Journal: Disease Markers

Article Title: Leptin Promotes HTR-8/SVneo Cell Invasion via the Crosstalk between MTA1/WNT and PI3K/AKT Pathways

doi: 10.1155/2022/7052176

Figure Lengend Snippet: Leptin mediates β -catenin activation through the crosstalk between MTA1/WNT and PI3K/AKT pathways in HTR-8/SVneo cells. (a) HTR-8/SVneo cells were treated with exogenous leptin (0 and 200 ng/ml) for 24 h, and MTA1, WNT1, p-GSK3 β (Ser9), and p-AKT (Ser473) levels were detected by Western blot. Data are shown as mean ± SD; ∗ P < 0.01 vs. leptin (0 ng/ml). (b) The knockdown efficiencies of MTA1 and WNT1 were analyzed by Western blot analysis and qRT-PCR. ∗ P < 0.05 vs. control. (c) HTR-8/SVneo cells were transfected with MTA1 siRNA or scramble siRNA (Scr) in the presence or absence of 200 ng/ml leptin for 24 h, and Western blot analysis was performed to detect the expression of MTA1, WNT1, p-GSK3 β (Ser9), and nuclear β -catenin. ∗ P < 0.01 vs. control and # P < 0.01 vs. leptin. (d) HTR-8/SVneo cells were transfected with WNT1 siRNA or scramble siRNA (Scr) in the presence or absence of 200 ng/ml leptin for 24 h, and Western blot analysis was performed to detect the expression of WNT1, p-GSK3 β (Ser9), and nuclear β -catenin. ∗ P < 0.01 vs. control and # P < 0.01 vs. leptin. (e) The knockdown efficiencies of AKT and β -catenin were analyzed by Western blot analysis and qRT-PCR. ∗ P < 0.05 vs. control. (f) HTR-8/SVneo cells were transfected with AKT siRNA or scramble siRNA (Scr) in the presence or absence of 200 ng/ml leptin for 24 h, and Western blot analysis was performed to detect the expression of AKT, p-GSK3 β (Ser9), and nuclear β -catenin. ∗ P < 0.01 vs. control and # P < 0.01 vs. leptin. All experiments were performed in triplicate.

Article Snippet: The following primary antibodies were used: MMP9 (ab76003), MTA1 (ab71153), WNT1 (ab15251), AKT (ab179463), GSK3 β (ab32391), β -catenin (ab32572), histone 3 (ab1791), β -actin (ab6276) (Abcam, USA); p-AKT (Ser473) (#4060), p-GSK3 β (Ser9) (#9322) (Cell Signaling Technology, USA).

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

Journal: Cell Death Discovery

Article Title: Coal dust nanoparticles induced pulmonary fibrosis by promoting inflammation and epithelial-mesenchymal transition via the NF-κB/NLRP3 pathway driven by IGF1/ROS-mediated AKT/GSK3β signals

doi: 10.1038/s41420-022-01291-z

Figure Lengend Snippet: A , B The activation level of AKT/GSK3β pathway was detected by western blot in vitro and in vivo. C , D The expression levels of EMT marker molecules (N-cadherin, E-cadherin and TGFβ1), inflammatory pathway protein molecules (NLRP3, Cleaved Caspase1/Caspase1) and fibrosis marker molecules (COL-1, COL-3 and α-SMA) was detected by western blot. E Inflammatory factor IL-1β and inflammatory chemokine CXCL2 the supernatant of alveolar epithelial cells were detected by ELISA. Data were expressed as the mean ± SD, n = 3. ** P < 0.01 and *** P < 0.001. F The migration and invasion abilities of alveolar epithelial cells were detected by scratch healing assay and transwell assay.

Article Snippet: The information of antibodies is as follows: IGF1 (1: 1000, Abclonal, #A11985, China), IGF1R (1: 1000, CST, #9750, USA), p-IGF1R(Tyr1135/1136) (1: 1000, CST, #3024, USA), COL-1 (1: 2000, Abcam, ab260043, UK), COL-3 (1: 1000, Abcam, ab184993, UK), α-SMA (1:1000, CST, #19425, USA), heme oxygenase-1 (HO-1, 1: 1000, CST, #5853, USA), N-cadherin (1: 1000, CST, #49398, USA), E-cadherin (1: 1000, CST, #49398, USA), TGFβ1 (1: 1000, abclonal, #A2124, China), p-NF-kB p65(Ser536) (1: 1000, CST, #3033, USA), NF-kB p65 (1: 1000, CST, #8242, USA), NLRP3 (1: 1000, CST, #15101, USA), Cleaved Caspase1(Asp297) (1: 1000, CST, #4199, USA), Caspase1 (1: 1000, CST, #2225, USA), p-Akt(Ser473) (1: 1000, CST, #4060, USA), Akt (1: 1000, CST, #4691, USA), p-GSK3β(Ser9) (1: 1000, CST, #9322, USA), GSK3β (1: 1000, CST, #9369, USA) and β-actin (1: 1000, CST, #4970, USA).

Techniques: Activation Assay, Western Blot, In Vitro, In Vivo, Expressing, Marker, Enzyme-linked Immunosorbent Assay, Migration, Transwell Assay

Journal: Respiratory Research

Article Title: Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: I. Effects on lung remodeling and pathology

doi: 10.1186/1465-9921-14-113

Figure Lengend Snippet: Effect of repeated intranasal LPS challenge and treatment with the selective GSK-3 inhibitor SB216763 on extracellular matrix deposition in the lung. (A) Expression of fibronectin was evaluated in whole lung homogenates 24 hours after the last challenge by immunoblotting using specific antibodies. Equal protein loading was verified by the analysis of GAPDH. Effects of repeated LPS challenge and SB216763 treatment on fibronectin expression were quantified by densitometry, representing mean ± s.e.m. of 9 animals per group. (B) Histological staining of the extracellular matrix protein collagen using Sirius Red. The non-cartilaginous airways were digitally photographed (100-200 × magnification) and analysed by using ImageJ software. Effects of repeated LPS challenge and SB216763 treatment on airway collagen expression were quantified, representing mean ± s.e.m. of 9 animals per group. (C) The mean linear intercept (LMI), a measure for alveolar airspace size, was determined by staining the tissue-sections with haematoxylin and eosin. Data represent means ± s.e.m. of 9 animals per group. **p < 0.01 compared to control group and # p < 0.05 compared to LPS treated animals. Scale bar = 200 μm.

Article Snippet: Rabbit anti-phospho-Ser9/21-GSK-3 antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Western Blot, Staining, Software

Journal: Respiratory Research

Article Title: Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: I. Effects on lung remodeling and pathology

doi: 10.1186/1465-9921-14-113

Figure Lengend Snippet: Repeated LPS instillation and pharmacological inhibition of GSK-3 by SB216763 do not affect airway smooth muscle content. Immunohistological analysis of sm-MHC positive area in (A) large (cartilaginous) and (B) small (non-cartilaginous) airways. Effects of repeated LPS challenge and SB216763 treatment on airway smooth muscle sm-MHC expression were quantified. Data represent means ± s.e.m. of 9 animals per group. Scale bar = 200 μm.

Article Snippet: Rabbit anti-phospho-Ser9/21-GSK-3 antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Inhibition, Expressing

Journal: Respiratory Research

Article Title: Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: I. Effects on lung remodeling and pathology

doi: 10.1186/1465-9921-14-113

Figure Lengend Snippet: Right ventricle hypertrophy after repeated intranasal LPS instillation is attenuated by GSK-3 inhibition. Effect of repeated LPS instillation and GSK-3 inhibition by SB216763 on right ventricle hypertrophy. Effects of repeated LPS challenge and SB216763 treatment on size of right ventricle were quantified as right ventricle weight over total heart weight, representing mean ± s.e.m. of 9 animals per group. **p < 0.01 compared to control group and # p < 0.05 compared to LPS treated animals.

Article Snippet: Rabbit anti-phospho-Ser9/21-GSK-3 antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Inhibition

Journal: Respiratory Research

Article Title: Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: I. Effects on lung remodeling and pathology

doi: 10.1186/1465-9921-14-113

Figure Lengend Snippet: GSK-3 inhibition does not inhibit LPS-induced pulmonary inflammation. Effect of repeated LPS instillation and GSK-3 inhibition by SB216763 on inflammatory cell infiltration in the airways. Cells within 50 μm of the basement membrane were quantified and expressed relative to basement membrane length, representing mean ± s.e.m. of 9 animals per group. *p < 0.05 compared to control group. Scale bar = 200 μm.

Article Snippet: Rabbit anti-phospho-Ser9/21-GSK-3 antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Inhibition

Journal: Respiratory Research

Article Title: Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: I. Effects on lung remodeling and pathology

doi: 10.1186/1465-9921-14-113

Figure Lengend Snippet: Activation of β-catenin in response to repeated intranasal LPS challenge is prevented by treatment with the selective GSK-3 inhibitor SB216763. (A) Expression of active β-catenin, phosphorylated GSK-3 (ser9/21 GSK-3) and total GSK-3 was evaluated in whole lung homogenates 24 hours after the last challenge by immunoblotting using specific antibodies. Equal protein loading was verified by the analysis of GAPDH. (B,C) Responses of repeated LPS challenge and SB216763 treatment on active β-catenin expression (B) and GSK-3 phosphorylation (C) were quantified by densitometry, representing mean ± s.e.m. of 9 animals per group. (D) Correlation between pulmonary expression of fibronectin (data from Figure ) and active β-catenin in all guinea pigs. R = 0.552; p < 0.001. (E) Immunofluorescence analysis of active β-catenin (red) in large airways counterstained with Hoechst 3342 to stain nuclei (blue). *p < 0.05 compared to control group and # p < 0.05 compared to LPS treated animals.

Article Snippet: Rabbit anti-phospho-Ser9/21-GSK-3 antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activation Assay, Expressing, Western Blot, Immunofluorescence, Staining

Journal: BioMed Research International

Article Title: The Effects of Omega-3 Fatty Acid Supplementation on Dexamethasone-Induced Muscle Atrophy

doi: 10.1155/2014/961438

Figure Lengend Snippet: Protein expression in GA muscle samples from rats treated with n-3 PUFA or vehicle for 40 days and dexamethasone or vehicle (last 10 days). Relative optical density of total Akt (tAkt) and phosphorylated Akt (P-Akt) ((a) and (c)), total GSK3 β (tGSK3 β ) and phosphorylated GSK3 β (P-GSK3 β ) ((b) and (d)), and tAkt/P-Akt and tGSK3 β /P-GSK3 β ratios ((e) and (f), resp.). Bands representing tAkt, P-Akt, tGSK3, and P-GSK3 β are illustrated in (g). α -Tubulin (55 kDa) was used as control. The numbers represent the mean ± S.E.M. CT = control group ( n = 6), DX = dexamethasone group ( n = 5), n-3 = n-3 PUFA group ( n = 6), and DX + n-3 = dexamethasone + n-3 PUFA group ( n = 6). Values represent means ± S.E.M., analyzed by Student's t -test, # P < 0.05.

Article Snippet: The primary antibodies used were Akt (1 : 2,000, Cell Signaling #4691), P-Akt (1 : 2,000, Cell Signaling #4060, Ser473); mTOR (1 : 500, Cell Signaling #2972), P-mTOR (1 : 500, Cell Signaling #2971, Ser2448), GSK3 β (1 : 2,000, Cell Signaling #9315), P-GSK3 β (1 : 2,000, Cell Signaling #9322, Ser9), FoXO3a (1 : 500, Cell Signaling #2497), P-FoXO3a (1 : 500, Cell Signaling #9466, Ser253), and α -tubulin (1 : 30,000, Hybridoma bank) in 5% BSA/TBS-T.

Techniques: Expressing

Journal: Stem Cells International

Article Title: Angelica Sinensis Polysaccharide Prevents Hematopoietic Stem Cells Senescence in D-Galactose-Induced Aging Mouse Model

doi: 10.1155/2017/3508907

Figure Lengend Snippet: Effect of ASP on Wnt/ β -catenin signaling mediators of Sca-1 + HSC/HPCs in D-gal- induced aging mice. The Sca-1+ HSC/HPCs were collected after the treatment. (a) GSK-3 β , Ser9-phosphorylated GSK-3 β and TCF-4 protein expressions by Western blot. β -actin was used as the internal control. (b) The relative protein expression of GSK-3 β . (c) The relative protein expression of Ser9-phosphorylated GSK-3 β . (d) The relative protein expression of TCF-4. Different letters: P < 0.05.

Article Snippet: Membranes were incubated overnight at 4°C with the primary antibodies against β -catenin, GSK-3 β , Phospho-GSK-3 β , TCF-4, γ -H2A.X (all purchased from Cell Signaling Technology, USA, catalog number: 9562, 9832, 9336, 2566, 2577), p16 INK4a , Rb, p53, p21 Cip1/Waf1 , β -actin (all purchased from Santa Crus, USA, catalog number: sc-73434, sc-1538, sc-99, sc-397, sc-130656), and 4-HNE (ab46545, Abcam, USA), respectively.

Techniques: Western Blot, Expressing

Journal: PLoS ONE

Article Title: Augmented Cardiac Hypertrophy in Response to Pressure Overload in Mice Lacking ELTD1

doi: 10.1371/journal.pone.0035779

Figure Lengend Snippet: A, Western blots of MEK1/2, ERK1/2, p38, and JNK phosphorylation and their total protein levels 28 days after surgery in WT and KO mice. GAPDH was used as the sample loading control. (n = 6). Up, representative blots. Down, quantitative results. B, Western blots of Akt,GSK3β, and mTOR phosphorylation and their total protein levels 28 days after surgery. GAPDH was used as the sample loading control (n = 6). Up, representative blots. Down, quantitative results. For A and B, Data represent as the means ± SEM *P<0.01 vs. corresponding sham; #P<0.01 vs. WT/AB after AB.

Article Snippet: Antibodies against the following proteins were purchased from Cell Signaling Technology: p-MEK1/2 (#9154), T-MEK1/2 (#9122), p-ERK1/2 (#4370), T-ERK1/2 (#4695), p-p38 (#4511), T-p38 (#9212), p-JNK1/2 (#4668), T-JNK 1/2(#9258), p-Akt (#4060), T-Akt (#4691), p-GSK3β (#9322), T-GSK3β (#9315), p-mTOR (#2974), T-mTOR (#2972), and GAPDH (#2118).

Techniques: Western Blot

Journal: Mediators of Inflammation

Article Title: GSK-3 β Inhibition Attenuates CLP-Induced Liver Injury by Reducing Inflammation and Hepatic Cell Apoptosis

doi: 10.1155/2014/629507

Figure Lengend Snippet: GSK-3 β inhibition improves survival and protects against CLP-induced liver injury. The septic mice were treated with SB216763 (25 mg/kg, i.p.) or vehicle (DMSO) at 1 h, 6 h, and 12 h after CLP. (a) Liver samples were harvested at 6 h after CLP and were subjected to western blotting analysis of phosphorylated (serine 9) GSK-3 β and phosphorylated glycogen synthases (GS). (b) ALT and AST levels were analyzed as a measure of hepatocellular injury. Data are shown as mean ± SD. * P < 0.05, # P < 0.01 compared with vehicle-treated group. (c) The liver specimens were sampled at 20 h after CLP and stained with hematoxylin and eosin staining (original magnification ×400). Representative images from 6 mice/group were selected. (d) The Kaplan-Meier method was used to determine the difference of survival rate after CLP. P value was analyzed by log rank test. * P < 0.05 compared with vehicle-treated group.

Article Snippet: After blocking, the membranes were incubated with primary antibodies: rabbit anti-phospho-GSK-3 β (Ser9) (1 : 1000; Cell Signaling Technology, Beverly, MA), rabbit anti-GSK-3 β (1 : 1000, Cell Signaling Technology), rabbit anti-phospho-glycogen synthase (1 : 1000, Cell Signaling Technology), rabbit anti-cleaved caspase-3 (1 : 1000, Cell Signaling Technology), rabbit anti-cleaved caspase-7 (1 : 1000, Cell Signaling Technology), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1 : 20000; Sigma-Aldrich), at 4°C with gentle shaking overnight.

Techniques: Inhibition, Western Blot, Staining

Journal: Mediators of Inflammation

Article Title: GSK-3 β Inhibition Attenuates CLP-Induced Liver Injury by Reducing Inflammation and Hepatic Cell Apoptosis

doi: 10.1155/2014/629507

Figure Lengend Snippet: GSK-3 β inhibition attenuates the inflammatory cytokine production following CLP. (a) GSK-3 β inhibition altered mRNA expression of TNF- α , IL-6, IL-1 β , and IL-10 in the liver of septic mice. (b) Serum TNF- α and IL-6 levels were assessed by ELISA. Data are shown as mean ± SD. n = 6 per group. * P < 0.05, # P < 0.01 compared with vehicle-treated group.

Article Snippet: After blocking, the membranes were incubated with primary antibodies: rabbit anti-phospho-GSK-3 β (Ser9) (1 : 1000; Cell Signaling Technology, Beverly, MA), rabbit anti-GSK-3 β (1 : 1000, Cell Signaling Technology), rabbit anti-phospho-glycogen synthase (1 : 1000, Cell Signaling Technology), rabbit anti-cleaved caspase-3 (1 : 1000, Cell Signaling Technology), rabbit anti-cleaved caspase-7 (1 : 1000, Cell Signaling Technology), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1 : 20000; Sigma-Aldrich), at 4°C with gentle shaking overnight.

Techniques: Inhibition, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Mediators of Inflammation

Article Title: GSK-3 β Inhibition Attenuates CLP-Induced Liver Injury by Reducing Inflammation and Hepatic Cell Apoptosis

doi: 10.1155/2014/629507

Figure Lengend Snippet: GSK-3 β inhibition reduces the neutrophil infiltration in liver following CLP. (a) MPO staining was performed on liver sections from mice at 20 h after CLP. (b) The numbers of MPO-positive neutrophils that infiltrated the liver were determined (original magnification ×400). Representative images from 6 mice/group were selected. Data are shown as mean ± SD. n = 6 per group. # P < 0.01 compared with vehicle-treated group.

Article Snippet: After blocking, the membranes were incubated with primary antibodies: rabbit anti-phospho-GSK-3 β (Ser9) (1 : 1000; Cell Signaling Technology, Beverly, MA), rabbit anti-GSK-3 β (1 : 1000, Cell Signaling Technology), rabbit anti-phospho-glycogen synthase (1 : 1000, Cell Signaling Technology), rabbit anti-cleaved caspase-3 (1 : 1000, Cell Signaling Technology), rabbit anti-cleaved caspase-7 (1 : 1000, Cell Signaling Technology), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1 : 20000; Sigma-Aldrich), at 4°C with gentle shaking overnight.

Techniques: Inhibition, Staining

Journal: Mediators of Inflammation

Article Title: GSK-3 β Inhibition Attenuates CLP-Induced Liver Injury by Reducing Inflammation and Hepatic Cell Apoptosis

doi: 10.1155/2014/629507

Figure Lengend Snippet: GSK-3 β inhibition modulates the NF- κ B and CREB activation following CLP. Mice were subjected to CLP and treated with either SB216763 (25 mg/kg, i.p.) or vehicle (DMSO). Liver samples were harvested at 20 h after CLP. NF- κ B activity, as well as CREB activity, was assessed. Data are shown as mean ± SD. n = 6 per group. * P < 0.05, # P < 0.01 compared with vehicle-treated group.

Article Snippet: After blocking, the membranes were incubated with primary antibodies: rabbit anti-phospho-GSK-3 β (Ser9) (1 : 1000; Cell Signaling Technology, Beverly, MA), rabbit anti-GSK-3 β (1 : 1000, Cell Signaling Technology), rabbit anti-phospho-glycogen synthase (1 : 1000, Cell Signaling Technology), rabbit anti-cleaved caspase-3 (1 : 1000, Cell Signaling Technology), rabbit anti-cleaved caspase-7 (1 : 1000, Cell Signaling Technology), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1 : 20000; Sigma-Aldrich), at 4°C with gentle shaking overnight.

Techniques: Inhibition, Activation Assay, Activity Assay

Journal: Mediators of Inflammation

Article Title: GSK-3 β Inhibition Attenuates CLP-Induced Liver Injury by Reducing Inflammation and Hepatic Cell Apoptosis

doi: 10.1155/2014/629507

Figure Lengend Snippet: GSK-3 β inhibition reduces the proinflammatory cytokine expression in cultured macrophages stimulated by LPS. (a) RAW264.7 cells were stimulated with LPS for 6 h in the absence or presence of SB216763 (10 μ M). TNF- α , IL-6, IL-1 β , and IL-10 mRNA expression levels were measured by quantitative PCR. (b) TNF- α and IL-6 concentration in the cultured medium was measured by ELISA. (c) RAW264.7 cells were stimulated with LPS for 1 h in the absence or presence of SB216763 (10 μ M). NF- κ B activity, as well as CREB activity, was assessed. Data are shown as mean ± SD. n = 5 per group. * P < 0.05, # P < 0.01 compared with vehicle-treated group.

Article Snippet: After blocking, the membranes were incubated with primary antibodies: rabbit anti-phospho-GSK-3 β (Ser9) (1 : 1000; Cell Signaling Technology, Beverly, MA), rabbit anti-GSK-3 β (1 : 1000, Cell Signaling Technology), rabbit anti-phospho-glycogen synthase (1 : 1000, Cell Signaling Technology), rabbit anti-cleaved caspase-3 (1 : 1000, Cell Signaling Technology), rabbit anti-cleaved caspase-7 (1 : 1000, Cell Signaling Technology), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1 : 20000; Sigma-Aldrich), at 4°C with gentle shaking overnight.

Techniques: Inhibition, Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Mediators of Inflammation

Article Title: GSK-3 β Inhibition Attenuates CLP-Induced Liver Injury by Reducing Inflammation and Hepatic Cell Apoptosis

doi: 10.1155/2014/629507

Figure Lengend Snippet: GSK-3 β inhibition prevents the CLP-induced hepatic apoptosis. (a) Mice were subjected to CLP and treated with either SB216763 (25 mg/kg, i.p.) or vehicle (DMSO). Liver samples were harvested at 20 h after CLP. The caspase-3 activity was measured. Data are shown as mean ± SD. n = 6 per group. # P < 0.01 compared with vehicle-treated group. (b) The expression levels of the cleaved caspase-3 and the cleaved caspase-7 were examined by western blotting analysis.

Article Snippet: After blocking, the membranes were incubated with primary antibodies: rabbit anti-phospho-GSK-3 β (Ser9) (1 : 1000; Cell Signaling Technology, Beverly, MA), rabbit anti-GSK-3 β (1 : 1000, Cell Signaling Technology), rabbit anti-phospho-glycogen synthase (1 : 1000, Cell Signaling Technology), rabbit anti-cleaved caspase-3 (1 : 1000, Cell Signaling Technology), rabbit anti-cleaved caspase-7 (1 : 1000, Cell Signaling Technology), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1 : 20000; Sigma-Aldrich), at 4°C with gentle shaking overnight.

Techniques: Inhibition, Activity Assay, Expressing, Western Blot