stat3 promoter  (Igene Biotechnology Inc)

Journal: PLOS ONE

Article Title: Homocysteine causes neuronal leptin resistance and endoplasmic reticulum stress

doi: 10.1371/journal.pone.0278965

Figure Lengend Snippet: (A-B) Homocysteine inhibited leptin-induced STAT3 phosphorylation. SHSY5Y Ob-Rb cells were pretreated with homocysteine (Hcy; 3 and 10 mM) for 4 h and then stimulated with leptin (Lep; 0.01 μg/mL) for 15 min and leptin-induced STAT3 phosphorylation was analyzed by immunoblot. Statistical analysis used Dunnett’s post-hoc test following one-way ANOVA; *p< 0.05, n = 3–5. (C-D) Methionine and cysteine were slightly inhibited leptin-induced STAT3 phosphorylation. SHSY5Y Ob-Rb cells were treated with homocysteine (Hcy, 10 mM), methionine (Met, 10 mM) and cysteine (Cys, 10 mM) for 4 h and then stimulated with leptin (Lep; 0.01 μg/mL, 15 min), and leptin-induced STAT3 phosphorylation was analyzed. Statistical analysis used Turkey’s post-hoc test following one-way ANOVA; * p < 0.05, n = 3–5.

Article Snippet: SH-SY5Y Ob-Rb cells, transfected with STAT3 promoter-NanoLuc plasmid (Promega), were cultured at 96 well plate for 48 h. Cells were then treated with homocysteine (1–10 mM) and leptin (0.01μg/mL, equal to 0.6 nM) for 4 h, and luminescence signal was measured using Nano-Glo® Luciferase Assay System (Promega).

Techniques: Phospho-proteomics, Western Blot

Journal: PLOS ONE

Article Title: Homocysteine causes neuronal leptin resistance and endoplasmic reticulum stress

doi: 10.1371/journal.pone.0278965

Figure Lengend Snippet: SH-SY5Y-Ob-Rb cells transfected with the STAT3-promoter-Nluc construct were treated with homocysteine (Hcy; 1–10 mM) and leptin (Lep; 0.01 μg/mL), and the luminescence signal is measured 4 hours after treatments. Data were expressed as relative luciferase activity, normalized with the control condition. * p < 0.05, ** p < 0.01, n = 4.

Article Snippet: SH-SY5Y Ob-Rb cells, transfected with STAT3 promoter-NanoLuc plasmid (Promega), were cultured at 96 well plate for 48 h. Cells were then treated with homocysteine (1–10 mM) and leptin (0.01μg/mL, equal to 0.6 nM) for 4 h, and luminescence signal was measured using Nano-Glo® Luciferase Assay System (Promega).

Techniques: Transfection, Construct, Luciferase, Activity Assay, Control

Journal: PLOS ONE

Article Title: Homocysteine causes neuronal leptin resistance and endoplasmic reticulum stress

doi: 10.1371/journal.pone.0278965

Figure Lengend Snippet: (A-B) HEK293T cells were treated with homocysteine (Hcy) for 4 hours at 10 mM, then stimulated with IL-6 (100 ng/mL) for 15 minutes. The level of phospho-STAT3 was analyzed. n = 5. (C-D) HEK293T cells were treated with homocysteine (Hcy) for 4 hours at 10 mM, then stimulated with IL-6 (100 ng/mL) for 15 minutes. The level of HERP was analyzed. n = 5.

Article Snippet: SH-SY5Y Ob-Rb cells, transfected with STAT3 promoter-NanoLuc plasmid (Promega), were cultured at 96 well plate for 48 h. Cells were then treated with homocysteine (1–10 mM) and leptin (0.01μg/mL, equal to 0.6 nM) for 4 h, and luminescence signal was measured using Nano-Glo® Luciferase Assay System (Promega).

Techniques:

Journal: PLOS ONE

Article Title: Homocysteine causes neuronal leptin resistance and endoplasmic reticulum stress

doi: 10.1371/journal.pone.0278965

Figure Lengend Snippet: High concentrations of homocysteine trigger ER stress and inhibit the phosphorylation and transcriptional activity of STAT3 regulated by the anti-obesity hormone leptin. This mechanism may contribute to homocysteine-induced leptin resistance in neuronal cells.

Article Snippet: SH-SY5Y Ob-Rb cells, transfected with STAT3 promoter-NanoLuc plasmid (Promega), were cultured at 96 well plate for 48 h. Cells were then treated with homocysteine (1–10 mM) and leptin (0.01μg/mL, equal to 0.6 nM) for 4 h, and luminescence signal was measured using Nano-Glo® Luciferase Assay System (Promega).

Techniques: Phospho-proteomics, Activity Assay

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet: Abi1 modulates phosphorylation and activation of Jak2 and STAT3 (A and B) Abi1 KD attenuates the PDGF-induced Jak2 (A) and STAT3 (B) phosphorylation in HASM cells. Data are means ± SEM. n = 5. Two-way ANOVA was used for statistical analysis. (C) Abi1 deficiency inhibits the PDGF-induced Jak2 kinase activity. Data are means ± SEM. n = 5. Two-way ANOVA was used for statistical analysis. (D) The reporter activity of STAT3 is reduced by Abi1 KD. Data are means ± SEM. n = 5. Two-way ANOVA was used for statistical analysis. (E and F) Overexpression of Abi1 in HASM cells enhances Jak2 kinase activity and STAT3 activity. Data are means ± SEM. n = 5. The t test was used for statistical analysis. ∗p < 0.05; ∗∗p < 0.01. Data normalization is calculated as follows: Data of treated samples/Data of control samples.

Article Snippet: Lentivirus encoding STAT3 luciferase promoter , BPS Bioscience , #79744.

Techniques: Activation Assay, Activity Assay, Over Expression

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet: Abi1 regulates STAT3 nuclear import in smooth muscle cells (A) Cells with perinuclear STAT3 localization are considered “untranslocated cells.” Cells with concentrated STAT3 distribution in the nucleus are considered “translocated cells.” The white arrow points to the nucleus without STAT3 translocation. The green arrows point to the nuclei with STAT3 translocation. (B) PDGF stimulation induces STAT3 translocation to the nucleus. Data are means ± SEM. n = 5. (C) Abi1 KD inhibits the PDGF-induced STAT3 nuclear import. Ctrl and Abi1 KD cells were treated with PDGF for 2 h followed by immunostaining. Data are means ± SEM. n = 4. Two-way ANOVA was used for statistical analysis. (D) Overexpression (OE) of Abi1 is sufficient to induce STAT3 nuclear translocation. Cells were infected with lentivirus encoding the constructs for 2 days followed by staining. Data are means ± SEM. n = 4. The t test was used for statistical analysis. ∗∗p < 0.01. The percentage of cells with translocated STAT3 was calculated as follows: numbers of cells with translocated STAT3/numbers of total cells observed × 100.Scale bar, 10 μm

Article Snippet: Lentivirus encoding STAT3 luciferase promoter , BPS Bioscience , #79744.

Techniques: Translocation Assay, Immunostaining, Over Expression, Infection, Construct, Staining

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet: Abi1 knockdown inhibits the activity of Jak2/STAT3 and the proliferation in Abi1-up HASM cells (A and B) The basal and the PDGF-induced activity of Jak2 (A) and STAT3 (B) is higher in Abi1-up cells. Data are means ± SEM. n = 4 for each group. One-way ANOVA was used for statistical analysis. (C and D) Abi1 KD inhibits the basal and the PDGF-induced activity of Jak2 (C) and STAT3 (D) in Abi1-up cells. Data are means ± SEM. n = 4 for each group. Two-way ANOVA was used for statistical analysis (E and F) Abi1 KD reduced the PDGF-induced cell numbers (E) and BrdU incorporation (F). Data are means ± SEM. n = 4 for each group. The t test was used for statistical analysis. ∗p < 0.05. Data normalization is calculated as follows: Data of treated samples/Data of control samples.

Article Snippet: Lentivirus encoding STAT3 luciferase promoter , BPS Bioscience , #79744.

Techniques: Activity Assay, BrdU Incorporation Assay

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet: Proposed mechanism Stimulation with growth factors such as PDGF induces spatial distribution of Abi1, which facilitates the activation of the Jak2/STAT3 pathway. The association of Abi1 with Jak2 renders the receptor in active state. Abi1 is upregulated in ∼50% of asthmatic airway smooth muscle cultures, promoting the activation of Jak2 and STAT3, hyperplasia, and airway smooth muscle (ASM) thickening.

Article Snippet: Lentivirus encoding STAT3 luciferase promoter , BPS Bioscience , #79744.

Techniques: Activation Assay

Journal: iScience

Article Title: Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling

doi: 10.1016/j.isci.2022.103833

Figure Lengend Snippet:

Article Snippet: Lentivirus encoding STAT3 luciferase promoter , BPS Bioscience , #79744.

Techniques: Luciferase, shRNA, Recombinant, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Kinase Assay, Software, Confocal Microscopy, Microscopy

Journal: Scientific Reports

Article Title: Application of the antitussive agents oxelaidin and butamirate as anti-glioma agents

doi: 10.1038/s41598-021-89238-9

Figure Lengend Snippet: Screening of RRAD inhibitors for drug repositioning using LN229-RRAD human glioblastoma tumorspheres. ( A ) RRAD inhibitor screening was performed using the 2,261 clinical compound library in LN229 cells expressing RRAD (LN229-RRAD). LN229-RRAD cells were treated with 3 μM of each compound for 7 days and the number of tumorspheres counted under a phase-contrast microscope. ( B ) Chemical structure of oxelaidin. ( C ) Dose–response curves showing the survival effect of RRAD overexpression. Cells were treated with the indicated doses of oxelaidin for 7 days, followed by tumorsphere counting. ( D ) Effect of OXL on growth of glioblastoma cells (U87MG and T98G). Cells were treated with OXL for 7 days, followed by tumorsphere counting. ( E ) STAT3 activity in LN229-RRAD and U87MG is downregulated by OXL. A STAT3-dependent luciferase reporter system was established in LN229-Vector, LN229-RRAD, and U87MG. After OXL treatment (5 μM for 2 h), STAT3 activity was monitored using the luciferase assay. ( F ) qRT-PCR assay of endogenous STAT3 target gene expression in LN229-RRAD tumorspheres treated with OXL for 4 days using GAPDH as a loading control. (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).

Article Snippet: STAT3 promoter plasmid (4 × M67 pTATA TK-Luc, #8688) was acquired from Addgene (Cambridge, MA) and pRL-TK renilla luciferase plasmid from Promega (Madison, WI).

Techniques: Expressing, Microscopy, Over Expression, Activity Assay, Luciferase, Plasmid Preparation, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Application of the antitussive agents oxelaidin and butamirate as anti-glioma agents

doi: 10.1038/s41598-021-89238-9

Figure Lengend Snippet: Effects of oxelaidin on EGFR and STAT3 signaling in glioblastoma. ( A ) Kaplan–Meier survival curve for EGFR high patients with GBM according to high (n = 30) and low (n = 57) expression levels of RRAD. All glioma patient data were publicly available in the de-identified form and obtained from the NCI Repository for Molecular Brain Neoplasia Data (REMBRANDT). Data mining and statistical analyses were performed using Project Betastasis software ( http://www.betastasis.com/glioma/ ). ( B ) U87MG tumorspheres were treated with the indicated concentration (0, 1, 5 μM) of OXL for 3 h. ( C ) U87MG tumorspheres were treated with 5 μM of OXL for 0, 0.5, 1 and 3 h. Protein expression of pEGFR, pSTAT3, pAKT and pERK detected via western blot using actin as a loading control. ( D ) PLA of RRAD and activated EGFR or STAT3 performed using RRAD antibodies and either pEGFR Y1101 or pSTAT3 Y705 antibodies in U87MG. Cells were pretreated with OXL for 2 h and subsequently stimulated with EGF (50 ng/mL) for 15 min. Red dots represent interaction signals. Nuclei were stained with the DAPI. Fluorescent signals were detected and images captured at × 400 magnification (Error bars represent ± S.D.; ** P ≤ 0.01).

Article Snippet: STAT3 promoter plasmid (4 × M67 pTATA TK-Luc, #8688) was acquired from Addgene (Cambridge, MA) and pRL-TK renilla luciferase plasmid from Promega (Madison, WI).

Techniques: Expressing, Software, Concentration Assay, Western Blot, Staining

Journal: Scientific Reports

Article Title: Application of the antitussive agents oxelaidin and butamirate as anti-glioma agents

doi: 10.1038/s41598-021-89238-9

Figure Lengend Snippet: In silico prediction of docking simulation between RRAD and oxelaidin. The molecular docking study on OXL was based on X-ray crystal structures of RRAD (PDB code: 3Q72). ( A ) The pharmacophore features correspond to compound interactions with two binding poses of OXL (white and pink). Red: negative charge, blue: positive charge. Two potent docking simulation models are shown. Hydrogen bond interactions (H-bond) between Q250 (bold) and OXL (pink; model I, white; model II) are represented by a red dotted line. Pi-Pi stacking interactions (pi-pi) between K228 (model I) or R249 (model II) and OXL are represented with a blue dotted line. Energy profile of predicted binding poses obtained through molecular dynamic simulations and MM/GBSA calculations. ( B ) RRAD-FLAG expression was detected via western blot using tubulin as a loading control. LN229 cells were transfected with empty vector, wild-type RRAD or the RRAD mutated at the OXL interaction site (K228, R249, and Q250). ( C ) Mutation of the putative oxelaidin interaction sites of RRAD rescues the inhibitory effect of OXL (5 μM) on STAT3 activity of LN229 glioblastoma cells (** P ≤ 0.01). (D) Dose–response curves detailing the survival effects of mutant RRAD on tumorsphere formation. Cells were treated with the indicated doses of OXL for 7 days, followed by tumorspheres counting.

Article Snippet: STAT3 promoter plasmid (4 × M67 pTATA TK-Luc, #8688) was acquired from Addgene (Cambridge, MA) and pRL-TK renilla luciferase plasmid from Promega (Madison, WI).

Techniques: In Silico, Binding Assay, Expressing, Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Activity Assay

Journal: Scientific Reports

Article Title: Application of the antitussive agents oxelaidin and butamirate as anti-glioma agents

doi: 10.1038/s41598-021-89238-9

Figure Lengend Snippet: Comparison of the effects of oxelaidin with those of the structurally related antitussive agents. ( A ) Structures of antitussive agents, butamirate citrate, pre-084 and caramiphen. ( B ) U87MG, U87MG-TMZ-resistant (U87 TMZR), U87MG-lapatinib-resistant (U87 LAPAR) and T98G (TMZ resistant) tumorspheres were cultured in the presence of indicated drug for 7 days and the number of tumorspheres counted. ( C ) Dose–response curves showing the survival of LN229-RRAD after treatment with the indicated doses of oxelaidin or butamirate for 7 days, followed by tumorsphere counting. Western blot analysis of protein expression of pEGFR, pSTAT3, pAKT and pERK using actin as a loading control. ( F ) PLA of RRAD and activated STAT3 in U87MG performed using RRAD antibodies and pSTAT3 Y705 antibodies. U87MG cells were pretreated with butamirate citrate for 2 h, followed by stimulation with EGF (50 ng/mL) for 15 min. Red dots represent interaction signals. Nuclei were stained with DAPI. Representative images from analyses of two independent slides. Fluorescent signals were detected and images captured at × 400 magnification (** P ≤ 0.01 and ***P < 0.001). Error bars represent ± SD. ( D ) Butamirate downregulates STAT3 activity in LN229-RRAD but not in LN229-Vector cells. ( E ) Butamirate downregulates pEGFR, pSTAT3, pAKT and pERK Western blot analysis of protein expression of pEGFR, pSTAT3, pAKT and pERK using actin as a loading control. ( F ) Inhibitory effects of Butamirate on EGF-induced STAT3 activity and the RRAD-pSTAT3 interaction. PLA of RRAD and activated STAT3 in U87MG performed using RRAD antibodies and pSTAT3 Y705 antibodies. U87MG cells were pretreated with butamirate for 2 h, followed by stimulation with EGF (50 ng/mL) for 15 min. Red dots represent interaction signals. Nuclei were stained with DAPI. Representative images from analyses of two independent slides. Fluorescent signals were detected and images captured at × 400 magnification. Error bars represent ± SD. ( G ) Transwell migration assay. LN229 and U87MG cells were exposed to 5 μM oxelaidin or butamirate for 2 h before cell migration. Results are presented as means ± SD (n = 3). (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).

Article Snippet: STAT3 promoter plasmid (4 × M67 pTATA TK-Luc, #8688) was acquired from Addgene (Cambridge, MA) and pRL-TK renilla luciferase plasmid from Promega (Madison, WI).

Techniques: Cell Culture, Western Blot, Expressing, Staining, Activity Assay, Plasmid Preparation, Transwell Migration Assay, Migration