Assessment of the Anti-angiogenic Effect of VEGFR2 siRNA in Clonetics™ HUVEC using the Lonza 4D-Nucleofector™ System
Poster Apr 26, 2016
Srinivasan Kokatam1 , Kanchan Tiwari1 , Jenny Schroeder2 , Andrea Toell2 , Lubna Hussain3, Preeti Kapoor1
Angiogenesis is a hallmark of most cancers, and is thus an attractive target for the treatment of cancer. One of the easiest screening and target validation strategies for antiangiogenic target identification involves knocking down targets in Human Umbilical Vein Endothelial Cells (HUVEC) and assessing subsequent effects on tube formation in Corning’s Matrigel™ product.
The usage of small interfering RNA (siRNA) is one of the strategies to knock-down RNA, and thereby protein expression within cells. siRNA can be delivered within cells using either chemical transfection or electroporation-based strategies such as the one offered by the Lonza Nucleofector™ Technology.
In the current study we used the Lonza 4D-Nucleofector™ X-Unit to transfect single-donor Clonetics™ HUVEC cultured in EGM™ 2 Endothelial Cell Growth Media. Cells were transfected with siRNA directed against Vascular Endothelial Growth Factor Receptor 2 (VEGFR2). Transfection conditions were fine-tuned using pmaxGFP™ Vector, and it was shown that the Nucleofection process had no deleterious effect on the tube formation potential of HUVEC on Corning’s Matrigel™ product.
Efficient knock-down of VEGFR2 protein levels was demonstrated after the transfection of VEGFR2-siRNA. Best knock-down efficiency was observed 24 hours after transfection. VEGFR2-siRNA transfected cells demonstrated significant inhibition of tube formation on Corning’s Growth Factor Reduced Matrigel™ product in comparison to control samples. Since the efficient knock-down of the VEGFR2 protein in Clonetics™ HUVEC using the Nucleofector™ Technology can be demonstrated at time points as early as 24 hours after transfection, this is an efficient approach for target identification, validation and screening of siRNA based anti-angiogenesis therapeutics for cancer treatment.
We show that network analysis of co-localized ions from mass spectrometry imaging data provides a detailed chemo-spatial insight into the metabolic heterogeneity of tumors. Furthermore, module preservation analysis between colorectal cancer patients with and without metastatic recurrence suggests hypotheses on the nature of the different local metabolic pathways.READ MORE
Angioimmunoblastic T-cell lymphoma (AITL) is a common type of peripheral T-cell lymphoma. AITL can be missed until lymphadenopathy develops in patients initially presenting with skin lesions, as skin biopsy may lack conclusive findings. Our case highlights the extranodal presentation of AITL with cutaneous lesions displaying the TFH phenotype.READ MORE
Studies have shown that IFI16 acts as an antiviral restriction factor against a number of DNA viruses, by inhibiting viral replication or transcription through epigenetic modifications. However, till date, no specific epigenetic function of IFI16 has been identified. Here, we have discovered that IFI16 recruits two histone methyltransferases on the KSHV episome leading to altered Histone H3K9 methylation, thus regulating its lifecycle.READ MORE