Fig. 4.

Reduced JAGGED1 dependent Notch1 activation of SNAIL1 and SNAIL2 in LVOT-associated mutants. A. HMECs expressing WT or mutant rNOTCH1 were co-cultured with HMEC cells transfected with a control vector (HMEC-ctr) or a JAGGED1 expression vector (JAG1) for 24 h. Western blot analysis was performed using antibodies specific for SNAIL1, SNAIL2, HEY2, HEYL, or Tubulin. Representative results are shown indicating that these markers are upregulated when signal-receiving cells expressing wild-type NOTCH1 receptor are co-cultured with cells expressing JAGGED1, while activation of expression is reduced when signal-receiving cells express NOTCH1^A683T or NOTCH1^G661S. B. The induction of, SNAIL1, SNAIL2, HEYL and HEY2 by JAGGED1 was compared in HMEC cells expressing wild-type NOTCH1, NOTCH1^G661S or NOTCH1^A683T. Band intensities were quantified and normalized to the value for tubulin. Fold activation by JAGGED-expressing cells was calculated (level when co-cultured with JAGGED1 expressing cells/level when co-cultured with HMEC control cells), and the value for fold activation in cells expressing wild-type NOTCH1 was set to one for comparison across experiments (actual fold increases were 6.1±1.2 for SNAIL1 and 8.0±4.3 for SNAIL2, 3.2±1.1 for HEYL, and 2.5±1.2 for HEY2). We observe an overall 50–80% reduction in the expression levels of SNAIL1, SNAIL2, HEYL and HEY2 in cells expressing missense variants of NOTCH1 compared to cells expressing wild-type NOTCH1. Each experiment was performed at least three times in duplicate, and statistical analysis was performed (**=p<.01, *=p<.05 by ANOVA, Dunnett post hoc).