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: Annals of Translational Medicine

Article Title: LncRNA MALAT1 induced by hyperglycemia promotes microvascular endothelial cell apoptosis through activation of the miR-7641/TPR axis to exacerbate neurologic damage caused by cerebral small vessel disease

doi: 10.21037/atm-21-5997

Figure Lengend Snippet: Regulation of TPR expression affects the phosphorylation activity of the Akt pathway. (A) The expression level of TPR protein in each group detected by WB to verify the transfection effect; (B) the expression level of p-Akt/Akt, and p-GSK3β/GSK3β proteins in each group detected by WB; (C) lncRNA MATAL1/miR-7641/TPR pathway diagram. **P<0.01, ***P<0.001. WB, Western blot; TPR, translocated promoter region; lncRNA, long noncoding RNA; MALAT1, metastasis-associated lung carcinoma transcript 1; Nor, normal; Nor + TPR inhibitor, normal + TPR inhibitor; HG, high glucose; TPR oe, TPR over express; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p-GSK3β, phospho-glycogen synthase kinase-3β.

Article Snippet: The antibodies included anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody [6C5]-Loading Control (ab8245) (1:1,000), anti-von Willebrand factor (vWF) antibody [ {"type":"entrez-protein","attrs":{"text":"EPR12011","term_id":"523377596","term_text":"EPR12011"}} EPR12011 ] (ab174290) (1:2,000), anti-intercellular adhesion molecule-1 (ICAM1) antibody [ {"type":"entrez-protein","attrs":{"text":"EPR16608","term_id":"523382629","term_text":"EPR16608"}} EPR16608 ] (ab179707) (1:1,000), anti-Bcl-2 antibody (ab196495) (1:1,000), anti-caspase-3 antibody [31A1067] (ab13585) (1:50), anti-Akt antibody (ab8805) (1:500), anti-Akt (phospho T308) antibody (ab38449) (1:1,000), anti-GSK3 beta antibody (ab93926) (1:1,000), and phosphorylated glycogen synthase kinase 3 beta (p-GSK3β) (Ser9) (1:1,000, cat no. 9336, CST).

Techniques: Expressing, Activity Assay, Transfection, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: CD147 promotes epithelial-mesenchymal transition of prostate cancer cells via the Wnt/β-catenin pathway

doi: 10.3892/etm.2020.9058

Figure Lengend Snippet: CD147 regulates epithelial-mesenchymal transition via the Wnt/β-catenin pathway. (A and B) Expression of β-catenin, Snail and p-GSK-3β (Ser9) was determined in LNCaP/Scramble and LNCaP/shCD147 cells by western blotting. (C and D) Expression of β-catenin, Snail, p-β-catenin (Ser33/37/Thr41), vimentin and E-cadherin following treatment with or without LiCl in LNCaP/Scramble and LNCaP/shCD147 cells by western blotting. Values are presented as the mean ± standard deviation of three experiments. Lanes 1 and 3, LNCaP/Scramble group; Lanes 2 and 4, LNCaP/shCD147 group. * P<0.05, ** P<0.01 vs. LNCaP/Scramble; # P<0.05, ## P<0.01 vs. LNCaP/shCD147. LNCaP, lymph node carcinoma of the prostate; LiCl, lithium chloride; sh, short hairpin RNA; p-, phosphorylated; GSK-3β, glycogen synthase kinase-3β.

Article Snippet: The membranes were then blocked with 5% nonfat milk in PBS containing 0.05% Tween-20 at room temperature for 2 h. The membranes were incubated with rabbit monoclonal antibodies against CD147 (cat. no. 13287), human p-glycogen synthase kinase (GSK)-3β (Ser 9; cat. no. 9322), GSK-3β (cat. no. 9315), E-cadherin (cat. no. 3195), N-cadherin (cat. no. 13116), vimentin (cat. no. 5741), β-catenin (cat. no. 8480), p-β-catenin (Ser 33/37/Thr 41; cat. no. 9561) and Snail (cat. no. 3879) all at 1:1,000 dilution (Cell Signaling Technology, Inc.) overnight at 4˚C. β-actin (cat. no. 4970) and lamin B (cat. no. 13435) were used as loading controls (both 1:2,000; Cell Signaling Technology, Inc.).

Techniques: Expressing, Western Blot, Standard Deviation, shRNA