Journal: Frontiers in Endocrinology

Article Title: Culture in Glucose-Depleted Medium Supplemented with Fatty Acid and 3,3′,5-Triiodo- l -Thyronine Facilitates Purification and Maturation of Human Pluripotent Stem Cell-Derived Cardiomyocytes

doi: 10.3389/fendo.2017.00253

Figure Lengend Snippet: High purity of cardiomyocytes can be obtained under glucose-depleted and fatty acid (with or without T 3 )-supplemented conditions. (A) The schematic for purification and maturation using metabolic selection. Glycogen synthase kinase 3 inhibitor CHIR99021 and Wnt pathway inhibitor IWP2 (gray boxes) were added to the differentiation medium on differentiation days 1 and 4, respectively. On differentiation day 10, cells were dissociated and plated on glass cover slips. After 3 days recovery, cells were cultured with metabolic selection medium (No-glucose DMEM medium supplemented with lactate, fatty acid, and fatty acid + T 3 ). On differentiation day 18, cells were collected for patch clamping experiments. On differentiation day 21, cells were collected for other experiments. (B) Representative fluorescence-activated cell sorting (FACS) analyses of cardiac Troponin T expression in the human pluripotent stem cell (hPSC)-derived cells on day 0 with routine culture medium, and on day 8 with metabolic selection medium. In the control group, the isotype control (mouse IgG) was used instead of the primary antibody. (C) Time courses of selection efficiency using the lactate-supplemented condition (red), the fatty acid-supplemented condition (green), and the fatty acid with T 3 -supplemented condition (purple); cells cultured with routine medium (blue) were set up as the control groups ( n = 3). The asterisk means each test group is significantly different from controls, P < 0.01.

Article Snippet: Briefly, hPSC were treated with small molecule CHIR99021 (Tocris, 4423, final concentration 10 μM) in the RPMI-BSA medium [RPMI 1640 Medium (HyClone, SH30027.01) supplemented with 213 μg/ml AA2P ( l -ascorbic acid 2-phosphate magnesium) (A8960, Sigma) and 0.1% bovine serum albumin (BSA) (A1470, Sigma)] for 24 h, then were incubated with RPMI-BSA medium for 48 h. On differentiation day 4, cells were treated with the small molecule IWP2 (Tocris, 3533, final concentration 5 μM) in RPMI-BSA medium.

Techniques: Purification, Selection, Cell Culture, Fluorescence, FACS, Expressing, Derivative Assay, Control

Journal: Developmental Cell

Article Title: The RAB11A-Positive Compartment Is a Primary Platform for Autophagosome Assembly Mediated by WIPI2 Recognition of PI3P-RAB11A

doi: 10.1016/j.devcel.2018.03.008

Figure Lengend Snippet:

Article Snippet: Drug treatments used include: 250nM AP21967 (Clontech, 635057), 400 nM BAFA1 (Enzo, BML-CM110), 1mM DFP (Sigma, 379409), 5 μM CCCP (Sigma, C2759), 40 μM SMER28 (Tocris, 4297), 300nM VPS34-IN1 (Calbiochem, 532628) for 30 min. Microtubule depolymerization treatments include hypotonic medium (1:3 full medium:water) for 5 min or nocodazole (25 μM for 2 h).

Techniques: Transduction, Recombinant, Lactate Dehydrogenase Assay, CRISPR, Knock-Out, Mutagenesis, Software, Microscopy

Journal:

Article Title: Molecular blockade of VEGFR2 in human epithelial ovarian carcinoma cells

doi: 10.1038/labinvest.2010.52

Figure Lengend Snippet: Increased VEGF-A production in suspension conditions by VEGFR2 OVCAR-3 knockdown cells. (a) ELISA for VEGF-A (Mean ± SEM) detected no change in the amount of VEGF produced by the three OVCAR-3 transfected constructs growing in adhesive monolayer culture. The middle graph shows a significant increase in the production of VEGF-A by shRNAKDR1 clones growing in suspension and the bottom graph shows a significant increase in VEGF-A production by spheroids formed from shRNAKDR1 cells compared shRNAKDR5 and shKDRGAPDH. (b) Short term blockade of VEGFR2 signaling in OVCAR-3 cells with pharmacological inhibitor ZM323881 lead to reduced expression of VEGFR2, NRP-1 and VEGF. (c) Proliferation assay using AlamarBlue (Mean ± SEM) demonstrates significant cell growth in shRNAKDR1 cells compared to other clones. (d) Upper panel shows ascites (OVCAR-3 cells) collected from the intraperitoneal cavity of mice; bottom panel shows western blot of collected cells, demonstrating the stability of VEGFR2 knockdown in the recovered ascites cells. (e) ELISA results (Mean ± SEM) showing higher VEGF-A production in shRNAKDR1 ascites supernatant and lysed cells recovered from intraperitoneal cavities of injected mice, compared to samples from other stable lines.

Article Snippet: For short-term inhibition of VEGFR2 signaling, the small molecule tyrosine kinase inhibitor ZM323881 hydrochloride (Tocris Bioscience, Ellisville, MS, USA) was used as previously reported ( 21 ).

Techniques: Suspension, Knockdown, Enzyme-linked Immunosorbent Assay, Produced, Transfection, Construct, Adhesive, Clone Assay, Expressing, Proliferation Assay, Western Blot, Injection