across different stimulation. Bars show mean ± SEM. (D) Gene set enrichment analysis (GSEA) ( Subramanian et al., 2005 ) plots for STAT3 upregulated genes (GEO: GSE21670) comparing stimulated versus unstimulated Th-1 transcriptomes. NES, normalized enrichment score; FDR, false discovery rate. (E) Violin plot showing the mean STAT3 binding intensity in n = 2,585 STAT3-bound regions across different stimulations. Peaks are identified by comparing HyIL-6+MSC stimulation and input. The p values were determined by two-tailed Wilcoxon rank-sum test ( ∗∗∗∗ p < 0.0001). (F) Representative loci showing STAT3 binding across different stimulations. The height of the tracks are indicated at bottom-right corner of the plots. (G) GSEA plots for 475 STAT3-bound genes comparing stimulated versus unstimulated Th-1 transcriptomes.

Article Snippet: ATP was purchased from Sigma (Cat# A2383-10G), human recombinant CDKs were purchased from Thermo (CDK7/CyclinH/MNAT1 Cat# PV3868, CDK8/CyclinC Cat# PV4402 and CDK9/CyclinK Cat# PV4335) and human recombinant STAT3 was purchased from NovusBiologicals (Cat# H0000677-P01-10 μg).

Techniques: Expressing, Binding Assay, Two Tailed Test

Journal: Cell Reports

Article Title: CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci

doi: 10.1016/j.celrep.2020.108545

Figure Lengend Snippet: Role of CDK8 Ser727 Phosphorylation of STAT3 in Th-17 Differentiation In Vitro (A) Experimental workflow for human Th-17 differentiation in vitro from isolated human resting CD4 + T cells. (B and C) Dot plot representations of IL-17- and IFNγ-positive cells in populations grown in the presence of HyIL-6 (B) or HyIL-6 + MSC2530818 (C). (D) IL-17-positive cells were identified by flow cytometry in untreated cells or cells treated with 2 μM MSC2530818. Data are percentage of positive cells ± SEM in four biological replicates; p values were calculated using a paired t test. (E) As in (D) but for IFNγ-positive cells. (F) Amount of IL-17 ± SEM in four biological replicates detected in growth media following growth of cells minus or plus inhibitor. (G) Amount of IFNγ ± SEM in four biological replicates detected in growth media following growth of cells minus or plus inhibitor. Statistical significance was calculated by unpaired t test.

Article Snippet: ATP was purchased from Sigma (Cat# A2383-10G), human recombinant CDKs were purchased from Thermo (CDK7/CyclinH/MNAT1 Cat# PV3868, CDK8/CyclinC Cat# PV4402 and CDK9/CyclinK Cat# PV4335) and human recombinant STAT3 was purchased from NovusBiologicals (Cat# H0000677-P01-10 μg).

Techniques: In Vitro, Isolation, Flow Cytometry

Journal: Cell Reports

Article Title: CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci

doi: 10.1016/j.celrep.2020.108545

Figure Lengend Snippet:

Article Snippet: ATP was purchased from Sigma (Cat# A2383-10G), human recombinant CDKs were purchased from Thermo (CDK7/CyclinH/MNAT1 Cat# PV3868, CDK8/CyclinC Cat# PV4402 and CDK9/CyclinK Cat# PV4335) and human recombinant STAT3 was purchased from NovusBiologicals (Cat# H0000677-P01-10 μg).

Techniques: Purification, Recombinant, Software