Journal: Nucleic Acids Research

Article Title: Precise and efficient C-to-U RNA base editing with SNAP-CDAR-S

doi: 10.1093/nar/gkad598

Figure Lengend Snippet: Benchmark with Cas13-RESCUE-S on endogenous targets and applications. ( A ) Comparison of editing yields at various sites on various endogenous targets and one disease-relevant cDNA (APOE) comparing SNAP-CDAR-S with standard guide RNAs (30 nt, 6-C-23, 2′OMe gapmer) versus Cas13 RESCUE-S with plasmid-borne optimized Cas guide RNAs. Both editing enzymes were expressed from the same single genomic locus. ( B ) Comparing both tools, SNAP-CADR-S versus Cas RESCUE-S, for the activation of β-catenin by RNA editing. Given are C-to-U editing yields (T41I) and the luminescence-based read-out of pathway activation. For further controls, see . ( C ) Editing of the regulatory phospho-site serine 727-to-glycine in STAT3, read-out of editing yield by Sanger sequencing, and amount of total STAT3 and pS727 STAT3 protein by western blot. Data in (A), (B) and (C) are shown as the mean ± s.d. of N = 3 independent experiments.

Article Snippet: 30 μg of total protein was run on a NovexTM WedgeWellTM 8 to 16%, Tris-glycine, 1.0 mm, Mini Protein Gel (ThermoFisher Scientific) with 200 V for 60 min. Blotting was performed with a Mini Trans-Blot Cell ® (BioRad) at 100 V for 60 min. For protein detection, membranes were incubated with monoclonal anti-β-actin antibody produced in mouse (Sigma, 1:5000 dil.) and either Stat3 (DRZ2G) Rabbit mAb (CellSignaling, 1:1000 dil.) or P-Stat3 (S727) (D8C2Z) Rabbit mAb (CellSignaling, 1:1000 dil.).

Techniques: Comparison, Plasmid Preparation, Activation Assay, Sequencing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Negative Regulation of Stat3 by Activating PTPN11 Mutants Contributes to the Pathogenesis of Noonan Syndrome and Juvenile Myelomonocytic Leukemia

doi: 10.1074/jbc.m109.020495

Figure Lengend Snippet: FIGURE 1. Ablation of Stat3 in bone marrow cells gives rise to skewed myeloid cell differentiation and hypersensitivity to GM-CSF. A, morphological, histological, and flow cytometric analyses of spleens from Stat3f/f/btie2-cre and Stat3f/f littermate control mice. Panel a, splenomagaly was found in Stat3f/f/ btie2-cre mice; panel b, comparison of spleen weight to body weight ratio of Stat3f/f and Stat3f/f/btie2-cre mice, p 0.01 (Student’s t test, 2 tails); panels c and d, disrupted spleen architecture in Stat3f/f/btie2-cre mice (d) when compared with Stat3f/f controls (c); panel e, both F480Mac1 and GR-1Mac1 popula- tions were significantly increased in Stat3f/f/btie2-cre spleens compared with the Stat3f/f control spleens. B, bone marrow F480Mac1 and GR-1Mac1 populations were increased in the Stat3f/f/btie2-cre mice compared with Stat3f/f control mice. C, methylcellulose-based colony forming assays demonstrated increased bone marrow progenitor-derived colonies in response to increasing doses of GM-CSF from the Stat3f/f/btie2-cre mice compared with the Stat3f/f mice. D,increasedproliferativeactivityofStat3f/f/btie2-crebonemarrowcellsinresponsetovariouscytokines.Thefinalconcentrationofallcytokines,exceptIL-3(100u/ml), was 30 ng/ml. E, decreased apoptosis in Stat3f/f/btie2-cre bone marrow cells when cultured in a low serum in the presence of 0.1 or 1 ng/ml of GM-CSF. Error bars represent S.D.

Article Snippet: To analyze phospho-Stat3 expression in the developing heart valve, we used a rabbit monoclonal antibody against phosphor-Stat3 (Tyr705) (Cell Signaling, D3A7) and a Vector staining system (Vector, PK-2200) according to the manufacturer’s instructions.

Techniques: Cell Differentiation, Control, Comparison, Derivative Assay, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: Negative Regulation of Stat3 by Activating PTPN11 Mutants Contributes to the Pathogenesis of Noonan Syndrome and Juvenile Myelomonocytic Leukemia

doi: 10.1074/jbc.m109.020495

Figure Lengend Snippet: FIGURE 2. Shp2 gain-of-function mutants negatively regulate Stat3 acti- vationinmurinebonemarrowcellsandperipheralbloodnucleatedcells from individuals with NS. A, panel a, Western blot analyses examining GM- CSF-stimulated (20 ng/ml) Stat3 activation in the mouse bone marrow-de- rived macrophage progenitors transduced with MIEG3 (empty vector), WT Shp2, or Shp2E76K. Panel b, density quantification of protein band in A, panel a; B, panel a, representative flow cytometric analysis showing reduced Stat3 tyrosine phosphorylation in peripheral blood cells from individuals with NS in both the quiescent state and after IL-6 stimulation. Panel b, statistical analysis

Article Snippet: To analyze phospho-Stat3 expression in the developing heart valve, we used a rabbit monoclonal antibody against phosphor-Stat3 (Tyr705) (Cell Signaling, D3A7) and a Vector staining system (Vector, PK-2200) according to the manufacturer’s instructions.

Techniques: Western Blot, Activation Assay, Transduction, Plasmid Preparation, Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: Negative Regulation of Stat3 by Activating PTPN11 Mutants Contributes to the Pathogenesis of Noonan Syndrome and Juvenile Myelomonocytic Leukemia

doi: 10.1074/jbc.m109.020495

Figure Lengend Snippet: FIGURE 3. Stat3activationinvalvulogenesis,andShp2gain-of-functionmutantsinhibittheactivationofStat3inresponsetoEGF.A,immunohistochemistry staining of activated Stat3 (pY Stat3) in developing and mature PV of wild type mice. Nuclear brown signals are positive staining. Activated Stat3 is apparent in E14.5 embryonic heart and is restricted to the developing leaflets (A, panels a to c), and remains into adult stage (panel d). Activated Stat3 is present in the PV of control embryo hearts at E17.5 (panel e), absent in PV from Stat3f/f/nestin-cre embryo heart at the same stage (panel f). B, panel a, after serum deprivation overnight, Western blotanalysiswasusedtoexaminebasalandEGF-stimulated(50ng/ml)Stat3tyrosinephosphorylationinNIH3T3cellsexpressingShp2mutants(N308DandE76K)or controls(MIEG3orWTShp2).Panelb,densityquantificationoftheproteinbandinB,panela.C,EMSAtoexamineStat3DNAbindingactivityusingthem67-SIEprobe and nuclear extracts from NIH3T3 cells transduced with MIEG3 (M), WT Shp2 (WT), Shp2N308D (N308D), or Shp2E76K (E76K). Error bars represent S.D.

Article Snippet: To analyze phospho-Stat3 expression in the developing heart valve, we used a rabbit monoclonal antibody against phosphor-Stat3 (Tyr705) (Cell Signaling, D3A7) and a Vector staining system (Vector, PK-2200) according to the manufacturer’s instructions.

Techniques: Immunohistochemistry, Staining, Control, Western Blot, Transduction

Journal: Journal of Biological Chemistry

Article Title: Negative Regulation of Stat3 by Activating PTPN11 Mutants Contributes to the Pathogenesis of Noonan Syndrome and Juvenile Myelomonocytic Leukemia

doi: 10.1074/jbc.m109.020495

Figure Lengend Snippet: FIGURE 4. Conditional deletion of Stat3 with nestin-cre induces pulmonary stenosis due to the thicken- ing of leaflets. A, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside(X-gal) staining of nestin-cre/Rosa26R mouse embryos and newborns indicates the cre activity in the developing mouse embryos. A, panels a and b, central nervous system is positive for cre activity in the mouse embryos at E10.5. Panels c and d, cre activity was found in the pulmonary valves (PV) and the aortic valves (AV) at E14.5 (A, panel c) and P0 (B, panel d). B, ablation of Stat3 with nestin-cre leads to pulmonary stenosis caused by the thickening and enlargement of leaflets. B, panel a, newborn of Stat3f/f (f/f) and Stat3f/f/Nestin-cre (c;f/f) mice. B, panel b, hematoxylin and eosin staining of the transverse section of heart from control (f/f) and STAT3f/f/Nestin-cre (c;f/f) newborn mice (P0). Note that Stat3 ablation leads to right ventricle dilation in Stat3f/f/Nestin-cre mice. B, panels d–g, hematoxylin and eosin staining of sections of the pulmonary valve and aorta valve regions in control (B, panels d and f) and Stat3f/f/ Nestin-cre mutant (B, panels e and g) newborn mice. LV, left ventricle; RV, right ventricle. B, panel h, quantifica- tionofpulmonaryvalvethicknessinStat3f/f/Nestin-creandcontrolhearts(P0)(n6,p0.03,Student’spaired t test; 2 tails).

Article Snippet: To analyze phospho-Stat3 expression in the developing heart valve, we used a rabbit monoclonal antibody against phosphor-Stat3 (Tyr705) (Cell Signaling, D3A7) and a Vector staining system (Vector, PK-2200) according to the manufacturer’s instructions.

Techniques: Staining, Activity Assay, Control, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Negative Regulation of Stat3 by Activating PTPN11 Mutants Contributes to the Pathogenesis of Noonan Syndrome and Juvenile Myelomonocytic Leukemia

doi: 10.1074/jbc.m109.020495

Figure Lengend Snippet: FIGURE 5. Shp2 dephosphorylates Tyr(P)-Stat3 and Shp2-Stat3-medi- atedsignalingcriticallycontributestothepathogenesisofNS.A,Western blot analysis of Stat3-deficient bone marrow-derived macrophage progeni- tors treated with GM-CSF to assess Stat3 and ERK activation. B, panel a, puri- fied constitutively active Shp2 protein (catalytic domain) was capable of dephosphorylating Tyr(P)-Stat3. Stat3 protein was immunoprecipitated from IL-6-treated Raw 264.7 cell extract, and then incubated with purified recom- binant Shp2 protein (active form, 2 g) for the indicated time with or without preincubation of the small molecule Shp2 phosphatase inhibitor IIB-08 (100 M). Panel b, Shp2 inhibitor IIB-08 inhibits ERK activation, whereas enhancing the Tyr(P)-Stat3 level, in IL-6 induced Raw 264.7 cells. Raw 264.7 cells were pretreated with the Shp2 inhibitor IIB-08 (10 M), or dimethyl sulfoxide (DMSO) for 60 min, and then incubated with IL-6 (20 ng/ml) for 60 min before being subjected to lysis and Western blot analysis.

Article Snippet: To analyze phospho-Stat3 expression in the developing heart valve, we used a rabbit monoclonal antibody against phosphor-Stat3 (Tyr705) (Cell Signaling, D3A7) and a Vector staining system (Vector, PK-2200) according to the manufacturer’s instructions.

Techniques: Western Blot, Derivative Assay, Activation Assay, Immunoprecipitation, Incubation, Purification, Lysis

Journal: Journal of Biological Chemistry

Article Title: Negative Regulation of Stat3 by Activating PTPN11 Mutants Contributes to the Pathogenesis of Noonan Syndrome and Juvenile Myelomonocytic Leukemia

doi: 10.1074/jbc.m109.020495

Figure Lengend Snippet: FIGURE 7. Schematic diagram of dual-signaling pathways regulated by Shp2. In normal physiological conditions, Stat3 activity is under the positive and negative regulation of Janus kinases and Shp2, respectively (A). Shp2 gain-of-function mutations lead to the hyperactivation of the RAS/ERK signaling pathway and the excessive inactivation of Stat3, which synergistically promotes the pathogenesis of Noonan syndrome and/or JMML (B).

Article Snippet: To analyze phospho-Stat3 expression in the developing heart valve, we used a rabbit monoclonal antibody against phosphor-Stat3 (Tyr705) (Cell Signaling, D3A7) and a Vector staining system (Vector, PK-2200) according to the manufacturer’s instructions.

Techniques: Protein-Protein interactions, Activity Assay

Journal: bioRxiv

Article Title: Leptin Activated Hypothalamic BNC2 Neurons Acutely Suppress Food Intake

doi: 10.1101/2024.01.25.577315

Figure Lengend Snippet: a,c, Brain slices from adult male BNC2-Cre mice, injected with DIO-mCherry into ARC, were subjected to pSTAT3 (a) and cFos (c) immunostaining under three conditions: overnight fasting (FD), overnight fasting followed by refeeding with chow for 3 hours (Ref), and overnight fasting followed by leptin injection (3mg/kg) for 3 hours (Lep). Scale bar, 50µm. b,d, Percentage of pSTAT3/mCherry double-positive cells (b) or cFos/mCherry double-positive cells (d) in mCherry positive cells (N = 4 mice per group). e, Spontaneous APs of BNC2 neurons in ARC from overnight fasted adult male BNC2-Cre mice (injected with DIO-GFP). Leptin (100 nM) was added to the bath solution at the indicated time window. f,g, Spontaneous AP frequency (f) and RMP (g) of BNC2 neurons before, during, and after leptin application (N = 17 cells from 4 mice per group). h, Percentage of leptin-responsive BNC2 neurons under the overnight fasting condition and ad-libitum-fed condition. Data are presented as mean± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Details of the statistical analysis are provided as Source Data.

Article Snippet: The following antibodies were used: cFos antibody (1:1000, Synaptic systems, #226308), pSTAT3 antibody (1: 1000, Cell Signaling Technology, #9145s), GFP (1:1000, abcam, ab13970), RFP (1:1000, Rockland, #600-401-379).

Techniques: Injection, Immunostaining

Journal: bioRxiv

Article Title: Leptin Activated Hypothalamic BNC2 Neurons Acutely Suppress Food Intake

doi: 10.1101/2024.01.25.577315

Figure Lengend Snippet: Brain slices from overnight fasted adult male BNC2-Cre::LSL-Cas9-GFP mice, injected with sgCtrl or sglepr into the unilateral ARC, were subjected to mCherry and pSTAT3 immunostaining 3 hours after leptin injection. Scale bar, 50 µm.

Article Snippet: The following antibodies were used: cFos antibody (1:1000, Synaptic systems, #226308), pSTAT3 antibody (1: 1000, Cell Signaling Technology, #9145s), GFP (1:1000, abcam, ab13970), RFP (1:1000, Rockland, #600-401-379).

Techniques: Injection, Immunostaining

Journal: Frontiers in Pharmacology

Article Title: Oridonin Targets Multiple Drug-Resistant Tumor Cells as Determined by in Silico and in Vitro Analyses

doi: 10.3389/fphar.2018.00355

Figure Lengend Snippet: Molecular docking studies of oridonin and known inhibitors on proteins involved in EGFR signaling pathway. Proteins have been represented in new cartoon format with different colors, while oridonin was represented in yellow. (A) Docking poses in to the pharmacophore of Akt2 kinase domain (PDB code: 3E87 in green cartoon representation). GSK690693 was represented in blue. (B) Docking poses in to the pharmacophore of EGFR tyrosine kinase domain (PDB code: 1M17 in gray cartoon representation). Gefitinib was represented in green and erlotinib was represented in blue. (C) Docking poses in to the pharmacophore of mTOR (PDB code: 4JSP in orange representation). Sirolimus was represented in blue. (D) Docking poses in to the pharmacophore of STAT3 DNA-binding domain (homology model created by using the template PDB code: 1BG1 in pink cartoon representation). NSC74859 was represented in blue. (E) Docking poses in to the pharmacophore of VEGFR1 (PDB code: 3HNG in black cartoon representation). Axitinib was represented in blue.

Article Snippet: Briefly, 1 million cells per well were seeded in 12-well plate, next day treatment with oridonin was performed, total protein were extracted after 24 h. The following primary antibodies (Cell Signaling Technology, Frankfurt, Germany) were used: anti-rabbit EGFR, anti-rabbit phosphorylated EGFR (Tyr1068) (1:1000), anti-rabbit STAT3, anti-mouse phosphorylated STAT3 (Tyr705) (1:1000), anti-rabbit Akt, anti-rabbit phosphorylated Akt (Ser473) (1:1000), and anti-rabbit β-actin (1:2000).

Techniques: Binding Assay

Journal: Frontiers in Pharmacology

Article Title: Oridonin Targets Multiple Drug-Resistant Tumor Cells as Determined by in Silico and in Vitro Analyses

doi: 10.3389/fphar.2018.00355

Figure Lengend Snippet: Western blot analysis of oridonin on EGFR pathway proteins. The effects of oridonin on phosphorylation of ΔEGFR, STAT3, and Akt were evaluated. Bands were normalized to β-actin in order to obtain numerical values (mean ± SEM of three independent experiments). Total EGFR, STAT3, and Akt protein levels are also shown. A representative blot is shown and statistical analysis was done by paired Student’s t -test. ∗∗ p < 0.01, ∗ p < 0.05.

Article Snippet: Briefly, 1 million cells per well were seeded in 12-well plate, next day treatment with oridonin was performed, total protein were extracted after 24 h. The following primary antibodies (Cell Signaling Technology, Frankfurt, Germany) were used: anti-rabbit EGFR, anti-rabbit phosphorylated EGFR (Tyr1068) (1:1000), anti-rabbit STAT3, anti-mouse phosphorylated STAT3 (Tyr705) (1:1000), anti-rabbit Akt, anti-rabbit phosphorylated Akt (Ser473) (1:1000), and anti-rabbit β-actin (1:2000).

Techniques: Western Blot, Phospho-proteomics