Journal: BMC cancer

Article Title: Expression of STAT3 and hypoxia markers in long-term surviving malignant glioma patients.

doi: 10.1186/s12885-024-12221-w

Figure Lengend Snippet: Fig. 2 The expression of selected hypoxia-related markers (IDH1, IDH2, HIF1a, HIF1b, HIF2a, EGFR, PTEN, VEGFA, VEGFC and STAT3) in primary glioma cells GBMLe3, GBMLe4 and GBMDo2 (A) and in cryopreserved samples corresponding to the tumor used for particular primary glioma culture derivation (B) at mRNA level. The expression of mRNA was determined by RT-PCR. Data are expressed as fold increase ± SD of averages from two independent experi ments. Beta-2-microglobulin was used as a housekeeping gene. * p < 0.05 GBM26 vs. GBM43; # p < 0.05 GBM26 vs. GBM59

Article Snippet: Crispr/Cas STAT3 knockout cell model Glioma cells U87MG grown to 50–70% confluence were transfected with transfection mixture (gRNA vectors in Opti-MEM I, the donor DNA and Turbofectin 8.0 - the ratios of 3:1 for Turbofectin: DNA) as based on manufacturer’s protocol (STAT3 Human Gene Knockout Kit (CRISPR), CAT#: KN204922, Origene).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: BMC cancer

Article Title: Expression of STAT3 and hypoxia markers in long-term surviving malignant glioma patients.

doi: 10.1186/s12885-024-12221-w

Figure Lengend Snippet: Fig. 3 Comparison of tumor growth and drug accumulation in Foxn1-nu mice after implantation of glioma cell lines followed by TMZ treatment. Tumor size of implanted (n = 4) (A) U87MG IDH1wt, U87MG STAT3 KO with and without TMZ (0.9 mg/kg) treatment. Evaluation of accumulation of (B) TMZ and (C) its metabolites AIC inside the brain, tumor and plasma in tumor bearing mice with implanted U87MG IDH1wt, resp. U87MG STAT3 KO. The administration of drug (TMZ– 0.9 mg/kg) begins two weeks after implantation (from day 15. to day 28. daily). Organs were collected 15 min after last TMZ application. Confidence interval values of tumor size are shown as mean ± SD. The data of drug accumulation are expressed as ng per mg of tissue. Measurements were performed in two independent experiments

Article Snippet: Crispr/Cas STAT3 knockout cell model Glioma cells U87MG grown to 50–70% confluence were transfected with transfection mixture (gRNA vectors in Opti-MEM I, the donor DNA and Turbofectin 8.0 - the ratios of 3:1 for Turbofectin: DNA) as based on manufacturer’s protocol (STAT3 Human Gene Knockout Kit (CRISPR), CAT#: KN204922, Origene).

Techniques: Comparison, Clinical Proteomics

Journal: BMC cancer

Article Title: Expression of STAT3 and hypoxia markers in long-term surviving malignant glioma patients.

doi: 10.1186/s12885-024-12221-w

Figure Lengend Snippet: Fig. 4 The expression of selected markers related with hypoxia (IDH1, IDH2, HIF1a, HIF1b, HIF2a, EGFR, PTEN, VEGFA, VEGFC and STAT3) in glioma U87MG and U87MG STAT3 KO glioma cell lines (A) and glioma samples collected from Foxn1-nu mice with implanted U87MG and U87MG STAT3 KO glioma cells on mRNA level (B). Tumors were collected 28 days after glioma cell implantation and processed as described in Materials and methods section

Article Snippet: Crispr/Cas STAT3 knockout cell model Glioma cells U87MG grown to 50–70% confluence were transfected with transfection mixture (gRNA vectors in Opti-MEM I, the donor DNA and Turbofectin 8.0 - the ratios of 3:1 for Turbofectin: DNA) as based on manufacturer’s protocol (STAT3 Human Gene Knockout Kit (CRISPR), CAT#: KN204922, Origene).

Techniques: Expressing

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: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular vesicles secreted by hypoxia pre-challenged mesenchymal stem cells promote non-small cell lung cancer cell growth and mobility as well as macrophage M2 polarization via miR-21-5p delivery

doi: 10.1186/s13046-019-1027-0

Figure Lengend Snippet: N-EV and H-EV treatment promote macrophage M2 polarization by delivering miR-21-5p that targets PTEN. a , western blot analysis of PTEN protein expression level in induced macrophages. H/i-miR-EV, monocytes were induced with the presence of EV secreted by miR-21-5p-inhibited, hypoxia pre-challenged MSCs; H-EV + i-miR, monocytes were transfected with miR-21-5p inhibitor-expressing vector before induction with the presence of H-EV. Macrophages induced without MSC-EV were used as negative control (NC). b, c , flow cytometry determining the percentage of CD163 + CD206 + cells among total CD68 + cells after induction. N-EV + O/E PTEN or H-EV + O/E PTEN, monocytes were transfected with PTEN overexpressing vector before N-EV or H-EV treatment, respectively. d – f , western blot detecting Akt and STAT3 protein expression as well as their activating phosphorylation (p-Ser473 for Akt and p-tyr705 for STAT3) in macrophages after induction. g – i , ELISA evaluating IL-10, TGF-β and VEGF-α in macrophage culture medium after induction. Macrophages induced with the presence of N-EV were used as negative control in b – i . Tukey’s test was used for statistical analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001

Article Snippet: Protein level of N-cadherin, E-cadherin, and Vimentin (NBP1–48309, NBP2–19051 and NBP1–31327, respectively, Novus Biologicals), CD9 and CD81 (NBP2–22187 and NB100–65805, Novus Biologicals) Arginase-1 and iNOS (P05089 and MAB9502, R&D Systems), PTEN (4C11A11, BioLegend, San Diego, USA), PDCD4 (NBP2–26138, Novus Biologicals, Littleton, USA), RECK (MA5–14781, Invitrogen), AKT (ab8805, Abcam, Cambridge, USA), STAT3 (NBP2–22471, Novus Biologicals), GAPDH (NB300–221, Novus Biologicals) as well as Ser473 phosphorylation of Akt protein (649,001, BioLegend) and Tyr705 phosphorylation of STAT3 (AF4607, R&D Systems) in cell lysate was analyzed by western blot.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Negative Control, Flow Cytometry, Phospho-proteomics, Enzyme-linked Immunosorbent Assay

Journal: Virology Journal

Article Title: Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication

doi: 10.1186/s12985-023-02146-4

Figure Lengend Snippet: Oligonucleotides sequences used for quantifying miR-141 and mRNA of indicated genes

Article Snippet: For staining MxA and STAT3, the primary antibodies; rabbit polyclonal anti-MxA (Novus Biologicals, NBP132905) and mouse monoclonal anti-STAT3 (Abcam, ab119352) were used.

Techniques:

Journal: Virology Journal

Article Title: Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication

doi: 10.1186/s12985-023-02146-4

Figure Lengend Snippet: The correlation between miR-141 level and the expression profile of MxA and STAT3 in infected A549 cells. (A) Quantification of steady-state miR-141 in infected A549 cells with MOI of 0.5 and transfected with either pre-miR-141 or miR-141 inhibitor compared with noninfected cells (control) using qRT-PCR. (B) Relative gene expression of MxA and STAT3 in infected A549 cells transfected with either specific inhibitor against miR-141 or pre- miR-141 compared with control-transfected cells using qRT-PCR. Error bars indicate the STD of three independent experiments. Student two-tailed t -test used for statistical analysis, (*) indicates P-values ≤ 0.05, and (**) indicates P ≤ 0.01. (C) Flow cytometric assay quantifies the kinetic proteins expression profile of MxA (in blue dots) and STAT3 (in red dots) in infected and transfected A549 cells compared with control cells. (D) Western blot analysis reveals the protein expression level of MxA and STAT3 in infected and transfected cells compared to control cells, β-actin expression profile severed as an internal control

Article Snippet: For staining MxA and STAT3, the primary antibodies; rabbit polyclonal anti-MxA (Novus Biologicals, NBP132905) and mouse monoclonal anti-STAT3 (Abcam, ab119352) were used.

Techniques: Expressing, Infection, Transfection, Control, Quantitative RT-PCR, Gene Expression, Two Tailed Test, Flow Cytometry, Western Blot

Journal: Virology Journal

Article Title: Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication

doi: 10.1186/s12985-023-02146-4

Figure Lengend Snippet: Quantification analysis of miR-141, MxA, and STAT3 in transfected and infected A549 cells

Article Snippet: For staining MxA and STAT3, the primary antibodies; rabbit polyclonal anti-MxA (Novus Biologicals, NBP132905) and mouse monoclonal anti-STAT3 (Abcam, ab119352) were used.

Techniques: Transfection, Infection, Expressing, Control

Journal: Brain research

Article Title: STAT3 protects dopaminergic neurons against degeneration in animal model of Parkinson's disease.

doi: 10.1016/j.brainres.2023.148691

Figure Lengend Snippet: Fig. 1. STAT3 reduced 6-OHDA neurotoxicity in N27 cell lines. Control N27 cells were examined in the absence (A) or presence (B) of 50 µM 6-OHDA for 24 h. N27 cells expressing caSTAT3 were examined in the absence (C) or presence (D) of 50 µM 6-OHDA for 24 h E) Graphical plot illustrating that caSTAT3 transfected N27 cells reduced percentage of cell deaths under 6-OHDA induced neurotoxicity. Three different batches of cell culture used. Result in mean ± SE. For each sample a minimum of 10,000 cells were recorded. The pink box represents the FVD + population of dead cells. The cells were double stained with FVD eFluor 660 and Annexin V-PE. The FVD-/Annexin V- population is regarded as normal healthy cells, while FVD-/AnnexinV + population is early apoptotic cells, and FVD+/AnnexinV+/- population represents necrotic/late apoptotic like cell death. Expression of caSTAT3 greatly reduced the percentage of dead cells 24 h after 6-OHDA treatment. F) Western blot analysis showed expression of phosphorylated STAT3 (pSTAT3) at Tyr705 (lane 3). An upregulation of pSTAT3 expression was found when compared to non-infected (lane 1) or GFP (lane 2) transfected N27 cell lysates. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Recombinant adeno-associated virus 2 (AAV2) carrying GFP or caRheb (kindly provided by Zhigang He, Harvard University, Boston, USA) was tagged with HA or caSTAT3 (pMXs-Stat3-C was a gift from Shinya Yamanaka (Addgene plasmid #13373) by helper virus-free system (Ayuso et al., 2010).

Techniques: Control, Expressing, Transfection, Cell Culture, Staining, Western Blot, Infection

Journal: Brain research

Article Title: STAT3 protects dopaminergic neurons against degeneration in animal model of Parkinson's disease.

doi: 10.1016/j.brainres.2023.148691

Figure Lengend Snippet: Fig. 2. caSTAT3 attenuated 6-OHDA-induced mitochondrial dysfunction. Control fluorescence, caSTAT3-expressing, and caRheb-expressing N27 cells were plated on coverslips and treated with indicated concentrations of 6-OHDA (µM) for 24 hrs prior to imaging (A); cells were loaded with 0.05 nM MitoSOX Red. B) The mean fluorescence intensity (F580 nm) of multiple cells (6 – 50 per condition) was quantified and plotted ± S.E.M. Two-way analysis of variance (ANOVA, F (2, 14) = 9.646, p = 0.0023) indicates significantly decreased mitochondrial superoxide production in caRheb or caSTAT3-expressing cells treated with either 6-OHDA dose compared to control. Sidak post hoc values for individual data points ** p < 0.01, *** p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Recombinant adeno-associated virus 2 (AAV2) carrying GFP or caRheb (kindly provided by Zhigang He, Harvard University, Boston, USA) was tagged with HA or caSTAT3 (pMXs-Stat3-C was a gift from Shinya Yamanaka (Addgene plasmid #13373) by helper virus-free system (Ayuso et al., 2010).

Techniques: Control, Fluorescence, Expressing, Imaging

Journal: Brain research

Article Title: STAT3 protects dopaminergic neurons against degeneration in animal model of Parkinson's disease.

doi: 10.1016/j.brainres.2023.148691

Figure Lengend Snippet: Fig. 4. caSTAT3 protects dopaminergic neurons from 6-OHDA induced neurodegeneration. Data shows dopaminergic neuron survival 4 weeks after unilateral in jections of 6-OHDA in rats pre-injected with AAV/GFP (A,D), AAV/caSTAT3 (B,E,G) and AAV/caRheb (C,F) into the substantia nigra (SN). Coronal brain section (A,B, C) showing the extent of dopaminergic axon terminals with the striatum and the survival of dopaminergic neurons within the substantia nigra (D, E, F). Dramatic protection of striatal terminals and neurons is apparent in animals treated with either caSTAT3 (compare B & E to A & D, respectively) or caRheb (compare C & F to A & D, respectively). High magnification of TH + neurons from panel E shows neurons with a normal morphology (G). Quantitative analysis shows caRheb + caSTAT3, caSTAT3, or caRheb to statistically [ANOVA, F(3, 55) = 7.93, bonferroni post-hoc, p < 0.0001] preserve high density of dopaminergic nerve terminals within the striatum when compared to controls (H). Likewise, caRheb + caSTAT3, caSTAT3, or caRheb resulted in significantly increased survival [ANOVA, F(3, 81) = 21.16 bonferroni post-hoc, p < 0.0001] of dopaminergic neurons within the substantia nigra (I). Scale bar: A-F 500 µm; G 50 µm.

Article Snippet: Recombinant adeno-associated virus 2 (AAV2) carrying GFP or caRheb (kindly provided by Zhigang He, Harvard University, Boston, USA) was tagged with HA or caSTAT3 (pMXs-Stat3-C was a gift from Shinya Yamanaka (Addgene plasmid #13373) by helper virus-free system (Ayuso et al., 2010).

Techniques: Injection

Journal: Brain research

Article Title: STAT3 protects dopaminergic neurons against degeneration in animal model of Parkinson's disease.

doi: 10.1016/j.brainres.2023.148691

Figure Lengend Snippet: Fig. 5. Neuroprotection was observed in caSTAT3 transfected nigral dopami nergic neurons after 6-OHDA insult: A) Neurons are labelled with TH. B) Neurons are labelled with cMyc and C) neurons are merged for TH and cMyc. Data shows dopaminergic neuron survival 4 weeks after unilateral injections of 6-OHDA in rats pre-injected with AAV/caSTAT3. Scale bar = 50 µm.

Article Snippet: Recombinant adeno-associated virus 2 (AAV2) carrying GFP or caRheb (kindly provided by Zhigang He, Harvard University, Boston, USA) was tagged with HA or caSTAT3 (pMXs-Stat3-C was a gift from Shinya Yamanaka (Addgene plasmid #13373) by helper virus-free system (Ayuso et al., 2010).

Techniques: Transfection, Injection

Journal: Brain research

Article Title: STAT3 protects dopaminergic neurons against degeneration in animal model of Parkinson's disease.

doi: 10.1016/j.brainres.2023.148691

Figure Lengend Snippet: Fig. 6. caSTAT3 protects against 6-OHDA motor behavioral impairment: A) Amphetamine-induced rotational behavior was significantly decreased in caRheb + STAT3, caSTAT3, and caRheb expressing rats 4 weeks after 6-OHDA lesioning [ANOVA, F(3, 23) = 9.51, p = 0.0003] when compared to controls. B) After unilateral 6-OHDA lesions GFP treated animals showed a deficit in the use of the right (contralateral) paw, however, those treated with caRheb + caSTAT3, caSTAT3, or caRheb showed significant [ANOVA, F(3,17) = 8.29, p = 0.0013] use of the right paw in glass cylinder searching when compared to controls. Bonferroni post-hoc values for individual data points * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant adeno-associated virus 2 (AAV2) carrying GFP or caRheb (kindly provided by Zhigang He, Harvard University, Boston, USA) was tagged with HA or caSTAT3 (pMXs-Stat3-C was a gift from Shinya Yamanaka (Addgene plasmid #13373) by helper virus-free system (Ayuso et al., 2010).

Techniques: Expressing

Journal: Nature Communications

Article Title: CD44 connects autophagy decline and ageing in the vascular endothelium

doi: 10.1038/s41467-023-41346-y

Figure Lengend Snippet: a Fluorescence analysis of the colocalization of CD44ICD with endogenous STAT3 and core proteins of the PtdIns3K complexes in HUVECs transduced with Ev or CD44ICD. Nuclei were stained with DAPI. Bar = 10 μm. The line scanned profiles at the right of confocal images show the distribution of fluorescence for each channel in the white line in the corresponding confocal images. b Reactivity of CD44ICD or STAT3 with PtdIns3K complex proteins, and STAT3 with CD44ICD in a yeast two-hybrid system. Yeast strain AH109 was co-transformed with a bait plasmid, BD-CD44ICD or BD-STAT3, and a prey plasmid, pGADT7-PtdIns3K complex proteins (AD-ATG14L, AD-BECN1, AD-UVRAG, AD-PIK3C3, and AD-PIK3R4) or pGADT7-CD44ICD, which encodes PtdIns3K complex proteins or CD44ICD fused to the Gal4 activation domain. Co-transformation of BD-Lam/AD-T and BD-P53/AD-T was used as negative and positive controls, respectively. c BiFC analysis of CD44ICD-STAT3 and STAT3-PIK3R4 interactions. HUVECs were transfected with indicated combinations of constructs. Co-transfection of VN-Jun and VC-Fos was used as positive control. Bar = 30 μm. d , e GST-pull-down of recombinant STAT3 with a GST-CD44ICD fusion protein ( d ) or a GST-P150 domain fusion protein ( e ). f The binding mode of full-sequence structures of STAT3 and CD44ICD. Colors indicate: STAT3, green; CD44ICD, red. The key residues interacting between STAT3 and CD44ICD are indicated as gray and yellow, respectively. Hydrogen bond is described by pink dash lines. g Western blots showing co-IP of CD44ICD_ΔN35 with endogenous STAT3 and core proteins of the PtdIns3K complexes in HUVECs transduced with Ev or CD44ICD_ΔN35 (DDK tag). Lysates, whole cell lysates; IP, immunoprecipitates. h Proposed schematic diagram of CD44ICD-mediated autophagy decline. CD44ICD suppresses the levels of PIK3C3 and PIK3R4 and the kinase activity of PIK3C3, activates STAT3, and disrupts the assembly of the PtdIns3K complex by interacting with STAT3. Three biologically independent experiments. Source data are provided as a Source data file.

Article Snippet: For the BiFC assay, the sequences of CD44ICD, PIK3R4 and STAT3 were cloned and inserted into both VN155 and VC155 (Addgene, MA, USA).

Techniques: Fluorescence, Transduction, Staining, Transformation Assay, Plasmid Preparation, Activation Assay, Transfection, Construct, Cotransfection, Positive Control, Recombinant, Binding Assay, Sequencing, Western Blot, Co-Immunoprecipitation Assay, Activity Assay