akt inhibitor  (Santa Cruz Biotechnology)

Journal: Cancers

Article Title: LDOC1 Suppresses Microbe-Induced Production of IL-1β in Human Normal and Cancerous Oral Cells through the PI3K/Akt/GSK-3β Axis

doi: 10.3390/cancers12113148

Figure Lengend Snippet: LDOC1 downregulation leads to inhibitory phosphorylation of GSK-3β S9 by pAkt S473 and GSK-3β regulates the clonogenicity of the CGHNK2-shLDOC1 cell line. ( a ) Results of the human phosphokinase array analysis using CGHNK2-shLDOC1 and CGHNK2-shCtrl cells. The bar charts represent the relative protein phosphorylation levels for the top four kinases and the transcription factor STAT3 exhibiting different phosphorylation states in CGHNK2-shLDOC1 and CGHNK2-shCtrl cells (the array contained some non-kinase phosphoprotein spots). Experiments were performed according to the instructions provided by the manufacturer, as described in Materials and Methods. ( b ) Western blotting analysis of pGSK-3β S9 , GSK-3β, pAkt S473 , and Akt in CGHNK2-shLDOC1 and CGHNK2-shCtrl cells. GAPDH was used as a loading control for each protein sample. ( c ) The serine kinase activity of Akt is required for the phosphorylation of GSK-3β S9 in CGHNK2-shLDOC1 and TW2.6-GFP cells. Cellular protein lysate was harvested after a 6 h treatment with inhibitors of Akt (SH-6, 5 μM) or PI3K (LY294002, 10 μM). Western blotting analyses of pGSK-3β S9 , GSK-3β, pAkt S473 , Akt, and GAPDH were then performed. Cells treated with PBS (0.1%) were used as controls. The pixel intensity ratio for each band was shown. There are technical duplicates for the human phosphokinase array analysis; Western blotting analysis under each condition was performed at least twice. ( d ) Ectopic expression of GSK-3β inhibits the acquired clonogenicity of CGHNK2-shLDOC1 cells. Cells were subjected to a colony-forming assay. The bar chart represents the mean values of triplicate tests (mean ± SD). Representative images are presented.

Article Snippet: The PI3K inhibitor LY294002 and Akt inhibitor SH-6 were obtained from Enzo Life Sciences (Farmingdale, NY, USA).

Techniques: Western Blot, Activity Assay, Expressing

Journal: Cancers

Article Title: LDOC1 Suppresses Microbe-Induced Production of IL-1β in Human Normal and Cancerous Oral Cells through the PI3K/Akt/GSK-3β Axis

doi: 10.3390/cancers12113148

Figure Lengend Snippet: The PI3K/Akt signaling pathway involved in CA -induced IL-1β production in LDOC1 -deficient oral cells. ( a , b ) Effect of CA SC5314 on the phosphorylation of Akt S473 in LDOC1 -deficient or LDOC1 -expressing oral cell lines. Cells cultured in a normal growth medium were treated with CA SC5314 (MOI = 0.5) for the times indicated. Equal amounts of whole-cell lysates were subjected to Western blotting analysis with antibodies specific for pAKT S473 , Akt, and GAPDH. A representative blot is presented in the upper panels. As presented in the bottom panels, densitometric analyses were performed to quantify the fold change (FC) in the intensity of the pAKT S473 blots with untreated controls (time 0) set as 1. Data are expressed as mean ± SD ( n = 2, biological duplicates). * p < 0.05, ** p < 0.01 vs. basal activation. ( c , d ) Levels of IL-1β induced by CA SC5314 were suppressed by inhibitors of Akt and PI3K (SH-6 and LY294002, respectively) in LDOC1 -deficient CGHNK2-shLDOC1 ( c ) and TW2.6-GFP ( d ) cell lines. Concentrations of IL-1β in the conditioned medium of cells were measured by CBA after coculture with live CA SC5314 for 16 h, with or without pretreatment of SH-6 (2.5 µM and 5 µM for CGHNK2- and TW2.6-derived cell lines, respectively) or LY294002 (10 µM) for 6 h. Data are presented as mean ± SD ( n = 3), analyzed using the ANOVA test. * p < 0.05 and ** p < 0.01.

Article Snippet: The PI3K inhibitor LY294002 and Akt inhibitor SH-6 were obtained from Enzo Life Sciences (Farmingdale, NY, USA).

Techniques: Expressing, Cell Culture, Western Blot, Activation Assay, Derivative Assay