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: Oncology Letters

Article Title: MicroRNA-367-3p induces apoptosis and suppresses migration of MCF-7 cells by downregulating the expression of human choline kinase α

doi: 10.3892/ol.2021.12444

Figure Lengend Snippet: Features of miR-367-3p and chka mRNA 3′-UTR pairing. The shaded sequence represents Watson-Crick base pairing at the seed region of miRNA. miR-367-3p binds the chka mRNA transcript (NM_001277.2) at nucleotides 1804–1825. miR, microRNA; chka , choline kinase α; UTR, untranslated region.

Article Snippet: The firefly luciferase reporter construct containing the 3′-UTR of the chka gene (pMirTarget- chka −3′-UTR) was purchased from OriGene Technologies, Inc. (cat. no. SC212759).

Techniques: Sequencing

Journal: Oncology Letters

Article Title: MicroRNA-367-3p induces apoptosis and suppresses migration of MCF-7 cells by downregulating the expression of human choline kinase α

doi: 10.3892/ol.2021.12444

Figure Lengend Snippet: Target validation of miR-367-3p. MCF-7 cells transfected with the pMirTarget- chka −3′-UTR were treated individually or in combination with the specified miRNAs and miRNA inhibitors. Error bars indicate standard error of the mean from triplicate experiments. *P<0.05, compared with cells treated with the negative control miRNA. miR, microRNA; chka , choline kinase α; UTR, untranslated region.

Article Snippet: The firefly luciferase reporter construct containing the 3′-UTR of the chka gene (pMirTarget- chka −3′-UTR) was purchased from OriGene Technologies, Inc. (cat. no. SC212759).

Techniques: Transfection, Negative Control

Journal: Oncotarget

Article Title: The H3K27me3-demethylase KDM6A is suppressed in breast cancer stem-like cells, and enables the resolution of bivalency during the mesenchymal-epithelial transition

doi: 10.18632/oncotarget.19214

Figure Lengend Snippet: (A) HMLE cells were transduced with either an empty vector (HMLE-Vector), or genes encoding the EMT-inducing transcription factors Snail (HMLE-Snail) or Twist (HMLE-Twist) . Whole-cell lysates were analyzed by immunoblotting for KDM6A. β-actin was used as a loading control. (B-F) MCF10A cells were treated with sterile 4 mM HCl containing 0.1% bovine serum albumin (Vehicle) or TGFB for 5 days (TGFB+). In parallel, MCF10A cells were exposed to TGFB for 5 days in order to induce EMT, and subsequently subjected to TGFB-withdrawal, for a further 5 days, to elicit MET (TGFB+/-). (B-C) MCF10A cells, treated as described, were fixed and immunostained for E-cadherin (B) and N-cadherin (C). Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. (D) MCF10A cells, treated as described, were analyzed by immunoblotting for KDM6B and vimentin (Vim). COX IV was used as a loading control. (E) MCF10A cells, treated as described, were fixed and immunostained for KDM6A. Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. (F) Cells were analyzed by immunoblotting for KDM6A, EZH2 and RING1A. β-actin was used as a loading control.

Article Snippet: Primary antibodies, used for immunoblotting and immunofluorescence, were raised against the following antigens: KDM6A (A302-374A; Bethyl Laboratories, Montgomery, TX, USA, and A302-374A; NBP1-80628, Novus Biologicals, Danvers, MA, USA), vimentin (10082-248; Proteintech, Rosemont, IL, USA), N-cadherin (D4R1H; Cell Signaling Technology, Danvers, MA, USA), E-cadherin (61081; BD Biosciences, San Jose, CA, USA), COX IV (926-42214; Licor, Lincoln, NE, USA), KDM6B (NBP1-06640; Novus Biologicals), EZH2 (5246; Cell Signaling, Danvers, MA, USA), RING1A (13069; Cell Signaling) and β-actin (ab8227; Abcam, Cambridge, MA, USA).

Techniques: Transduction, Plasmid Preparation, Western Blot, Control, Sterility