Journal: PLoS ONE

Article Title: Loss of the Promyelocytic Leukemia Protein in Gastric Cancer: Implications for IP-10 Expression and Tumor-Infiltrating Lymphocytes

doi: 10.1371/journal.pone.0026264

Figure Lengend Snippet: (A) SNU-638 gastric cancer cells were transiently transfected with Pml siRNA or control siRNA. Two days after transfection, cells were treated with or without interferon-gamma (IFN-γ (10 ng/ml) for 8 h and lysed and analyzed by immunoblotting. Effective siRNA-mediated suppression of PML protein expression was verified for each assay by immunoblotting. (B) Jurkat cells and culture supernatants from SNU-638 cells which were transiently transfected with either control siRNA or Pml siRNA were analyzed by a transwell migration assay. After transfection, SNU-638 cells were incubated with IFN-γ (10 ng/ml) for 8 h or left untreated, and the collected supernatants were placed in the lower chambers. The upper chambers received 5×10 5 Jurkat cells in a volume of 100 µl and the transwell migration units were incubated at 37°C for 2 h. Migration of Jurkat cells from the upper to lower chamber was analyzed by examining cells harvested from the lower chambers by fluorescent count-bead mediated counting on a flow cytometer. Relative cell migration was determined by the number of migrated cells normalized to the number of cells migrating to supernatants from SNU-638 cells transiently transfected with control siRNA without IFN-γ, and the value from parental cells was arbitrarily set at 1. (C) SNU-638 gastric cancer cells were transiently transfected with either empty vector (Mock) or PML IV expression vector (PML IV). Day after transfection, cells were treated with or without IFN-γ (10 ng/ml) for 8 h and culture supernatants were obtained and subjected to transwell migration assay as described in B. PML protein expression was verified for each assay by immunoblotting. Data shown are representative of at least three experiments. Bars, SD. * P <0.05.

Article Snippet: NIH3T3 cells were transiently transfected using Mirus transfection reagent (Mirus Bio LLC, Madison, WI, USA) or Lipofectamine™ RNAiMAX (Invitrogen, Carlsbad, CA, USA) with either 300 nmol of mPML siRNA or negative control siRNA (Samchully Pharm, Co. Ltd., Seoul, Korea), according to the manufacturer's instructions.

Techniques: Transfection, Western Blot, Expressing, Transwell Migration Assay, Incubation, Migration, Flow Cytometry, Plasmid Preparation

Journal: PLoS ONE

Article Title: Loss of the Promyelocytic Leukemia Protein in Gastric Cancer: Implications for IP-10 Expression and Tumor-Infiltrating Lymphocytes

doi: 10.1371/journal.pone.0026264

Figure Lengend Snippet: −/− MEFs compared to Pml +/+ wild-type cells. (A) RNA from Pml +/+ and Pml −/− MEFs treated with IFN-γ (10 ng/ml) for 0-24 h was subjected to ribonuclease protection assay (RPA). Quantification of IP-10, RANTES and STAT-1 mRNA (FOLD INCREASE) was calculated by dividing the densitometric value of each lane by the corresponding GAPDH value. (B) Pml +/+ and Pml −/− MEFs cells were incubated in the absence or presence of IFN-γ (10 ng/ml) for 12 h. IP-10 protein from the culture supernatants were analyzed by ELISA and normalized by the cell protein concentration. The relative concentration was calculated as the normalized amount divided by the normalized amount of untreated Pml −/− MEFs cells. (C) NIH3T3 fibroblasts were transiently transfected with either Pml siRNA or control siRNA. Two days after transfection, cells were treated with or without IFN-γ for 0-24 h, and analyzed by immunoblotting for detection of PML protein expression and RT-PCR for IP-10 mRNA. Quantification (Quant) of IP-10 mRNA was calculated by dividing the densitometric value of each lane by the corresponding actin mRNA value. Data shown are representative of at least three experiments. Bars, SD. * P <0.01.

Article Snippet: NIH3T3 cells were transiently transfected using Mirus transfection reagent (Mirus Bio LLC, Madison, WI, USA) or Lipofectamine™ RNAiMAX (Invitrogen, Carlsbad, CA, USA) with either 300 nmol of mPML siRNA or negative control siRNA (Samchully Pharm, Co. Ltd., Seoul, Korea), according to the manufacturer's instructions.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Protein Concentration, Concentration Assay, Transfection, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Loss of the Promyelocytic Leukemia Protein in Gastric Cancer: Implications for IP-10 Expression and Tumor-Infiltrating Lymphocytes

doi: 10.1371/journal.pone.0026264

Figure Lengend Snippet: (A) Immunohistochemical staining for PML and IP-10 protein expression was performed for 49 cases of stage IV advanced gastric carcinomas. Immunopositivity for PML protein was categorized as diffuse positivity (DP; nuclear immunoreactivity in ≥50% of tumor cells), focal positivity (FP; in ≥10% but <50%), or complete loss (CL; in <10%). For quantitative analysis of immunoreactivity of IP-10, positively stained areas were measured using an ImageJ software program as described in . Representative images show PML and IP-10 protein expression in advanced gastric carcinoma tissues (right). (B) SNU-638 cells were transiently transfected with Pml siRNA or control siRNA. Two days after transfection, cells were incubated in the absence or presence of IFN-γ (10 ng/ml) for 8 h. IP-10 protein from the culture supernatants was analyzed by ELISA and normalized by the cell protein concentration. The relative concentration was calculated as the normalized amount divided by the normalized amount of SNU-638 cells which were transiently transfected with control siRNA in the presence of IFN-γ, and the concentration from parental cells was arbitrarily set at 100%. (C) SNU-638 gastric cancer cells were transiently transfected with either empty vector or PML IV expression vector. Day after transfection, cells were treated with or without IFN-γ (10 ng/ml) for 8 h and culture supernatants were obtained and subjected to ELISA as described in B. PML protein expression was verified for each assay by immunoblotting. Data shown are representative of at least three experiments. Bars, SD. * P <0.05, ** P <0.01.

Article Snippet: NIH3T3 cells were transiently transfected using Mirus transfection reagent (Mirus Bio LLC, Madison, WI, USA) or Lipofectamine™ RNAiMAX (Invitrogen, Carlsbad, CA, USA) with either 300 nmol of mPML siRNA or negative control siRNA (Samchully Pharm, Co. Ltd., Seoul, Korea), according to the manufacturer's instructions.

Techniques: Immunohistochemistry, Staining, Expressing, Software, Transfection, Incubation, Enzyme-linked Immunosorbent Assay, Protein Concentration, Concentration Assay, Plasmid Preparation, Western Blot

Journal: PLoS ONE

Article Title: Loss of the Promyelocytic Leukemia Protein in Gastric Cancer: Implications for IP-10 Expression and Tumor-Infiltrating Lymphocytes

doi: 10.1371/journal.pone.0026264

Figure Lengend Snippet: (A) The murine IP-10 promoter contains a putative GAS-like element (underlined). (B) NIH3T3 cells were co-transfected with the IP-10-luc reporter containing a GAS-like element and/or Pml siRNA, or the PML IV protein expression vector. Cells were either untreated or treated with murine IFN-γ (10 ng/ml) for 24 h, and then luciferase activity was determined. The pCMV-β-galactosidase vector was included to normalize transfection efficiency. Luciferase activity is normalized to the activity in the absence of IFN-γ and the activity from parental cells was arbitrarily set at 100% (RLA; relative luciferase activity). Increased or reduced PML protein expression in cell lysates transfected with either PML IV expression vector or Pml siRNA was verified by immunoblotting, respectively. (C) SNU-638 gastric cancer cells were co-transfected with the IP-10-luc reporter containing a GAS-like element and/or Pml siRNA, or the PML IV protein expression vector and analyzed as described in B. Values are the mean ± SD of three separate experiments. Bars, SD. * P <0.05, ** P <0.001.

Article Snippet: NIH3T3 cells were transiently transfected using Mirus transfection reagent (Mirus Bio LLC, Madison, WI, USA) or Lipofectamine™ RNAiMAX (Invitrogen, Carlsbad, CA, USA) with either 300 nmol of mPML siRNA or negative control siRNA (Samchully Pharm, Co. Ltd., Seoul, Korea), according to the manufacturer's instructions.

Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Western Blot

Journal: Scientific Reports

Article Title: Novel small molecule 11 β -HSD1 inhibitor from the endophytic fungus Penicillium commune

doi: 10.1038/srep26418

Figure Lengend Snippet: ( A ) Compound 3 was observed to occupy the active site with significant scores of ICM docking score and IMC docking mfScore, and adopted a conformation similar to that of known inhibitors. ( B ) Compound 3 had the ability to form key hydrophobic interaction with residues Leu126, Tyr177, Tyr183, and Leu171.

Article Snippet: Using the Molsoft ICM method (ICM-Pro 3.8 molecular docking software), we optimized the geometry of compound 3 and determined the binding site with the lowest-energy and the most favorable orientation of the compound.

Techniques:

Journal: Cell

Article Title: Development of a Novel Lead that Targets M. tuberculosis Polyketide Synthase 13

doi: 10.1016/j.cell.2017.06.025

Figure Lengend Snippet: Structural Features of the Pks13-TE Crystal Structure, Related to Figure 1 (A) Surface representation of the Pks13-TE structure colored by electrostatic potential (contoured at ± 5 kT/e; red for negative and blue for positive) to illustrate the substrate binding groove (∼30 Å, double-headed yellow arrow) on the lid domain. The zoomed view shows the bound PPG fragment (cyan) in the catalytic pocked formed by residues Ser1533, His1699 and Asp1560 along with the residues of the oxyanion-hole (Leu1534 and Ala1477) rendered as sticks; catalytic water shown as red sphere. The 2Fo-Fc electron density map contoured at 1.2σ is shown for the bound PPG fragment. Hydrogen bond interactions are shown as black dashed lines. (B) Predicted tunnels in Pks13-TE structure by CAVER analysis ( Chovancova et al., 2012 ). The three potential tunnels are shown in pink, blue and green surface rendering. The largest of the tunnels (pink) opens onto the substrate binding surface groove and contained the bound PPG fragment. (C) Docking of mycolic acid on Pks13-TE lid domain. A molecule of mycolic acid (shown as yellow sticks) was docked using Molsoft ICM-Pro software to determine a possible binding mode in the substrate binding groove. The zoomed view of the docking indicates that the surface groove can accommodate acyl chains of the mycolic acid precursor attached to the C-terminal ACP domain and position the thioester for cleavage near the catalytic Ser1533 residue.

Article Snippet: A molecule of mycolic acid (shown as yellow sticks) was docked using Molsoft ICM-Pro software to determine a possible binding mode in the substrate binding groove.

Techniques: Binding Assay, Software

Journal: Cell

Article Title: Development of a Novel Lead that Targets M. tuberculosis Polyketide Synthase 13

doi: 10.1016/j.cell.2017.06.025

Figure Lengend Snippet:

Article Snippet: A molecule of mycolic acid (shown as yellow sticks) was docked using Molsoft ICM-Pro software to determine a possible binding mode in the substrate binding groove.

Techniques: Recombinant, Mutagenesis, Purification, Plasmid Preparation, Software