Journal: Journal of Virology

Article Title: Human Choline Kinase-α Promotes Hepatitis C Virus RNA Replication through Modulation of Membranous Viral Replication Complex Formation

doi: 10.1128/JVI.00960-16

Figure Lengend Snippet: Assessment of the interaction of hCKα with NS5A. (A) Huh7 cells coexpressing T7 RNA polymerase and NS3-NS5B were lysed and subjected to co-IP with an anti-NS5 MAb or an isotype-matched control mouse IgG. The precipitates were analyzed by SDS-PAGE, followed by immunoblotting analysis to detect each of the indicated proteins. A portion of aliquots containing 5% of the total proteins in the lysates used for precipitation was loaded as the input control. (B) 293T cells were cotransfected with plasmids encoding each of HCV genotype 1b Con1 strain NS proteins, as indicated, with a pCMV6 plasmid encoding hCKα-Flag. Cell lysates were pulled down with rabbit anti-HA, and the precipitated proteins were subjected to immunoblot analysis with the indicated MAb antibodies. (C) Lysates from Huh7 cells expressing the indicated proteins were precipitated with rabbit anti-Flag, and the coprecipitated proteins were subjected to immunoblot detection with the indicated MAbs. (D) Lysates from Huh7 cells coexpressing GFP-tagged hCKα and different Myc-tagged NS5A domains were pulled down with rabbit anti-GFP, followed by immunoblot analysis using the indicated MAbs.

Article Snippet: Plasmids pCMVΔR8.91 and pMD.G and the pLKO-based short hairpin RNA (shRNA) constructs TRC0005 and TRCN0000284352 were obtained from the National RNAi Core Facility at Academia Sinica. pCMV6-hCKα (RC207209), which encodes hCKα tagged with Myc and DDDK (or Flag) tags at the C terminus, was purchased from Origene, while pEGFP-C1-hCKα encodes hCKα fused with EGFP at the N terminus.

Techniques: Co-Immunoprecipitation Assay, SDS Page, Western Blot, Plasmid Preparation, Expressing

Journal: Breast Cancer Research : BCR

Article Title: Integrative analyses identify modulators of response to neoadjuvant aromatase inhibitors in patients with early breast cancer

doi: 10.1186/s13058-015-0532-0

Figure Lengend Snippet: Integrative analysis of microarray-based comparative genomic hybridization, gene expression and Ki67-based response data. (A) Matched heatmaps of gene expression and aCGH within two amplified loci; 11q13.2-q13.4 and 17q12-q21.1. Bar plots show the result of a Mann-Whitney U -test for expression as a continuous variable and gene amplification as the grouping variable. Bars in red show adjusted P -values <0.05. aCGH, green copy number loss; black, no copy number change; dark red, copy number gain; bright red, gene amplification; gene expression: green, downregulation; red, upregulation; MWU, Mann-Whitney U -test; adjp, adjusted P -value. (B) Venn diagram shows the intersect between the list of genes that are overexpressed when amplified and those genes that are associated with a poor response to AI when amplified. The call-out box lists these genes and their loci, highlighting that only three genes are upregulated in long-term estrogen deprived (LTEDs). (C) Scatter plots demonstrating that for each of the three genes selected for functional validation, significant negative correlation was identified between the aCGH-derived cbs ratios and the percentage decrease in Ki67 following 2 weeks of AI therapy. In each plot, cbs-smoothed ratios are plotted on the y-axis while the percentage decrease in Ki67 at 2 weeks is plotted on the x-axis. Red, CHKA, Blue, LRP5, Green, SAPS3.

Article Snippet: Assays for CHKA (Hs03778879_cn), LRP5 (Hs06321584_cn), SAPS3 (Hs06297988_cn) and reference gene TERT were purchased from Life Technologies.

Techniques: Microarray, Hybridization, Gene Expression, Amplification, MANN-WHITNEY, Expressing, Functional Assay, Biomarker Discovery, Derivative Assay

Journal: Breast Cancer Research : BCR

Article Title: Integrative analyses identify modulators of response to neoadjuvant aromatase inhibitors in patients with early breast cancer

doi: 10.1186/s13058-015-0532-0

Figure Lengend Snippet: Mechanistic assessment of the effect of CHKA in modulating response to aromatase inhibitor (AI) therapy. (A) Following RNA-interference-mediated silencing of CHKA, SUM44 cells were transfected with an estrogen receptor (ER)/ERE luciferase reporter construct, and then treated with E2 or dextran charcoal-stripped media (DCC) for 2 days before reading luciferase activity. Data are normalized to the activity in the DCC-treated control transfected cell lines. *Significant P -value (<0.05) between the indicated column and corresponding siControl-equivalent. (B) To validate effects seen with the ER-ERE reporter assay, expression levels of two well known ER-regulated genes ( TFF1 and GREB1 ) were assessed by quantitative real-time PCR following RNA-interference-induced silencing of CHKA. Data normalized to DCC-treated control transfected cell lines; * P -value <0.01 between indicated column and corresponding siCON equivalent. (C) Following RNA-interference-induced silencing of CHKA, cell lysates were subjected to gel electrophoresis and western blotting using indicated antibodies. Cells were treated with 1nM E2 for 1 h or 24 h following transfection, to represent the two phases of ER dynamics (early active- and late turnover phase). Blots are representative of at least two independent experiments; numbers below each band represent densitometry analysis of intensity, measured as a ratio of the siControl with no siCHKA or E2 treatments.

Article Snippet: Assays for CHKA (Hs03778879_cn), LRP5 (Hs06321584_cn), SAPS3 (Hs06297988_cn) and reference gene TERT were purchased from Life Technologies.

Techniques: Transfection, Luciferase, Construct, Activity Assay, Control, Reporter Assay, Expressing, Real-time Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: Integrative analyses identify modulators of response to neoadjuvant aromatase inhibitors in patients with early breast cancer

doi: 10.1186/s13058-015-0532-0

Figure Lengend Snippet: The effect of siRNA (CHKA) on cell cycle as determined by flow cytometry. (A) Flow cytometry was used to compare DNA content in Sum44 WT cells treated with dextran charcoal-stripped media (DCC) and DCC + E2. There was no significant difference between the cell cycle phases in the cells treated with DCC. CHKA arrests cells at the G1/S, but not at the G2/M phase of the cell cycle in cells treated with DCC + E2. (B) The percentage of cells at each cell phase: G0/G1, S, G2/M are shown on the bar chart as the mean ± SD. The experiment was performed in triplicate; ** P <0.005 and * P <0.01.

Article Snippet: Assays for CHKA (Hs03778879_cn), LRP5 (Hs06321584_cn), SAPS3 (Hs06297988_cn) and reference gene TERT were purchased from Life Technologies.

Techniques: Flow Cytometry