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: Figure 1. The acyloxy nitroso compounds, NCA and NCP, and the prototypical HNO donor, Angel’s salt, target STAT3. Chemical structures of (A) 1-Nitrosocyclohexyl acetate, NCA, (B) 1-nitrosocyclohexyl pivalate, NCP, and (C) Angeli’s salt (AS). doi:10.1371/journal.pone.0043313.g001

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

Techniques:

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: Figure 2. NCA and NCP inhibit LIF-induced STAT3 activation in human microvascular endothelial cells. HMEC-1 were pretreated for 1 h with vehicle (0.04% v/v DMSO), (A) 100 mM NCP, or (C) 100 mM 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. doi:10.1371/journal.pone.0043313.g002

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

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Control

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: Figure 3. NCP inhibits LIF-induced STAT3 activation in cardiac myocytes. Neonatal rat ventricular myocytes (A & B) were pretreated for 1 h with 100 mM 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 mM NCP (+) or vehicle (2). 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). doi:10.1371/journal.pone.0043313.g003

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

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Control

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: Figure 4. Dose response curves for inhibition of LIF-induced STAT3 activation by NCA, NCP and Angeli’s salt (AS). HMEC-1 were pretreated (A) for 1 h with various doses (02100 mM) of NCA or NCP and the same amount of vehicle (0.04% v/v DMSO) or (B) for 30 min with 02500 mM Angeli’s salt and the same amount of vehicle (50 mM 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. doi:10.1371/journal.pone.0043313.g004

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

Techniques: Inhibition, Activation Assay, Western Blot, Phospho-proteomics

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: Figure 8. STAT3 possesses redox-sensitive cysteines. (A) NCA and NCP block thiolate labeling. Recombinant human STAT3 was treated with vehicle (DMSO), NCA (100 mM) or NCP (100 mM) 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 4uC. Immunoprecipitates were processed as described under ‘‘Materials and Methods’’ 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. doi:10.1371/journal.pone.0043313.g008

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, Control

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: Figure 9. NCP enhances STAT3 glutathionylation and dimerization. HL-1 cells were treated for 30 min with vehicle (control), 500 mM NCP, 1 mM diamide, or 500 mM 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). doi:10.1371/journal.pone.0043313.g009

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

Techniques: Control, SDS Page, Produced, Comparison

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: Figure 10. Oxidative stress, NCP and diamide alter the Western blot profile of STAT3 under nonreducing conditions. (A & B) Aliquots of a cleared mouse heart homogenate were incubated for 30 min with vehicle, 500 mM NCP, 1 mM diamide, or 500 mM 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% b-mercaptoethanol (b-ME)) conditions. Protein loads were normalized using the direct blue 71 stained membranes (DB71). *P,0.05 (Student t-test). doi:10.1371/journal.pone.0043313.g010

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, Incubation, SDS Page, Control, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

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: Figure 3. 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 (Weinheim, Baden-Wurttemberg, Germany)

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: Figure 4. 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 (Weinheim, Baden-Wurttemberg, Germany)

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: Figure 5. 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 (Weinheim, Baden-Wurttemberg, Germany)

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: Figure 6. 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 (Weinheim, Baden-Wurttemberg, Germany)

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: Figure 7. 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 (Weinheim, Baden-Wurttemberg, Germany)

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: Figure 8. Schematic representation of the molecular mechanism that C21orf58 played oncogenic adaptor role on promoting cell growth and so- rafenib 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: Molecular cancer research : MCR

Article Title: Constitutive Phosphorylation of STAT3 by the CK2-BLNK-CD5 Complex

doi: 10.1158/1541-7786.MCR-16-0291

Figure Lengend Snippet: (A) CK2 and serine pSTAT3 are expressed in CLL cells. CLL cells from the PB of seven patients were lysed and subjected to Western immunoblotting using anti-CK2, anti-STAT3, and anti–serine pSTAT3 antibodies. β-actin served as a loading control, and HeLa cells were used as a positive control. (B) STAT3 and CK2 co-immunoprecipitate. CLL cell lysates were immunoprecipitated either with anti-STAT3 (upper panel) or with anti-CK2 (lower panel) antibodies. The immune complex was separated using SDS-PAGE. STAT3, serine pSTAT3, and CK2 were detected in CLL cell lysates that were immunoprecipitated with anti-STAT3 antibodies (upper panel). Similarly, CK2, STAT3, and serine pSTAT3 were detected in CLL cell lysates that were immunoprecipitated with anti-CK2 antibodies (lower panel) using Western immunoblotting. HeLa and Jurkat cells were used as controls. I.P., immunoprecipitate; B, beads. (C) CK2 phosphorylates STAT3 on serine 727 residues. Recombinant human STAT3 was incubated with (treated) or without (control) active CK2 in adenosine 5′-triphosphate (ATP)– or guanosine 5′-trisphosphate (GTP)-supplemented buffer for 30 minutes and analyzed by western immunoblotting. As shown, maximal phosphorylation of STAT3 occurred in the presence of CK2 and ATP. (D) CK2-siRNA reduces the phosphoserine STAT3 levels in CLL cells. CLL PB cells from two patients were transfected with CK2-siRNA using electroporation. After 48 hours, the cells were harvested and processed. Transfection efficiency was 30%, as assessed by flow cytometry detecting cells with intracellular GFP-conjugated siRNA. Left panel: CK2-siRNA significantly reduced CK2 mRNA levels. qRT-PCR was used to detect CK2 transcripts. The δ-δ cycle threshold method was used to determine the relative fold change in CK2 transcripts after transfection with CD5-siRNA. Right panel: Western immunoblotting of CLL cells from two patients transfected with CK2-siRNA or GAPDH were analyzed using Western immunoblotting. As shown, CK2-siRNA, but not GAPDH, significantly reduced the protein levels of CK2 and serine pSTAT3 compared with levels in untreated CLL cells. HeLa cells were used as positive controls.

Article Snippet: Recombinant STAT3 from Novus Biologicals and active CK2 from New England Biolabs were used according to the manufacturers’ instructions.

Techniques: Western Blot, Control, Positive Control, Immunoprecipitation, SDS Page, Recombinant, Incubation, Phospho-proteomics, Transfection, Electroporation, Flow Cytometry, Quantitative RT-PCR

Journal: Molecular cancer research : MCR

Article Title: Constitutive Phosphorylation of STAT3 by the CK2-BLNK-CD5 Complex

doi: 10.1158/1541-7786.MCR-16-0291

Figure Lengend Snippet: (A) CD5 is constitutively phosphorylated in CLL cells. Western immunoblotting detected CD5 and tyrosine pCD5 in CLL cells from the PB of eight randomly selected CLL patients. C, control (Jurkat cells). (B) CD5, CK2, and serine pSTAT3 co-immunoprecipitate. CLL cell lysates from three patients were immunoprecipitated with anti-CD5 antibodies. The immune complex was separated using SDS-PAGE, and serine pSTAT3, CK2, and CD5 were detected in the immunoprecipitate by Western immunoblotting. HeLa and Jurkat cells were used as controls. I.P., immunoprecipitate; B, beads. (C) CD5-neutralizing antibodies reduce serine pSTAT3 levels in CLL cells. CLL cells were incubated with CD5-neutralizing antibodies, isotype antibodies, or culture media. After 2 hours, the cells were harvested and subjected to Western immunoblotting. As shown, compared with untreated cells, the levels of CD5 and serine pSTAT3 were lower in cells that were incubated with CD5-neutralizing antibodies, but not in cells incubated with the isotype control antibodies, whereas the levels of STAT3 were unchanged by any treatment. Densitometry analysis was used to quantify protein levels. Abs.: antibodies. (D) CD5-siRNA reduces the phosphoserine STAT3 levels in CLL cells. CLL PB cells from two patients were transfected with CD5-siRNA using electroporation. After 48 hours, the cells were harvested and processed. Transfection efficiency was 35%, as assessed by flow cytometry detecting cells with intracellular GFP-conjugated siRNA. Left panel: CD5-siRNA significantly reduced CD5 mRNA levels. qRT-PCR was used to detect CD5 transcripts. The δ-δ cycle threshold method was used to determine the relative fold change in CD5 transcripts after transfection with CD5-siRNA. Right panel: Western immunoblotting of CLL cells from two patients transfected with CD5-siRNA or GAPDH were analyzed using Western immunoblotting. As shown, CD5-siRNA, but not GAPDH, significantly reduced the protein levels of CD5 and serine pSTAT3 compared with levels in untreated CLL cells. K-562, HeLa, and Jurkat cells were used as positive controls.

Article Snippet: Recombinant STAT3 from Novus Biologicals and active CK2 from New England Biolabs were used according to the manufacturers’ instructions.

Techniques: Western Blot, Control, Immunoprecipitation, SDS Page, Incubation, Transfection, Electroporation, Flow Cytometry, Quantitative RT-PCR

Journal: Molecular cancer research : MCR

Article Title: Constitutive Phosphorylation of STAT3 by the CK2-BLNK-CD5 Complex

doi: 10.1158/1541-7786.MCR-16-0291

Figure Lengend Snippet: (A) BLNK is constitutively phosphorylated in CLL cells. We obtained PB CLL cells from seven randomly selected CLL patients and, using Western immunoblotting, detected tyrosine pBLNK in all samples. Equal loading was confirmed by Ponceau staining (not shown). Untreated and IgM-treated RAMOS cells were used as controls. IgM: immunoglobulin M. (B) CK2, CD5, STAT3, and serine pSTAT3 co-immunoprecipitated with BLNK. CLL cell lysates were immunoprecipitated with anti-BLNK antibodies. The immune complex was separated using SDS-PAGE, and STAT3, serine pSTAT3, CK2, and CD5 were detected in the immunoprecipitate by Western immunoblotting. I.P., immunoprecipitate; B, beads. (C) BLNK-siRNA inhibits the phosphorylation of STAT3 on serine residues. CLL cells from three different patients were transfected by electroporation with BLNK-siRNA or GAPDH or were left untreated (controls). Left panel: BLNK-siRNA significantly reduced BLNK mRNA levels. qRT-PCR was used to detect BLNK transcripts. The δ-δ cycle threshold method was used to determine the relative fold change in BLNK transcripts after treatment with BLNK-siRNA. Right panel: CLL cells transfected with BLNK-siRNA or GAPDH were analyzed using Western immunoblotting. As shown, BLNK-siRNA, but not GAPDH, significantly reduced the protein levels of BLNK and serine pSTAT3, whereas the levels of STAT3 remained unchanged by either treatment. HeLa cells were used as positive controls.

Article Snippet: Recombinant STAT3 from Novus Biologicals and active CK2 from New England Biolabs were used according to the manufacturers’ instructions.

Techniques: Western Blot, Staining, Immunoprecipitation, SDS Page, Phospho-proteomics, Transfection, Electroporation, Quantitative RT-PCR

Journal: Molecular cancer research : MCR

Article Title: Constitutive Phosphorylation of STAT3 by the CK2-BLNK-CD5 Complex

doi: 10.1158/1541-7786.MCR-16-0291

Figure Lengend Snippet: (A) CLL cell lysates from two randomly selected CLL patients were immunoprecipitated with anti-STAT3 antibodies. The immune complex was separated using SDS-PAGE, and STAT3, serine pSTAT3, CK2, BLNK, and CD5 were detected in the immunoprecipitate by Western immunoblotting. HeLa and Jurkat cells were used as controls. I.P., immunoprecipitate; B, beads. (B) Similarly, CLL cell lysates were immunoprecipitated with anti-CD5 antibodies. The immune complex was separated using SDS-PAGE, and STAT3, serine pSTAT3, BLNK, CD5, and CK2 were detected in the immunoprecipitate by Western immunoblotting. HeLa, Jurkat, and HEK293 cells were used as controls.

Article Snippet: Recombinant STAT3 from Novus Biologicals and active CK2 from New England Biolabs were used according to the manufacturers’ instructions.

Techniques: Immunoprecipitation, SDS Page, Western Blot

Journal: Molecular cancer research : MCR

Article Title: Constitutive Phosphorylation of STAT3 by the CK2-BLNK-CD5 Complex

doi: 10.1158/1541-7786.MCR-16-0291

Figure Lengend Snippet: (A) CD5, a crucial member of the STAT3 phosphorylation complex, is expressed on the cell membrane but not in the cytosol or nucleus of CLL cells. Confocal microscopy images (400×) of freshly isolated CLL cells were stained with anti-CD5 antibodies for 1 hour. CD5 was detected on the cell surface but not the cytoplasm or nucleus of CLL cells. DAPI was used to detect the nucleus and S6 the cytoplasm of CLL cells. (B) The BLNK/CD5/CK2/STAT3 phosphorylation complex is de-assembled following the phosphorylation of STAT3. Cytosolic and nuclear fractions from PB CLL cells of two patients were analyzed using Western immunoblotting. As shown, the proteins participating in the formation of the STAT3 phosphorylation complex—BLNK, CD5, and CK2—were detected only in the cytosolic fractions whereas STAT3 and serine pSTAT3 were detected in both the cytosol and the nucleus of CLL cells. Lamin B was used as the nuclear fraction control and S6 as the cytoplasmic fraction control. HeLa, HEK293, and Jurkat cells were used as controls.

Article Snippet: Recombinant STAT3 from Novus Biologicals and active CK2 from New England Biolabs were used according to the manufacturers’ instructions.

Techniques: Phospho-proteomics, Membrane, Confocal Microscopy, Isolation, Staining, Western Blot, Control

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: ACS Central Science

Article Title: Discovery of the Highly Selective and Potent STAT3 Inhibitor for Pancreatic Cancer Treatment

doi: 10.1021/acscentsci.3c01440

Figure Lengend Snippet: YY002 selectively targets STAT3. (A) Structure of YY002. (B) YY002 inhibited the STAT3 luciferase reporter activity. IL-6 was used as an activator of STAT3 ( n = 2). (C) YY002 inhibited the ATP production ( n = 2). (D) YY002 inhibited the OXPHOS rate (OCR = oxygen consumption rate, n = 2). (E) Direct binding of YY002 to STAT3 SH2 or STAT3 127–722 was determined by MST experiments ( n = 3). (F) The binding affinities between YY002 and STAT3 was detected by surface plasmon resonance (SPR) assay. (G) YY002 selectively bound to STAT3 and other STAT members ( n = 3). (H) The shRNA knock-down efficiency in PANC-1 and Capan-2 was confirmed by Western Blot. (I, J) The pancreatic cancer cell lines shNC, shSTAT3-1# and shSTAT3-2# of PANC-1 (I) and Capan-2 (J) were treated with YY002, and the proliferation was determined after 72 h of treatment ( n = 3). (K) BxPC3 cells were treated with 50 nM YY002 for 24 h, and then RNA-Seq assay was performed. The results were analyzed by gene set enrichment analysis (GSEA). (L) Capan-2 cells were treated with different concentrations of STAT3 inhibitors or YY002, and the proliferation was tested by MTS ( n = 2). (M) YY002 and Stattic directly bound to STAT3 127–722 , The binding affinities were determined by MST experiments ( n = 3). Data shown as mean ± SD ns, P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 by one-way ANOVA following multiple comparison.

Article Snippet: Recombinant full-length human STAT3 (Creative Biomart, Catalog: STAT3–001H, Batch number: PSS1072207) was immobilized on the sensor chip (CM5) using the amine-coupling method according to standard protocols.

Techniques: Luciferase, Activity Assay, Binding Assay, SPR Assay, shRNA, Knockdown, Western Blot, RNA Sequencing, Comparison

Journal: ACS Central Science

Article Title: Discovery of the Highly Selective and Potent STAT3 Inhibitor for Pancreatic Cancer Treatment

doi: 10.1021/acscentsci.3c01440

Figure Lengend Snippet: YY002 directly binds to STAT3 SH2 domain. (A) Computer docking model predicting that YY002 bound to the STAT3-SH2 domain. (B, C) YY002 binding to STAT3-SH2 and its indicated mutants. The binding affinities were measured by MST experiments ( n = 3). (D) PANC-1 shSTAT3-1# cells that were infected with indicated lentivirus vectors to reintroduce specific SH2 mutants were treated with indicated YY002 concentrations, and cell viability was measured ( n = 3). Data shown as mean ± SD. (E) Re-expression of STAT3 mutant in STAT3 knockdown PDAC cells. The shSTAT3-1# PANC-1 cells that were infected with the indicated lentivirus expression vectors, and the transfection efficacy was detected by Western blots. (F) In vitro enzyme inhibition assays.

Article Snippet: Recombinant full-length human STAT3 (Creative Biomart, Catalog: STAT3–001H, Batch number: PSS1072207) was immobilized on the sensor chip (CM5) using the amine-coupling method according to standard protocols.

Techniques: Binding Assay, Infection, Expressing, Mutagenesis, Knockdown, Transfection, Western Blot, In Vitro, Enzyme Inhibition Assay

Journal: ACS Central Science

Article Title: Discovery of the Highly Selective and Potent STAT3 Inhibitor for Pancreatic Cancer Treatment

doi: 10.1021/acscentsci.3c01440

Figure Lengend Snippet: YY002 inhibits pancreatic cancer cell growth, STAT3 Tyr705 phosphorylation and STAT3 nuclear function. (A) YY002 inhibited the proliferation of pancreatic cancer cell lines but only had a limited effect on normal cells ( n = 2). (B) The protein expression level of STAT3, p-STAT3 Tyr705 and p-STAT3 Ser727 in different cell lines. (C) STAT3 phosphorylation and downstream gene expression were measured by Western Blot in Capan-2, HPAC and BxPC-3 cells after treatment with YY002. (D) Down-stream gene expression was measured by quantitative real-time PCR in Capan-2, HPAC and BxPC-3 cells after treatment with YY002 ( n = 2). (E) YY002 inhibited the entry of STAT3 into the nucleus of BxPC3, Capan-2 and PANC-1 cells. Scale bar, 50 μm. Data shown as mean ± sd. ns, P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 by one-way ANOVA following multiple comparison.

Article Snippet: Recombinant full-length human STAT3 (Creative Biomart, Catalog: STAT3–001H, Batch number: PSS1072207) was immobilized on the sensor chip (CM5) using the amine-coupling method according to standard protocols.

Techniques: Phospho-proteomics, Expressing, Gene Expression, Western Blot, Real-time Polymerase Chain Reaction, Comparison

Journal: ACS Central Science

Article Title: Discovery of the Highly Selective and Potent STAT3 Inhibitor for Pancreatic Cancer Treatment

doi: 10.1021/acscentsci.3c01440

Figure Lengend Snippet: YY002 inhibits STAT3 Ser727 phosphorylation and mitochondrial OXPHOS. (A) Oxygen consumption rate (OCR) was evaluated by the Seahorse XF96 extracellular flux analyzer and OXPHOS was inhibited in BxPC-3 ( n = 2), CFPAC-1 ( n = 3) and Capan-2 ( n = 4) cells treated with YY002. (B) Knockdown of STAT3 in CFPAC-1 inhibited OXPHOS ( n = 3). (C) CFPAC-1 shSTAT3-1# cells were treated with YY002 or vehicle (DMSO), and the level of OXPHOS was measured ( n = 3). (D, E) The glycolysis level of BxPC-3 (D) and CFPAC-1 (E) cells treated with different concentrations of YY002 was determined ( n = 3). (F) A brief description of the mitochondrial electron transport chain. (G, H) Capan-2 cells were treated with 50 nM YY002 or vehicle control and injected with the indicated drugs 1, 2, 3, 4 sequentially, and the mitochondrial respiration was determined by Seahorse instrument assay ( n = 3). Data shown as mean ± sd.

Article Snippet: Recombinant full-length human STAT3 (Creative Biomart, Catalog: STAT3–001H, Batch number: PSS1072207) was immobilized on the sensor chip (CM5) using the amine-coupling method according to standard protocols.

Techniques: Phospho-proteomics, Knockdown, Control, Injection

Journal: ACS Central Science

Article Title: Discovery of the Highly Selective and Potent STAT3 Inhibitor for Pancreatic Cancer Treatment

doi: 10.1021/acscentsci.3c01440

Figure Lengend Snippet: YY002 inhibits pancreatic cancer growth in vivo . (A, B) The PANC-1 tumor volumes of mice were recorded every 4–5 days ( n = 8). Data shown as mean ± SEM * P < 0.05, * * P < 0.01 and **** P < 0.0001 by One-way ANOVA followed multiple comparison. (C) At the end of the experiment, the tumors in each group were excised, weighed and counted. (D-E) MIA PaCa-2 cells were injected into mice which were treated with different concentrations of YY002 orally ( n = 8). (F) The tumor volume was recorded every 4 days and after 24 days of administration, the tumors were harvested. Data shown as mean ± SEM * P < 0.05, * * P < 0.01 and **** P < 0.0001 by One-way ANOVA followed multiple comparison. At the end of the experiment, the tumors in each group were excised, weighed and counted. (G) Quantification of STAT3, pSTAT3 Tyr705 , and pSTAT3 Ser727 immuno-staining and representative images of PANC-1 tumor.

Article Snippet: Recombinant full-length human STAT3 (Creative Biomart, Catalog: STAT3–001H, Batch number: PSS1072207) was immobilized on the sensor chip (CM5) using the amine-coupling method according to standard protocols.

Techniques: In Vivo, Comparison, Injection, Immunostaining

Journal: ACS Central Science

Article Title: Discovery of the Highly Selective and Potent STAT3 Inhibitor for Pancreatic Cancer Treatment

doi: 10.1021/acscentsci.3c01440

Figure Lengend Snippet: YY002 inhibits metastasis of pancreatic cancer in vivo . (A) PAN02-Luciferase cells were implanted orthotopically into the pancreas tails of male C57/BL6 mice. Different concentrations of YY002 were given orally for 4 weeks. Quantification of bioluminescence in pancreatic orthotopic xenograft model( n = 7). (B) PAN02-Luciferase cells were inoculated intravenously into male C57/BL6 mice. Quantification of bioluminescence in pancreatic cancer liver metastasis mouse model ( n = 8). (C) Overall survival rates of the additional independent orthotopic of pancreatic cancer. Log-rank (mantel-cox) test was used ( n = 9 in Control, 10 mg/kg, 20 mg/kg groups; n = 8 in 5 mg/kg group). (D) Overall survival rates of the additional independent liver metastatic models of pancreatic cancer. Log-rank (mantel-cox) test was used ( n = 8 in each group). (E) AsPC-1 cells were implanted orthotopically into the pancreas tails of mice, and then mice were treated with different concentrations of YY002 for 4 weeks, The survival time was counted continuously ( n = 9 in Control, 10 mg/kg, 20 mg/kg groups; n = 8 in 5 mg/kg group). Data shown as mean ± sd. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 by One-way ANOVA followed multiple comparison. (F-J). YY002 induced the death of other STAT3-dependent tumors. (F) YY002 induced various STAT3-dependent cancer cells death. The tumor cells treated with YY002 in different concentrations, the cell viabilities were determined by MTS assays ( n = 2). (G, H) The STAT3 phosphorylation and the expression of STAT3 were measured by Western Blot in MDA-MB-231 and SUDHL-1 cells after treatment with YY002. (I) MDA-MB-231 cells were injected into mice which were treated with YY002 or BBI608 orally ( n = 10). (J) SUDHL-1 cells were injected into mice which were treated with YY002 or BBI608 orally ( n = 10).

Article Snippet: Recombinant full-length human STAT3 (Creative Biomart, Catalog: STAT3–001H, Batch number: PSS1072207) was immobilized on the sensor chip (CM5) using the amine-coupling method according to standard protocols.

Techniques: In Vivo, Luciferase, Control, Comparison, Phospho-proteomics, Expressing, Western Blot, Injection

Journal: Cancer Cell International

Article Title: A novel chalcone derivative suppresses melanoma cell growth through targeting Fyn/Stat3 pathway

doi: 10.1186/s12935-020-01336-2

Figure Lengend Snippet: Lj-1-60 is a novel inhibitor targeting Fyn protein kinase. a HEK293T cells transfected with 2 μg and 4 μg of Fyn-flag plasmids for 36 h, it was then harvested and assessed by pull-down assay. Cell lysates incubated with Sepharose coupled with Lj-1-60 were subjected to SDS-PAGE, and analyzed by immunoblotting with antibody anti-flag. b Melanoma cell lysates for pull-down assay was detected by immunoblotting with anti-Fyn antibody. c In vitro kinase assay. A reaction mixture of substrate human recombinant protein Stat3 with myc-tag (676-770 aa), Fyn kinase and Lj-1-60 were incubated at 30 °C for 40 min and subjected to immunoblotting with indicated antibodies. Data are representative of three independent experiments. d Knocked down of Fyn in Sk-Mel-5 and Sk-Mel-28 cell lines by two independent shRNA. Total cell lysates were subjected to immunoblotting using indicated antibodies. Data are representative of three independent experiments. e Lj-1-60 treated in Sk-Mel-5 and Sk-Mel-28 cell lines for 48 h with indicated concentration and analyzed by immunoblot using antibodies p-Stat3(Tyr 705), Stat3, and GAPDH as loading control

Article Snippet: Recombinant human Stat3 was purchased from CUSABIO.

Techniques: Transfection, Pull Down Assay, Incubation, SDS Page, Western Blot, In Vitro, Kinase Assay, Recombinant, shRNA, Concentration Assay, Control

Journal: Cancer Cell International

Article Title: A novel chalcone derivative suppresses melanoma cell growth through targeting Fyn/Stat3 pathway

doi: 10.1186/s12935-020-01336-2

Figure Lengend Snippet: Analysis of gene expression profiles involved in melanoma cells altered by Lj-1-60. a The mRNA expression of CDKN1A, GADD45A, MCM3 in melanoma cells Sk-Mel-5 and Sk-Mel-28 was measured by RT-PCR. Total RNA was extracted from the cells treated with 2 μM Lj-1-60 for 24, 48 h. Data were expressed as mean (n = 3) ± SD, *P < 0.05, ***P < 0.001, ****P < 0.0001. b The mRNA expression of CDKN1A, GADD45A, MCM3 in melanoma cells Sk-Mel-5 and Sk-Mel-28 was examined by RT-PCR. Total RNA was extracted from cells knocked down of Fyn with two independent shRNA. Data were expressed as mean (n = 3) ± SD, *P < 0.05, ***P < 0.001, ****P < 0.0001. c Structural model. Lj-1-60 inhibits the proliferation of melanoma and induces cell cycle arrested in G2/M phase and apoptosis by targeting Fyn through inhibiting the phosphorylation of Stat3

Article Snippet: Recombinant human Stat3 was purchased from CUSABIO.

Techniques: Gene Expression, Expressing, Reverse Transcription Polymerase Chain Reaction, shRNA, Phospho-proteomics

Journal: Cancers

Article Title: An FDA-Approved Antifungal, Ketoconazole, and Its Novel Derivative Suppress tGLI1-Mediated Breast Cancer Brain Metastasis by Inhibiting the DNA-Binding Activity of Brain Metastasis-Promoting Transcription Factor tGLI1

doi: 10.3390/cancers14174256

Figure Lengend Snippet: KCZ and the novel derivative KCZ-7 inhibit tGLI1 transcriptional activity leading to downregulation of validated tGLI1-mediated stemness genes Nanog and OCT4 . ( a ) Representative Western blots of GLI1 and tGLI1 expression in isogenic SKBRM cell lines following 24 h treatment with vehicle, 1 μM KCZ, or 1 μM KCZ-7. The same membrane was probed to assess the loading control. ( b ) Western blots of recombinant GLI1 and N-tGLI1 (left). A tGLI1-selective Ab was used to detect tGLI1. Binding of recombinant GLI1 and N-tGLI1 to a dsDNA oligonucleotide containing the consensus GLI1/tGLI1-binding site (right). STAT3 was used as a negative control. ( c ) The DNA-binding ability of recombinant N-tGLI1, but not GLI1, is disrupted by KCZ or KCZ-7 treatment. ( d ) Relative binding of GLI1 or tGLI1 to the GLI1-binding sites in SKBRM cells, as determined by chromatin immunoprecipitation; qPCR was performed using primers spanning the GLI1 binding site. ( e , f ) Inhibition of GLI1- and tGLI1-mediated promoter transactivation by KCZ ( e ) and KCZ-7 ( f ). SKBR3 cells were transiently transfected with 8 × 3′GLI1 luciferase reporter and vector, GLI1, or tGLI1 plasmids, then treated with increasing doses of KCZ ( e ) or KCZ-7 ( f ) for 48 h and stimulated with SHH ligand (100 ng/mL) for 4 h. Right: Relative luciferase activity normalized to vehicle treatment. ( g , h ) Selective reduction of tGLI1-mediated stemness genes Nanog ( g ) and OCT4 ( h ) mRNA as assessed by RT-qPCR in isogenic SKBRM cell lines treated with vehicle, 1 μM KCZ, or 1 μM KCZ-7 for 24 h. ( i ) Nanog and OCT4 protein expression following treatment with vehicle, 1 μM KCZ, or 1 μM KCZ-7 in isogenic SKBRM cell lines. The same membrane was probed to assess the loading control. ( j , k ) Overexpression of Nanog ( j ) or OCT4 ( k ) rescues SKBRM-tGLI1 mammospheres from KCZ and KCZ-7 treatment. Scale bars represent 200 μm. N-tGLI1, N-terminal tGLI1; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; two-way ANOVA with post hoc Dunnett’s ( d – f ) or Bonferroni’s ( g , h , j , k ) multiple comparison test was used to calculate p -values. The uncropped blots are shown in page 2 of .

Article Snippet: Approximately 600 ng of recombinant STAT3 (Creative Biomart, STAT3-29823TH) (Shirley, NY, USA), GLI1 (Creative Biomart, GLI1-312H), or N-tGLI1 protein was mixed with 5X binding buffer (50 mM Tris pH 7.5, 50 mM NaCl, 200 mM KCl, 5 mM MgCl 2 , 10 mM EDTA, 5 mM DTT, 250 μg/mL BSA, 25% glycerol), 50 ng/μL poly dI·dC (Sigma P4929), and 5 pmol 6FAM-labeled dsDNA oligo (Integrated DNA Technologies, Coralville, IA, USA) in a total reaction volume of 20 μL.

Techniques: Activity Assay, Western Blot, Expressing, Membrane, Control, Recombinant, Binding Assay, Negative Control, Chromatin Immunoprecipitation, Inhibition, Transfection, Luciferase, Plasmid Preparation, Quantitative RT-PCR, Over Expression, Comparison

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

Journal: Journal of Neuroinflammation

Article Title: Inhibitory effect of ent -Sauchinone on amyloidogenesis via inhibition of STAT3-mediated NF-κB activation in cultured astrocytes and microglial BV-2 cells

doi: 10.1186/1742-2094-11-118

Figure Lengend Snippet: Structural interaction between ent -Sauchinone and STAT3. ent -Sauchinone was prepared from 4-(oxiran-2-yl) phenyl acetate, Pd(OAc)2 and tributyl phosphine (A) . Pull-down assay identifies an interaction between the ent -Sauchinone and STAT3. ent -Sauchinone was conjugated with cyanogen bromide Epoxy-activated Sepharose 6B (B) . Docking model of ent -Sauchinone with STAT3 as described in Materials and Methods (C and D).

Article Snippet: The cell lysate or STAT3 recombinant protein (Abnova, Taipei, Taiwan) were mixed with ent -Sauchinone conjugated Sepharose 6B or Sepharose 6B at 4 C for 24 hours.

Techniques: Pull Down Assay

Journal: Journal of Neuroinflammation

Article Title: Inhibitory effect of ent -Sauchinone on amyloidogenesis via inhibition of STAT3-mediated NF-κB activation in cultured astrocytes and microglial BV-2 cells

doi: 10.1186/1742-2094-11-118

Figure Lengend Snippet: Effects of ent -Sauchinone on STAT3 DNA binding activity and protein expressions of STAT3 were detected by western blotting using specific antibodies in astrocytes and microglial BV-2 cells. Astrocytes (A) and microglial BV-2 cells (B) were treated with 1 mg/ml of LPS alone or with LPS plus different concentrations (1, 5, and 10 μM) of ent -Sauchinone at 37°C for 1 hour. DNA binding activity of STAT1 and STAT3 was investigated using EMSA as described in Materials and Methods. Nuclear extracts were subjected to DNA binding reaction with 32 P end-labeled oligonucleotide specific to STAT3. Specific DNA binding of the STAT3 complex is indicated by an arrow. Similar results were treated with 1 μg/ml of LPS alone, or with LPS plus different concentrations (1, 5, and 10 μM) of ent -Sauchinone at 37°C for 1 hour. Equal amounts of total proteins (40 μg/lane) were subjected to 10% SDS-PAGE, and activation of STAT3 (phosphorylation) was detected by western blotting using specific antibodies in astrocytes (C) and in microglial BV-2 cells (D) . Values below or above of each figures mean quantified relative expression of the proteins or DNA binding activity.

Article Snippet: The cell lysate or STAT3 recombinant protein (Abnova, Taipei, Taiwan) were mixed with ent -Sauchinone conjugated Sepharose 6B or Sepharose 6B at 4 C for 24 hours.

Techniques: Binding Assay, Activity Assay, Western Blot, Labeling, SDS Page, Activation Assay, Phospho-proteomics, Expressing

Journal: Journal of Neuroinflammation

Article Title: Inhibitory effect of ent -Sauchinone on amyloidogenesis via inhibition of STAT3-mediated NF-κB activation in cultured astrocytes and microglial BV-2 cells

doi: 10.1186/1742-2094-11-118

Figure Lengend Snippet: Effects of inhibition of STAT3 on the effect of ent -Sauchinone on NF-κB activation and A level in cultured astrocytes and in microglial BV-2 cells. To block the STAT3 pathway, cells were treated with the STAT3 inhibitor AG490 (50 μM) or with 50 nM siRNA for STAT3 for 1 hour prior to treatment with ent -Sauchinone. Abolished effect of STAT3 inhibition on the inhibitory effect of ent -Sauchinone on NF-κB DNA binding activity in astrocytes (A) and microglial BV-2 cells (B) , and A 1-42 level in astrocytes (C) and microglial BV-2 cells (D) . For determination of DNA binding activity of NF-κB and Aβ 1-42 levels, cells were treated with 1 μg/ml of LPS alone, or with LPS plus different concentrations (1, 5, and 10 μM) of ent -Sauchinone at 37°C for 24 hours (1 hour for NF-κB) in the absence or presence of STAT3 inhibitor or siRNA. Values are mean ± S.E. for three experiments performed in triplicate. *indicates significantly different from the control group ( P <0.05.. # indicates significantly different from the LPS-treated group ( P <0.05.. & indicates significantly different from the LPS and ent -Sauchinone treated group ( P <0.05.. Values below each figure mean quantified relative DNA binding activity.

Article Snippet: The cell lysate or STAT3 recombinant protein (Abnova, Taipei, Taiwan) were mixed with ent -Sauchinone conjugated Sepharose 6B or Sepharose 6B at 4 C for 24 hours.

Techniques: Inhibition, Activation Assay, Cell Culture, Blocking Assay, Binding Assay, Activity Assay, Control