Figure 3.

PTPN11 variant cell models reveal MAPK pathway activation in HEK293 cells and disruption of angiogenesis in a HUVEC primary endothelial cell line. (a) PTPN11 mRNA expression is significantly increased in WT and all PTPN11-variant cells compared with that in untransfected controls, and in PTPN11^T468M and PTPN11^A461T variants, PTPN11 mRNA expression is significantly increased compared with WT overexpression (mean with SD of samples in quadruplicate). (b, c) Expression of phosphorylated ERK is significantly increased in all PTPN11-variant cells but not in WT cells, compared with that in untransfected controls, and PTPN11^T468M-induced ERK phosphorylation is further significantly increased compared with the WT (mean with SD of samples in triplicate). (d) Still images from IncuCyte live-cell analysis during endothelial cell tube formation in vitro show significantly disrupted angiogenesis in HUVEC lines transfected with variant PTPN11 relative to that in WT and untransfected and mock-transfected cells. Bar = 500 ‒m. (e‒g) Quantification of angiogenesis assay (error bars ± SEM) shows a significant difference in variance in (e) mean total isolated branch length, (f) mean number of isolated segments, and (g) mean mesh size. See also Supplementary Movie S1. ERK, extracellular signal‒regulated kinase; HEK293, human embryonic kidney 293; HUVEC, human umbilical vein endothelial cell; ns, not significant; WT, wild type.