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

Journal: International Journal of Molecular Medicine

Article Title: RFC3 induces epithelial-mesenchymal transition in lung adenocarcinoma cells through the Wnt/β-catenin pathway and possesses prognostic value in lung adenocarcinoma

doi: 10.3892/ijmm.2019.4386

Figure Lengend Snippet: Effects of RFC3 on the regulation of proteins associated with epithelial-mesenchymal transition and the Wnt/β-catenin signaling pathway. (A) RFC3 overexpression plasmid and empty vector were transfected into the lung cancer A549 cell line. (B) RFC3-specific siRNA and non-specific control siRNA were transfected into the lung cancer H1299 cell line. Total protein was extracted and subjected to immunoblotting of Wnt1, p-GSK3-β (Ser9), GSK3-β, β-catenin, c-MYC, E-cadherin, N-cadherin and Vimentin. Actin was used as a loading control. (A and B) Representative image data of immunoblotting, semi-quantitative representation of protein expression and ratios of p-GSK3-β/GSK3-β from three separate experiments. * P<0.05, ** P<0.01. GSK3, glycogen synthase kinase 3; p-, phosphorylated; RFC3, replication factor C subunit 3; siRNA, small interfering RNA.

Article Snippet: PVDF membranes were blocked with 5% milk in TTBS for 1 h at room temperature, and were probed with primary antibodies against RFC3 (1:500; cat. no. sc-390293; Santa Cruz Biotechnology, Inc.), Vimentin (1:1,000, cat. no. 5741S; Cell Signaling Technology, Inc.), N-cadherin (1:1,000, cat. no. 13116S, Cell Signaling Technology, Inc.), E-cadherin (1:1,000, cat. no. 14472S; Cell Signaling Technology, Inc.), phosphorylated (p)-glycogen synthase kinase 3 (GSK3)-β (Ser9) (1:1,000, cat. no. 5558S; Cell Signaling Technology, Inc.), β-catenin (1:1,000, cat. no. 8480S, Cell Signaling Technology, Inc.), GSK3-β (1:1,000, cat. no. 12456S; Cell Signaling Technology, Inc.), c-MYC (1:1,000, cat. no. 13987S; Cell Signaling Technology, Inc.), Wnt1 (1:1,000, cat. no. 27935-1-AP; Proteintech Group, Inc.), and GAPDH (1:1,000, cat. no. 5174S; Cell Signaling Technology, Inc.) or actin (1:2,000, cat. no. 3700S; Cell Signaling Technology, Inc.) overnight at 4°C.

Techniques: Over Expression, Plasmid Preparation, Transfection, Western Blot, Expressing, Small Interfering RNA

Journal: Experimental and Therapeutic Medicine

Article Title: Effect of type 2 diabetes mellitus caveolin-3 K15N mutation on glycometabolism

doi: 10.3892/etm.2019.7840

Figure Lengend Snippet: K15N mutation effect on protein expression in the AKT signaling pathway. Western blot and densitometry analysis of (A) CAV3-eGFP, (B) CAV1, (C) p-AKT, (D) AKT1 and AKT2, (E) GLUT-4 and (F) p-GSK3β expression in WT and K15N cells. All data are presented as the mean ± standard deviation. *P<0.05 vs. WT. CAV3, caveolin-3; eGFP, enhanced green fluorescent protein; CAV-1, caveolin-1; p, phosphorylated; GLUT-4, glucose transporter type 4; GSK3β, glycogen synthase kinase 3β; WT, wild-type; K15N, CAV3 K15N mutation.

Article Snippet: Membranes were blocked with 5% dried skim milk and TBS plus 0.01% Tween 20 for 1 h at room temperature and subsequently incubated overnight at 4°C with mouse anti-GFP monoclonal antibody (1:500; cat. no. J20625; Beijing Transgen Biotech Co., Ltd.), mouse anti-CAV3 monoclonal antibody (1:100; cat. no. sc-5310; Santa Cruz Biotechnology, Inc.), rabbit anti-caveolin-1 (CAV1) polyclonal antibody (1:1,000; cat. no. sc-894; Santa Cruz Biotechnology, Inc.), mouse anti-phosphorylated (p)-AKT ser473 monoclonal antibody (1:1,000; cat. no. 12694; Cell Signaling Technology, Inc.), mouse anti-AKT monoclonal antibody (1:1,000; cat. no. 2920; Cell Signaling Technology, Inc.), goat anti-AKT1 polyclonal antibody (1:1,000; cat. no. sc-1618; Santa Cruz Biotechnology, Inc.), mouse anti-AKT2 monoclonal antibody (1:1,000; cat. no. 5239; Cell Signaling Technology, Inc.), goat anti-GLUT-4 polyclonal antibody (1:100; cat. no. sc-01608; Santa Cruz Biotechnology, Inc.), rabbit anti-p glycogen synthase kinase 3 beta (GSK3β) monoclonal antibody (1:1,000; cat. no. 9323; Cell Signaling Technology, Inc.) or rabbit anti-GSK3β monoclonal antibody (1:1,000; cat. no. 9315; Cell Signaling Technology, Inc.).

Techniques: Mutagenesis, Expressing, Western Blot, Standard Deviation