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: World Journal of Gastroenterology

Article Title: γ-aminobutyric acid B2 receptor: A potential therapeutic target for cholangiocarcinoma in patients with diabetes mellitus

doi: 10.3748/wjg.v29.i28.4416

Figure Lengend Snippet: Baclofen inhibits the glycogen synthase kinase 3/β-catenin and signal transducer and activator of transcription 3 pathways. A and B: Phospho-kinase arrays reveal the inhibition of phosphorylation in multiple kinases and signal transducers; C: Glycogen synthase kinase 3 (GSK3)/β-catenin and signal transducer and activator of transcription 3 (STAT3) are included for further analysis as they are key pathways in cholangiocarcinoma (CCA) progression in which β-catenin and STAT3 are common targets of GSK3. RNA expression of γ-aminobutyric acid B2 receptor and that of STAT3 are significantly correlated in clinical CCA samples from Thai patients. HG: High glucose; GABBR2: γ-aminobutyric acid B2 receptor; GSK3: Glycogen synthase kinase 3; STAT3: Signal transducer and activator of transcription 3.

Article Snippet: Primary antibodies used to detect the proteins by western blot were: GABBR2 (1:500, Proteintech, Rosemont, IL), pSTAT3 (Y705) (1:500, Cell Signaling Technology, Danvers, MA), pSTAT3 (S727) (1:500, Cell Signaling Technology), STAT3 (1:1000, Cell Signaling Technology), p-glycogen synthase kinase 3 (GSK3)α/β (1:1000, Cell Signaling Technology), GSK3α/β (1:1000, Cell Signaling Technology), β-catenin (1:1000, Cell Signaling Technology), cyclin D1 (1:1000, Cell Signaling Technology), c-Myc (1:500, Santa Cruz Biotechnology, Dallas, TX), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10000, Millipore Sigma, Burlington, MA).

Techniques: Inhibition, RNA Expression

Journal: World Journal of Gastroenterology

Article Title: γ-aminobutyric acid B2 receptor: A potential therapeutic target for cholangiocarcinoma in patients with diabetes mellitus

doi: 10.3748/wjg.v29.i28.4416

Figure Lengend Snippet: Baclofen suppresses the glycogen synthase kinase 3/β-catenin and signal transducer and activators of transcription 3 pathways. A and B: Phosphorylation of glycogen synthase kinase 3 (GSK3) and signal transducer and activators of transcription 3 is decreased after baclofen treatment in both cholangiocarcinoma cell lines, both cultured in normal glucose and high glucose conditions. Total β-catenin protein is also decreased consistently with the decreased phosphorylated GSK3α/β. Western blots show the representative of three biological replications with the same trends of results. Band intensities are the average of three biological replications which are normalized using the intensities of glyceraldehyde-3-phosphate dehydrogenase for each experiment. The levels of phosphorylated forms are normalized with the total forms of their corresponding proteins. NG: Normal glucose; HG: High glucose; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; GSK3: Glycogen synthase kinase 3; STAT3: Signal transducer and activator of transcription 3.

Article Snippet: Primary antibodies used to detect the proteins by western blot were: GABBR2 (1:500, Proteintech, Rosemont, IL), pSTAT3 (Y705) (1:500, Cell Signaling Technology, Danvers, MA), pSTAT3 (S727) (1:500, Cell Signaling Technology), STAT3 (1:1000, Cell Signaling Technology), p-glycogen synthase kinase 3 (GSK3)α/β (1:1000, Cell Signaling Technology), GSK3α/β (1:1000, Cell Signaling Technology), β-catenin (1:1000, Cell Signaling Technology), cyclin D1 (1:1000, Cell Signaling Technology), c-Myc (1:500, Santa Cruz Biotechnology, Dallas, TX), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10000, Millipore Sigma, Burlington, MA).

Techniques: Cell Culture, Western Blot

Journal: World Journal of Gastroenterology

Article Title: γ-aminobutyric acid B2 receptor: A potential therapeutic target for cholangiocarcinoma in patients with diabetes mellitus

doi: 10.3748/wjg.v29.i28.4416

Figure Lengend Snippet: Schematic summary of the effects of high glucose on γ-aminobutyric acid B2 receptor expression and the effects of baclofen on cholangiocarcinoma cells. High glucose induces the expression of γ-aminobutyric acid B2 receptor (GABBR2) in cholangiocarcinoma (CCA) cells. The treatment of baclofen, a GABBR2 agonist, to CCA cells inhibits phosphorylation of glycogen synthase kinase 3, resulting in the activation of the kinase activity which further phosphorylates β-catenin. Phosphorylated β-catenin is subjected to degradation preventing its function on promoting cell proliferation via c-Myc and cyclin D1 expression. On the other hand, activated GABBR2 by baclofen also inhibits phosphorylation of signal transducer and activator of transcription 3 (STAT3). The inhibition of STAT3 phosphorylation also suppresses its functions as a transcription factor for c-Myc and cyclin D1 expression. Activating GABBR2 by baclofen, thus, suppresses the proliferation of CCA cells. GABBR2: γ-aminobutyric acid B2 receptor; GSK3: Glycogen synthase kinase 3; STAT3: Signal transducer and activator of transcription 3.

Article Snippet: Primary antibodies used to detect the proteins by western blot were: GABBR2 (1:500, Proteintech, Rosemont, IL), pSTAT3 (Y705) (1:500, Cell Signaling Technology, Danvers, MA), pSTAT3 (S727) (1:500, Cell Signaling Technology), STAT3 (1:1000, Cell Signaling Technology), p-glycogen synthase kinase 3 (GSK3)α/β (1:1000, Cell Signaling Technology), GSK3α/β (1:1000, Cell Signaling Technology), β-catenin (1:1000, Cell Signaling Technology), cyclin D1 (1:1000, Cell Signaling Technology), c-Myc (1:500, Santa Cruz Biotechnology, Dallas, TX), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10000, Millipore Sigma, Burlington, MA).

Techniques: Expressing, Activation Assay, Activity Assay, Inhibition

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Preparation and Multitarget Anti-AD Activity Study of Chondroitin Sulfate Lithium in AD Mice Induced by Combination of D-Gal/AlCl 3

doi: 10.1155/2022/9466166

Figure Lengend Snippet: Effects of CS-Li on tau phosphorylation in the hippocampi of mice. Western blotting of GSK-3 β , p-GSK-3 β (Ser9), p-tau (Ser396), p-tau (Ser404), and tau, GAPDH was used as the internal control; Western blotting of PP2A, β -actin was used as the internal control. # p < 0.05, ## p < 0.01, and ### p < 0.001 vs. the control group; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 vs. the model group.

Article Snippet: Rabbit anti-TNF- α , anti-IL-6, anti-IL-1 β , anti-p-p38MAPK, anti-p-Erk1/2, anti-p-NF- κ B, anti-p-GSK3 β (Ser9), and anti-PP2A antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA).

Techniques: Western Blot

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: PLoS ONE

Article Title: Heme Oxygenase-1 Prevents Cardiac Dysfunction in Streptozotocin-Diabetic Mice by Reducing Inflammation, Oxidative Stress, Apoptosis and Enhancing Autophagy

doi: 10.1371/journal.pone.0075927

Figure Lengend Snippet: A. Apoptosis assessed by TUNEL assay in the respective groups (left) with quantification on the right. First column: Nuclei of apoptotic cells TUNEL stained in green. Second column: The DAPI-stained nuclei appear in blue. Third column: merged picture of first and second column (scale bar, 20µm). B. Representative immunoblot for p53 and Bcl-2 in the myocardial tissues from the respective groups (left) and densitometric quantification (right). C. Flow cytometric analysis using Annexin V-FITC and propidium iodide stains to evaluate the effects of transfection with HO-1 or empty vector (PC) under control and high-glucose culture conditions (left) with quantification on the right. D. Representative immunoblot for p-Akt, Akt, p-GSK3 and GSK3 in the myocardial tissues from the respective groups (left) and densitometric quantification (right). (n = 5 in each group) Columns and errors bars represent mean ± SD. #p<0.01 vs. Wt/Con; *p<0.01 vs. Wt/DM; *** p<0.001 vs. PC/Con; ##p<0.01 vs. PC/Glu.

Article Snippet: The membrane was blocked with 5% non-fat milk in PBS containing 0.1% Tween 20 (PBS-T) and incubated for 2 h. Membranes were probed with antibodies against p-Akt, p-GSK3, p-AMPK, p53, Beclin-1, LC3- II (1:1000 dilution, Cell Signaling Technology, CA, USA) or Bcl-2 (1:500 dilution, Santa Cruz Biotechnology, CA, USA).

Techniques: TUNEL Assay, Staining, Western Blot, Transfection, Plasmid Preparation