Journal: Pharmaceuticals

Article Title: Design and Synthesis of a Novel 4-aryl-N-(2-alkoxythieno [2,3- b ]pyrazine-3-yl)-4-arylpiperazine-1-carboxamide DGG200064 Showed Therapeutic Effect on Colon Cancer through G2/M Arrest

doi: 10.3390/ph15050502

Figure Lengend Snippet: c-Jun stabilization by DGG200064 : ( a ) HCT116 and DLD1 treatment with DGG200064 in a dose-dependent manner (0, 10, 50, 100 nM). The effect of G2/M cell cycle arrest induced by DGG200064 treatment for 6 h in HCT116 and DLD1 cells. Expression was investigated with the following antibodies: c-Jun, p-c-Jun, cyclin B1, and β-actin; ( b ) c-Jun stabilization expression in HCT116 cells exposed to DGG200064 (50 nM) for 6 h. Immunocytochemistry analysis in HCT116 cells using anti c-Jun primary antibody, fluorophore-conjugated anti-mouse immunoglobulin secondary antibody and counterstained with DAPI to visualize nuclei. The scale bar was 20 μM. ( c ) After HCT116 and DLD1 cells were transfected with siRNA targeting c-Jun for 24 h, the cells were treated with DGG200064 (50 nM) for 6 h. Whole-cell lysates were subjected to immunoblotting with the indicating antibodies to c-Jun, cyclin B1, β-actin. ( d ) The siRNA targeting FBXW7 transfected in HCT116 and DLD1 cells for 24 h. Then these cells were treated with DGG200064 (50 nM) for 6 h in HCT116 and DLD1 cell. Whole-cell lysates were analyzed by immunoblotting with antibodies to FBXW7, c-Jun, cyclin B1, and β-actin.

Article Snippet: A small interfering RNA (siRNA) duplex targeting human c-Jun and FBXW7 (Genolution, Seoul, Korea) were introduced into cells using Lipofector-EXT (AptaBio, Yongin, Korea), according to the manufacturer’s instructions.

Techniques: Expressing, Immunocytochemistry, Transfection, Western Blot

Journal: Pharmaceuticals

Article Title: Design and Synthesis of a Novel 4-aryl-N-(2-alkoxythieno [2,3- b ]pyrazine-3-yl)-4-arylpiperazine-1-carboxamide DGG200064 Showed Therapeutic Effect on Colon Cancer through G2/M Arrest

doi: 10.3390/ph15050502

Figure Lengend Snippet: Inhibition of the c-Jun ubiquitination through interruption of FBXW7/c-Jun interaction in ( a ) HCT116 and ( b ) DLD1 cells. Cells were treated with DGG200064 (50 nM) for 18 h; then incubated with or without MG132 (10 μM) for 6 h. The protein was immunoprecipitated from cell lysates using a c-Jun antibody, followed by immunoblotting of c-Jun, Ubiquitin, and FBXW7.

Article Snippet: A small interfering RNA (siRNA) duplex targeting human c-Jun and FBXW7 (Genolution, Seoul, Korea) were introduced into cells using Lipofector-EXT (AptaBio, Yongin, Korea), according to the manufacturer’s instructions.

Techniques: Inhibition, Ubiquitin Proteomics, Incubation, Immunoprecipitation, Western Blot

Journal: Pharmaceuticals

Article Title: Design and Synthesis of a Novel 4-aryl-N-(2-alkoxythieno [2,3- b ]pyrazine-3-yl)-4-arylpiperazine-1-carboxamide DGG200064 Showed Therapeutic Effect on Colon Cancer through G2/M Arrest

doi: 10.3390/ph15050502

Figure Lengend Snippet: Identification of the interaction inhibition site between FBXW7 and c-Jun: ( a ) Docking modes and interactions of c-Jun, 7a , 7e , 7f, and DGG200064 at the binding site of FBXW7. Electrostatic, hydrogen bond, alkyl, and pi-sigma interaction are displayed as orange, green, pink, and purple dashed lines, respectively; ( b ) HCT116 cells transfected with MYC-FBXW7 WT , MYC-FBXW7 R689G&T628A for 24 h, and then treated with DGG200064 for 6 h. The cell extracts were immunoprecipitated with an MYC-tag antibody and subjected to immunoblotting with antibodies to c-Jun and Myc-tag.

Article Snippet: A small interfering RNA (siRNA) duplex targeting human c-Jun and FBXW7 (Genolution, Seoul, Korea) were introduced into cells using Lipofector-EXT (AptaBio, Yongin, Korea), according to the manufacturer’s instructions.

Techniques: Inhibition, Binding Assay, Transfection, Immunoprecipitation, Western Blot