S2510

siRNA experiments in human umbilical vein endothelial cells (HUVECs) were performed as previously described^{12 (link)}. HUVECs from Lonza were authenticated and tested negative for mycoplasma. siRNAs directed against KRIT1 (s2510, Invitrogen), KLF2 (s20270, Invitrogen), KLF4 (s17793, Invitrogen), STK24 (s15993, Invitrogen) or STK25 (s20570, Invitrogen) were used for the knockdown experiments. For overexpression studies, human microvascular endothelial cells (HMVECs) were infected with adenovirus encoding either mouse KLF2 (Penn Vector Core), human KLF4 (Vector BioLabs), or a LacZ control (Penn Vector Core). HMVECs from Lonza were authenticated and tested negative for mycoplasma. Cells were harvested 48 hours post-infection and total RNA was isolated using TRIzol Reagent (Life Technologies). cDNA was generated from 1 μg total RNA using SuperScript First-strand Synthesis System (Invitrogen) and real time qPCR was performed using Power SYBR Green PCR Master Mix (Applied Biosciences).
Primers for qPCR:

Cell culture: Pooled human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (#CC- 5 2519) and cultured in endothelial basal medium (EBM, Lonza) supplemented with hydrocortisone (1μg/ml), bovine brain extract (12 μg/ml), gentamicin (50 μg/ml), human recombinant epidermal growth factor (10 ng/ml), and 10% fetal bovine serum (FBS, Life Technologies). HUVECs were tested negative for mycoplasma and cultured until the fourth passage. Cells were maintained at 37 °C in a humidified atmosphere with 5% CO2.
siRNAs directed against KRIT1 (s2510, Invitrogen) were used for the knockdown experiments at a concentration of 10nM. For overexpression studies, HUVECs were infected with a control lentivirus encoding either GFP, or mutant human PIK3CA with the H1047R mutation (constructed from pBabe puro HA PIK3CA H1047R from Jean Zhao (Addgene plasmid # 12524). Cells were harvested 48 hours post-infection or post-transfection and total RNA was isolated using TRIzol Reagent (Life Technologies). cDNA was generated from 500ng total RNA using the SuperScript IV VILO Master Mix (Thermo Fisher, 11756050) and real time qPCR was performed using Power SYBR Green PCR Master Mix (Applied Biosciences). Protein was harvested with RIPA buffer with complete protease inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitor cocktail (Roche).