Journal: Oncotarget

Article Title: Anti-tumor activity of selective inhibitors of XPO1/CRM1-mediated nuclear export in diffuse malignant peritoneal mesothelioma: the role of survivin

doi:

Figure Lengend Snippet: A. Representative western immunoblotting showing nuclear and cytosolic fractions of XPO1/CRM1, p53, CDKN1a and survivin in DMPM cells exposed to selinexor (IC 50 ). β-actin and TBP were used to confirm equal protein loading on the gel and to show the relative purity of the nuclear fractions. B. Quantification of nuclear and cytosolic survivin protein levels by ELISA assay in DMPM cells exposed to selinexor (IC 50 ) alone or in the presence of subtoxic concentrations of Bortezomib. Data are reported as amount (pg) of survivin normalized to total (mg) protein, and represent the mean values ±SD of at least three independent experiments. C. Representative western immunoblotting showing the expression of survivin, STAT3 and Ac-STAT3 in DMPM cells exposed to selinexor (IC 50 ). β-actin was used to confirm equal protein loading on the gel. D. Quantification of survivin protein levels by ELISA assay in DMPM cells exposed to selinexor alone (IC 50 ) or in the presence of subtoxic concentrations of Bortezomib (1 nmol/L). Data are reported as the percentage of survivin expression in selinexor-treated cells compared with cells exposed to 0.01% DMSO (ctr), and represent the mean values ±SD of at least three independent experiments. E. Quantification of survivin mRNA expression levels by qRT-PCR in DMPM cells exposed to selinexor (IC 50 ). Data are reported as log10-transformed relative quantity (RQ) in selinexor-treated cells with respect to cells exposed to 0.01% DMSO (ctr), and represent the mean values ±SD of at least three independent experiments. Dashed line: relative survivin mRNA expression level in the ctr. F. Representative IP experiment showing increased ubiquitination of nuclear survivin in STO cells exposed to selinexor (IC 50 ) *** P < 0.001, ** P < 0.01, * P < 0.05, vs ctr; °°° P < 0.001, °° P < 0.01, ° P < 0.05, cells exposed to selinexor vs cells exposed to selinexor+Bortezomib.

Article Snippet: The antibodies used in the study were CDKN1a (#ab7960), p53 (#ab26), ubiquitin (#ab7780), survivin (#ab469), β-actin (#ab8227), TBP (#ab818) (Abcam Inc.), STAT3 (#4904) and STAT3 Ac (#2523) (Cell Signaling Technology).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Transformation Assay, Ubiquitin Proteomics

Journal: Molecular Medicine Reports

Article Title: Traditional Chinese medicine, Fuzheng Kang-Ai decoction, inhibits metastasis of lung cancer cells through the STAT3/MMP9 pathway

doi: 10.3892/mmr.2017.6905

Figure Lengend Snippet: STAT3 regulates MMP9 activity in lung cancer cells treated with FZKA. (A) Protein expression levels of STAT3 were reduced following treatment with FZKA (2 mg/ml; 0, 0.5, 2, 4 and 8 h). *P<0.05 vs. 0 h. (B) To overexpress STAT3, cells (H1650, A549 and PC9) were seeded into 6-well plates, and transfected with pCMV6-AC (negative control) and pCMV6-AC-STAT3 DNA constructs, prior to treatment with FZKA. STAT3 protein expression was then measured by western blot analysis. (C) MMP9 activity was increased by STAT3 overexpression. Following treatment with FZKA, the FZKA-mediated inhibition of MMP9 activity was significantly suppressed by STAT3 overexpression. *P<0.05 and **P<0.01 vs. control (Ctrl). FZKA, Fuzheng Kang-Ai; MMP9, matrix metalloproteinase 9; STAT5, signal transducer and activator of signaling 3; Ctrl, control.

Article Snippet: The control (pCMV6-AC) and STAT3 overexpression (pCMV6-AC-STAT3) vectors were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Activity Assay, Expressing, Transfection, Negative Control, Construct, Western Blot, Over Expression, Inhibition

Journal: Carcinogenesis

Article Title: Aberrantly expressed Fra-1 by IL-6/STAT3 transactivation promotes colorectal cancer aggressiveness through epithelial–mesenchymal transition

doi: 10.1093/carcin/bgv017

Figure Lengend Snippet: Fra-1 is transcriptionally regulated by STAT3 in response to IL-6 stimulation. ( A ) Western blots with the indicated antibodies from HT-29 cells pretreated for 1h with LY294002, U0126, or Stattic (specific inhibitors of PI3K, MEK and STAT3, respectively) and exposed to IL-6 for 12h. ( B ) Western blots of Fra-1 and STAT3 from HT-29 cells transfected with control or STAT3 siRNAs and incubated with IL-6 for 12h (GAPDH was used as a loading control). ( C ) Schematic representation of Fra-1 promoter with seven potential SIEs and the primer pair used in ChIP-PCR assays. The reporter construct Fra-1-Luc and its truncated and mutated derivatives are also shown. ( D ) Transcription activity in response to IL-6 treatmemt for 6h measured by luciferase assay in 293T cells with a series of deletion mutants of Fra-1-luc (internal control, pRL-TK). * P < 0.05. ( E ) Relative luciferase activity 6h after IL-6 incubation in 293T cells transfected with the wild-type or SIE mutated Fra-1 promoter reporter construct. * P < 0.05. ( F ) Chromatin prepared from HT-29 cells stimulated with IL-6 for 1h was immunoprecipitated with the indicated antibodies; PCR was performed on immunoprecipitated DNAs or soluble chromatin using specific primer pair for the Fra-1 promoter.

Article Snippet: The ZEB1, Snail, Slug, ERK1/2, p-ERK1/2, AKT, p-AKT, STAT3, p-STAT3 (Y705) and ac-STAT3 (K685) antibodies were from Cell Signaling Technology (Beverly, MA).

Techniques: Western Blot, Transfection, Control, Incubation, Construct, Activity Assay, Luciferase, Immunoprecipitation

Journal: Carcinogenesis

Article Title: Aberrantly expressed Fra-1 by IL-6/STAT3 transactivation promotes colorectal cancer aggressiveness through epithelial–mesenchymal transition

doi: 10.1093/carcin/bgv017

Figure Lengend Snippet: Acetylation and phosphorylation are both required for STAT3 activation to transactivate the Fra-1 gene. ( A ) Western blots of STAT3 and its PTM status with site-specific antibodies in serum-starved HT-29 cells treated with 50ng/ml IL-6 for the indicated times. ( B ) Western blots for the STAT3-null PC3 cells transfected with empty vector (EV), wild-type (WT) or mutated STAT3 and stimulated with IL-6 for 1h. ( C ) Western blots with specific antibodies from whole-cell extracts of HT-29 cells pretreated with AG490, anacardic acid or trichostatin A (inhibitors of JAK2, histone acetyltransferases and histone deacetylases, respectively) and further stimulated with IL-6 for 12h. ( D ) Relative luciferase activity in PC3 cells co-transfected with wild-type or mutated STAT3, Fra-1 promoter reporter (−720/+173) and an internal control reporter pRL-TK and administered with IL-6 24h later. * P < 0.05. ( E ) Upon IL-6 stimulation for 1h, ChIP assays were performed in PC3 cells reintroduced with wild-type STAT3 or derived mutants using the indicated antibodies. The Fra-1 promoter region containing STAT3 binding sites was amplified by PCR. ( F ) PC3 cells transfected with STAT3-WT or its mutant were treated with IL-6 for 1h, the binding ability of STAT3 to the biotin-labeled Fra-1 promoter probe (−760/−524) was analyzed by DNA pull-down assay. Unlabeled Fra-1 promoter probe (cold-probe) was used for competitive inhibition. Ku80 served as a control.

Article Snippet: The ZEB1, Snail, Slug, ERK1/2, p-ERK1/2, AKT, p-AKT, STAT3, p-STAT3 (Y705) and ac-STAT3 (K685) antibodies were from Cell Signaling Technology (Beverly, MA).

Techniques: Phospho-proteomics, Activation Assay, Western Blot, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Control, Derivative Assay, Binding Assay, Amplification, Mutagenesis, Labeling, Pull Down Assay, Inhibition

Journal: Redox Biology

Article Title: Gestational zinc deficiency impairs brain astrogliogenesis in rats through multistep alterations of the JAK/STAT3 signaling pathway

doi: 10.1016/j.redox.2021.102017

Figure Lengend Snippet: Gestational MZD affects different steps in the STAT3 pathway in the offspring brain cortex at different developmental stages. Dams were fed ad libitum either a control (C) or a marginal zinc diet (MZD) from gestation day 0 until E14, E19 and P2, at which time dams, and subsequently (after P20) the offspring were fed a control diet until P56. A) Experimental design. B-M) Brain cortex homogenates were prepared as described in the Materials and methods section. Western blots for B-D ) phosphorylated STAT3 at tyrosine-705 (p Y 705 -STAT3), total STAT3, and GAPDH; E-G ) phosphorylated JAK2 at tyrosine-1007/1008 (p Y 1007/1008 -JAK2), total JAK2, and GAPDH; H-J ) PTP1B, SHP2 and GAPDH; and K-M ) CT-1, LIF and GAPDH. GAPDH was used as loading control. After quantifications of bands, values were calculated as the ratios C ) p Y 705 -STAT3/STAT3, D ) STAT3/GAPDH, F ) p Y 1007/1008 -JAK2/JAK2, G ) JAK2/GAPDH, I ) PTP1B/GAPDH, J ) SHP2/GAPDH, L ) CT-1//GAPDH and M ) LIF/GAPDH. For all proteins values were normalized to those of the E14 control group. Results are shown as means ± S.E.M and are the average of 6 litters per group per developmental stage. *, p ≤ 0.05; **, p ≤ 0.01 are significantly different compared to the respective control at each developmental stage (Student's t -test).

Article Snippet: Primary antibodies for phospho-tyrosine 705 STAT3 (p Y705 -STAT3), acetyl-lysine 685 STAT3 (ac-STAT3), total STAT3, phospho-tyrosine 1007/1008 Janus kinase 2 (JAK2) (p Y1007/1008 -JAK2), total JAK2 (JAK2), and ubiquitin were purchased from Cell Signaling Technologies (Danvers, MA).

Techniques: Control, Western Blot

Journal: Redox Biology

Article Title: Gestational zinc deficiency impairs brain astrogliogenesis in rats through multistep alterations of the JAK/STAT3 signaling pathway

doi: 10.1016/j.redox.2021.102017

Figure Lengend Snippet: Gestational MZD alters the interactions of STAT3 with the tyrosine phosphatase PTP1B and cytoskeleton proteins in the E19 brain CT. A-C ) STAT3 was immunoprecipitated from E19 CT tissue lysates as described in the Materials and methods section. A) Western blots for PTP1B, SHP2, α-tubulin, β-actin and STAT3 in the STAT3 immunoprecipitates (IP). B ) Western blot for phosphotyrosine-705 STAT3 (p Y 705 -STAT3) and STAT3 I in the STAT3 immunoprecipitates (IP). C ) After quantifications of bands, values were calculated as the ratios p Y 705 -STAT3/STAT3, SHP2/STAT3, PTP1B/STAT3, α-tubulin/STAT3, and β-actin/STAT3, and normalized to control levels. D,E) Brain CT was subjected to non-reducing homogenization and SDS-PAGE. D ) Upper panel: Western blots for α-tubulin in control (C1,C2) and MZD (MZ1, MZ2) samples treated without (−) or with (+) addition of 1% (v/v) β-mercaptoethanol. St: molecular weight standard. HSC70 was assessed as loading control. E) After quantification, the ratio of intensity between bands of molecular weight higher than 100 kDa/50 kDa was calculated. Results are shown as means ± S.E.M. of six litters per group. *, p ≤ 0.05; **, p ≤ 0.01 are significantly different compared to controls (Student's t -test).

Article Snippet: Primary antibodies for phospho-tyrosine 705 STAT3 (p Y705 -STAT3), acetyl-lysine 685 STAT3 (ac-STAT3), total STAT3, phospho-tyrosine 1007/1008 Janus kinase 2 (JAK2) (p Y1007/1008 -JAK2), total JAK2 (JAK2), and ubiquitin were purchased from Cell Signaling Technologies (Danvers, MA).

Techniques: Immunoprecipitation, Western Blot, Control, Homogenization, SDS Page, Molecular Weight

Journal: Redox Biology

Article Title: Gestational zinc deficiency impairs brain astrogliogenesis in rats through multistep alterations of the JAK/STAT3 signaling pathway

doi: 10.1016/j.redox.2021.102017

Figure Lengend Snippet: Gestational MZD impaired STAT3-DNA binding and STAT3 acetylation in nuclear fractions form E19 and P2 brain CT. Nuclear and cytosolic fractions were isolated from E19 and P2 brain CT as described in the Materials and methods section. A,B) EMSA for STAT3 in nuclear (NF) and cytosolic fractions (CF) isolated from E19 and P2 CT. To determine the specificity of STAT3-DNA complex, a control NF was incubated in the presence of a 100-fold molar excess of unlabeled oligonucleotide containing the consensus sequence for an unspecific (U) transcription factor before the binding assay. A) Representative image for NF, B ) after the EMSA assay, bands were quantified and the ratio nuclear/cytosolic DNA binding (NF/CF) was calculated. Results were referred to the control P2 value (1 ). C–F) Western blots for ac K685 -STAT3, p Y 705 -STAT3, STAT3 and hnRNP in NF; and STAT3 and β-actin in CF from C,D ) E19 and E,F) P2 offspring CT. C,E show representative images. D,F ) After quantifications of bands, values were calculated as the ratios p Y 705 -STAT3/STAT3 and ac K685 -STAT3/STAT3 in NF and the ratio of STAT3 NF/STAT3 in CF and normalized to control levels. Results are shown as means ± S.E.M. of six litters per group. *, p ≤ 0.05; **, p ≤ 0.01 are significantly different compared to the respective control (Student's t -test).

Article Snippet: Primary antibodies for phospho-tyrosine 705 STAT3 (p Y705 -STAT3), acetyl-lysine 685 STAT3 (ac-STAT3), total STAT3, phospho-tyrosine 1007/1008 Janus kinase 2 (JAK2) (p Y1007/1008 -JAK2), total JAK2 (JAK2), and ubiquitin were purchased from Cell Signaling Technologies (Danvers, MA).

Techniques: Binding Assay, Isolation, Control, Incubation, Sequencing, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Resveratrol Alleviates 27-Hydroxycholesterol-Induced Senescence in Nerve Cells and Affects Zebrafish Locomotor Behavior via Activation of SIRT1-Mediated STAT3 Signaling

doi: 10.1155/2021/6673343

Figure Lengend Snippet: The 27HC induces STAT3 activation via SIRT1. (a) BV2 and PC12 cells were treated with 10 μ M 27HC and 10 nM E2. Western blot analysis was performed for SIRT1, Ac-STAT3Lys685, and β -Actin expression. CON group: ethanol treatment. (b) The quantitative analysis of protein bands of (a). ∗ P < 0.05, statistically significant difference from the CON group. SIRT1: (BV2: P = 0.0011, 27HC group; P = 0.0082, E 2 group) (PC12: P = 0.0248, 27HC group; P = 0.0947, E2 group); AC-STAT3 Lys685 : (BV2: P = 0.0005, 27HC group; P = 0.0009, E 2 group) (PC12: P = 0.0026, 27HC group; P = 0.4597, E 2 group). Data are expressed as the mean ± SD of three independent experiments. (c) BV2 and PC12 cells were treated with si-NC or siRNA-SIRT1 for 12 h. Western blot analysis was performed for SIRT1, p-STAT3 Tyr705 , Ac-STAT3 Lys685 , and β -actin expression. The two sets are experimental replicates. (d) The quantitative analysis of protein bands of (c). ∗ P < 0.05, statistically significant difference from the si-NC group. BV2: P < 0.0001, SIRT1; P < 0.0001, p-STAT3 Tyr705 ; P < 0.0001, Ac-STAT3 Lsy685 ; PC12: P < 0.0001, SIRT1; P = 0.0003, p-STAT3 Tyr705 ; P = 0.0001, Ac-STAT3 Lsy685 . (e) qRT-PCR analysis of IL-6 and SIRT1. ∗ P < 0.05, statistically significant difference from the si-NC group. (BV2: P = 0.0027, SIRT1; P = 0.0044, IL-6; PC12: P < 0.0001, SIRT1; P = 0.0013, IL-6). Data are expressed as the mean ± SD of three independent experiments.

Article Snippet: After blocking with 10% nonfat milk, membranes were incubated with the primary antibody at 4°C overnight, using antibodies for Ac-STAT3 Lys685 , SIRT1, DNMT1, and p-STAT3 Tyr705 (CST, 1 : 500 dilution) and β -actin (Beyotime Co., Ltd.; 1 : 500 dilution).

Techniques: Activation Assay, Western Blot, Expressing, Quantitative RT-PCR

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Resveratrol Alleviates 27-Hydroxycholesterol-Induced Senescence in Nerve Cells and Affects Zebrafish Locomotor Behavior via Activation of SIRT1-Mediated STAT3 Signaling

doi: 10.1155/2021/6673343

Figure Lengend Snippet: REV inhibits 27HC-induced senescence and STAT3 activation. BV2 and PC12 cells were treated with 10 μ M 27HC, 10 μ M 27HC + 20 μ M REV, or 20 μ M REV alone for 12 h. CON group: ethanol treatment. (a) Cells were subjected to an SA- β -Gal staining assay. Scale bars: 50 μ m. (b) The aged cells were stained in blue, and the number of stained cells from panels was counted. ∗ P < 0.05, statistically significant difference from the CON group. BV2: P < 0.0001; PC12: P < 0.0001. # P < 0.05, statistically significant difference from the 27HC-treated group. BV2: P < 0.0001; PC12: P < 0.0001. Data are expressed as the mean ± SD of three independent experiments. BV2 and PC12 cells were treated with 10 μ M 27HC or 10 μ M 27HC + 20 μ M REV for 12 h. CON group: ethanol treatment. (c) Western blot analysis of SIRT1, Ac-STAT3 Lys685 , p-STAT3 Tyr705 , and β -actin. (d) The quantitative analysis of protein bands of (c). ∗ P < 0.05, statistically significant difference from the CON group. BV2: (SIRT1: P = 0.0224; Ac-STAT3 Lys685 : P = 0.0053; p-STAT3 Tyr705 : P = 0.0129); PC12: (SIRT1: P = 0.0155; Ac-STAT3 Lys685 : P = 0.0011; p-STAT3 Tyr705 : P < 0.0001). # P < 0.05, statistically significant difference from the 27HC-treated group. BV2: (SIRT1: P = 0.0007; Ac-STAT3 Lys685 : P = 0.0144; p-STAT3 Tyr705 : P = 0.0176); PC12: (SIRT1: P < 0.0001; Ac-STAT3 Lys685 : P = 0.0053; p-STAT3 Tyr705 : P = 0.0368). (e) qRT-PCR analysis of IL-6. ∗ P < 0.05, statistically significant difference from the CON group. BV2: P = 0.0006; PC12: P < 0.0001. # P < 0.05, statistically significant difference from the 27HC-treated group. BV2: P = 0.0023; PC12: P < 0.0001. Data are expressed as the mean ± SD of three independent experiments.

Article Snippet: After blocking with 10% nonfat milk, membranes were incubated with the primary antibody at 4°C overnight, using antibodies for Ac-STAT3 Lys685 , SIRT1, DNMT1, and p-STAT3 Tyr705 (CST, 1 : 500 dilution) and β -actin (Beyotime Co., Ltd.; 1 : 500 dilution).

Techniques: Activation Assay, Staining, Western Blot, Quantitative RT-PCR

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Resveratrol Alleviates 27-Hydroxycholesterol-Induced Senescence in Nerve Cells and Affects Zebrafish Locomotor Behavior via Activation of SIRT1-Mediated STAT3 Signaling

doi: 10.1155/2021/6673343

Figure Lengend Snippet: Schematic representation of molecular mechanisms involved in 27HC-induced senescence. 27HC induces a high level of ROS generation, which can downregulate the expression of SIRT1 and subsequently activate the phosphorylation and acetylation of STAT3, which is regulated by SIRT1. Activation of STAT3 triggers aging-related changes in inflammation and the cell cycle leading to neural cell senescence and organism aging.

Article Snippet: After blocking with 10% nonfat milk, membranes were incubated with the primary antibody at 4°C overnight, using antibodies for Ac-STAT3 Lys685 , SIRT1, DNMT1, and p-STAT3 Tyr705 (CST, 1 : 500 dilution) and β -actin (Beyotime Co., Ltd.; 1 : 500 dilution).

Techniques: Expressing, Activation Assay