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: 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: Figure 6. 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 supplementary materials File S1.

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 MgCl2, 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: 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: 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

Journal: Molecular Cancer

Article Title: KIF5B-RET fusion kinase promotes cell growth by multilevel activation of STAT3 in lung cancer

doi: 10.1186/1476-4598-13-176

Figure Lengend Snippet: Signaling pathway of the proliferation is involved in KIF5B-RET positive cells. A & B . KIF5B-RET positively regulates the activation of the total STATs and ERK signaling pathways in positive cells. Control and enhanced KIF5B-RET expressing BEAS-2B (A) or A549 (B) cells were lysed and analyzed for phosphorylated Tyr 705 STAT3 and Thr 202 /Tyr 204 ERK 1/2 by western blots. C & D . The suppression of KIF5B-RET kinase activity by ZD6474 affects both STATs and ERK signaling pathways. Positive BEAS-2B or A549 cells were treated with ZD6474 at different doses for 48 h. Cells were lysed and analyzed by western blots for indicated marker antibodies. GAPDH was used as a loading control.

Article Snippet: STAT3 recombinant protein was purchased from Abnova.

Techniques: Activation Assay, Protein-Protein interactions, Control, Expressing, Western Blot, Activity Assay, Marker

Journal: Molecular Cancer

Article Title: KIF5B-RET fusion kinase promotes cell growth by multilevel activation of STAT3 in lung cancer

doi: 10.1186/1476-4598-13-176

Figure Lengend Snippet: KIF5B-RET phosphorylates and activates STAT3 at different levels in positive cell. A . KIF5B-RET phosphorylates and activates STAT3 both Tyr 705 and Ser 727 in positive cells. Western blots were performed on 293 T carrying KIF5B-RET or empty vector cells lysates using the indicated antibodies. B . JAK2 and c-Src kinase are involved in KIF5B-RET mediated-STAT3 Tyr 705 phosphorylation. 293 T cells carrying KIF5B-RET were pretreated with 15 μM AG490 (Jak2 inhibitor) or 5 μM PP1 (Src inhibitor) for the indicated times before cell lysis. Western blot was then performed using the indicated STAT3 antibodies. C . KIF5B-RET directly interacts STAT3 in positive cells. Enhanced KIF5B-RET expressing 293 T cell lysates were co-immunoprecipitated with control IgG or FLAG antibody. The immunoprecipitates were then subjected to western blot analysis with the indicated antibodies. D . KIF5B-RET directly phosphorylates STAT3 Tyr 705 . A bacterial-expressed GST- STAT3 fusion protein was incubated in vitro with KIF5B-RET IG that had been immunoprecipitated from 293 T carrying KIF5B-RET cell lysates. Western blots were then performed on the reaction mixtures using the indicated STAT3 antibodies. E . KIF5B-RET also induces a Ras/ERK 1/2 /STAT3 Ser 727 pathway. Enhanced KIF5B-RET expressing 293 T cells were treated overnight with U0126 (MEK inhibitor), and cell lysates were analyzed by western blot using the indicated antibodies. F . Expression of cyclin D1, VEGF, and ICAM-1 in KIF5B-RET positive cells. Two clones of the A549 and BEAS-2B cells, which exhibited stable KIF5B-RET expression, were analyzed cyclin D1, VEGF and ICAM-1 expression by RT-PCR, the products were separated by gel electrophoresis and visualized using ethidium bromide staining.

Article Snippet: STAT3 recombinant protein was purchased from Abnova.

Techniques: Western Blot, Plasmid Preparation, Phospho-proteomics, Lysis, Expressing, Immunoprecipitation, Control, Incubation, In Vitro, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Staining

Journal: Molecular Cancer

Article Title: KIF5B-RET fusion kinase promotes cell growth by multilevel activation of STAT3 in lung cancer

doi: 10.1186/1476-4598-13-176

Figure Lengend Snippet: Inhibition of STAT3 signaling reduces the proliferation of positive lung cancer cells. A . The inhibitors U0126, PP1 and S3I-201 reduce the colony-forming ability of KIF5B-RET positive cells. A549 cells carrying KIF5B-RET were diluted and seeded in 6-well plates, treated with DMSO, U0126 (MEK inhibitor) 10 μM, PP1 (SRC inhibitors) 5 μM or S3I-201(STAT3 inhibitors) 100 μM for 14 days. The total numbers of colonies, each containing more than 40 cells, were determined. ( * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t test). B . The suppression of STAT3 reduces the cell proliferation of KIF5B-RET positive cells. A549 cells carrying KIF5B-RET were seeded in 96-well plates and treated with DMSO (0.3%) or S3I-201 (100 μM) for 72 hours, and were analyzed by MTT assay at the indicated time ( *** P < 0.001, Student’s t test). C . Strategies to inhibit KIF5B-RET in lung cancer therapy. KIF5B-RET fusion kinase is constitutively active without GDNF stimulation. The fusion kinase induces STAT3 Tyr 705 phosphorylation directly, and also phosphorylates Tyr 705 indirectly through classical JAKs /STAT3 pathway, and STAT3 transcriptional activity is further enhanced by Ser 727 phosphorylation via a MEK/ERK 1/2 pathway. Inhibitors at different levels of KIF5B-RET-STAT3 signaling can suppress cell proliferation triggered by KIF5B-RET in lung cancer cells.

Article Snippet: STAT3 recombinant protein was purchased from Abnova.

Techniques: Inhibition, MTT Assay, Phospho-proteomics, Activity Assay