Journal: Oncogene

Article Title: Antiangiogenic and tumour inhibitory effects of downregulating tumour endothelial FABP4

doi: 10.1038/onc.2016.256

Figure Lengend Snippet: FABP4 knockdown reduces vascular sprout elongation and endothelial cell viability. Human umbilical vein endothelial cells (EC) were transfected with non-targeting control siRNA or two different sequences of siRNA targeting FABP4 (20 n m , pooled or single as indicated). To determine vascular sprouting, transfected or ECs were grown as spheroids in hanging drops and embedded in matrigel, and sprout number and length were quantified after 48 h. n =4 ( a ). To determine migration, scratched confluent monolayers were monitored for % wound closure over time ( b ). To measure cell number as an indicator for proliferation, ECs transfected with pooled siRNA sequences #1 and #2 targeting FABP4 were grown for 72 h, trypsinized and counted. n =3 ( c ). Cell viability was determined in the same samples based on cell membrane permeability for trypan blue. n =4 ( d ). * P <0.05; ** P <0.01; *** P <0.001. Error bars, s.d.

Article Snippet: Primary human umbilical ECs were purchased from Lonza, Visp, Switzerland and cultured in endothelial growth medium 2 (Lonza).

Techniques: Knockdown, Transfection, Control, Migration, Membrane, Permeability

Journal: Oncogene

Article Title: Antiangiogenic and tumour inhibitory effects of downregulating tumour endothelial FABP4

doi: 10.1038/onc.2016.256

Figure Lengend Snippet: FABP4 knockdown regulates fatty acid metabolism enzymes and increases fatty acid oxidation. Human umbilical vein endothelial cells (EC) were transfected with non-targeting control siRNA or two different sequences of siRNA targeting FABP4 (20 n m ) and mRNA expression of FABP4 and PPARG were determined and are expressed relative to β-actin (ACTB). n =5 ( a, b ). Protein levels of FABP4, phosphorylated HSL (Serine 565, p565, inhibitory; Serine 660, p660, activating) and total HSL were analysed in response to FABP4 knockdown by immunoblot analysis, using β-actin as a loading control. Band densitometry analysis of phosphorylated HSL was carried out relative to total HSL. n =3 ( c ). To measure lipid droplet accumulation, transfected ECs were exposed to media containing BSA or oleic acid (OA, serving as a positive control) conjugated to BSA for 16 h prior to staining lipid droplets with the fluorescent dye LD540 and measuring signal intensity in the fluorescein isothyanate (FITC) channel by FACS analysis. n =3 ( d ). Fatty acid uptake and oxidation were measured by detecting intracellular 14 C or 14 CO 2 derived from [U14C]-labelled OA in the cell culture media. n =4 ( e–g ). FAO rates were corrected for the levels of FAs taken up into the cells. Etomoxir (50 μ m , Eto) or dimethylsulphoxide (DMSO) were added 16 h after the transfection to inhibit fatty acid oxidation n =3 ( g ) * P <0.05; ** P <0.01; *** P <0.001. Error bars, s.d.

Article Snippet: Primary human umbilical ECs were purchased from Lonza, Visp, Switzerland and cultured in endothelial growth medium 2 (Lonza).

Techniques: Knockdown, Transfection, Control, Expressing, Western Blot, Positive Control, Staining, Derivative Assay, Cell Culture