Journal: Frontiers in Oncology

Article Title: Extracellular Vesicles Induce an Aggressive Phenotype in Luminal Breast Cancer Cells Via PKM2 Phosphorylation

doi: 10.3389/fonc.2021.785450

Figure Lengend Snippet: MDA-MB-231 cell–derived EVs impacts for activation of glucose uptake in MCF7 cells: (A) Confocal microscopy images of the microfluidic chip show tdTomato-labeled EVs signals inside MCF7 cells; (B) Nanoparticle tracking analysis (NTA) of isolated EVs from MDA-MB-231. The main peaks are located between 80-150nm; (C) Western blotting of EV-specific proteins, CD63, CD81, and TSG101. For verification of the isolated EVs, western blots were conducted in cell lysate and EV pellets; (D) FDG uptake of MCF7 cells increased in proportion to the dose after administration of the isolated EVs. Although there was no significant change until 20 µg/mL, it showed a significant increase in more than 100 µg/mL; (E) CCK8 assay shows increased cell proliferation of MCF7 cells after administration of the isolated EVs from MDA-MB-231; (F) The effect of MDA-MB-231-mediated EVs compared to control groups, EV-deprived conditioned medium and HFF-derived EVs. EVs of concentration of 100 ug/ml was used for the comparison. HFF-derived EVs failed to induce a significant increase of FDG uptake in MCF7 cells. Both EV-deprived conditioned media increased FDG uptake, but it was judged that there was a high possibility of change due to non-specific factors; EV_MDA, EVs isolated from MDA-MB-231; EV_HFF, EVs isolated from HFF; CM_MDA, EV-depleted conditioned media from MDA-MB-231; CM_HFF, EV-depleted conditioned media. Bars with standard deviation ( n = 3, biologically independent samples) indicate average FDG uptake or absorbance of each sample. Asterisks indicate P values * P < 0.05, ** P < 0.01, and **** P < 0.0001.

Article Snippet: The HepG2 (human hepatocellular carcinoma), Hep3B (human hepatocellular carcinoma), SK-OV-3 (human ovarian cancer), HT-1080 (human fibrosarcoma), HFF (human fibroblast), MDA-MB-231(human triple-negative breast cancer), and MCF7 (human luminal type breast cancer) cell lines were purchased from the Korean Cell Line Bank.

Techniques: Derivative Assay, Activation Assay, Confocal Microscopy, Labeling, Isolation, Western Blot, CCK-8 Assay, Control, Concentration Assay, Comparison, Standard Deviation

Journal: Oncotarget

Article Title: Tumor suppressor REIC/DKK-3 and co-chaperone SGTA: Their interaction and roles in the androgen sensitivity

doi: 10.18632/oncotarget.6488

Figure Lengend Snippet: A. The yeast two-hybrid analysis was conducted using pPC86 (AD)/full-length human SGTA (derived from a normal heart cDNA library) and pDBLeu (BD)/full-length human REIC/DKK-3 plasmids. The blue colonies indicate those with an interaction between the two proteins. B. For the pull-down (PD) assay, the full-length cDNA of human REIC/DKK-3 and SGTA was cloned into the pFN21A and pMACS Kk.HA-C plasmids, respectively. Cell lysates from Halo-tagged REIC/DKK-3- and/or HA-tagged SGTA-transfected 293T cells were analyzed. The sample pulled down using Halo-tagged REIC/DKK-3 was analyzed by Western blotting (WB) using anti-HA antibody. C. REIC/DKK-3 and SGTA protein expression in 293T, PC3 and Hs68 cells was analyzed by Western blotting. Coomassie Brilliant Blue (CBB) staining of the membrane is shown as a loading control. D. The co-localization of REIC/DKK-3 and SGTA was examined by double immunofluorescence staining and observed by fluorescence microscopy. The images in green and red show the intracellular localization of REIC/DKK-3 and SGTA, respectively. The areas of overlap between REIC/DKK-3 and SGTA are shown in yellow in the merged image.

Article Snippet: The normal human foreskin fibroblast cell line Hs68 was provided by JCRB Cell Bank (Osaka, Japan).

Techniques: Derivative Assay, cDNA Library Assay, Clone Assay, Transfection, Western Blot, Expressing, Staining, Membrane, Control, Double Immunofluorescence Staining, Fluorescence, Microscopy