Fig 1: JNK3 is expressed in regressing hyaloid vessels and mediates EphrinB2-dependent endothelial cell death. (a) STAT1 silencing augments cell viability in HUVEC expressing eB2-5Y. Results reflect the means±SD from 3 experiments; error bars: ±SD. (b,c) eB2-shRNA augments JNK3 mRNA (b; means±SD from 3 experiments; error bars: ±SD) and protein (c) expression in HUVEC. (d) Transduction of eB2-5Y but not eB2-WT augments JNK3 expression in HUVEC; means±SD from 3 experiments; error bars: ±SD (e) Time-dependent JNK3 and cJUN activation in serum-starved HUVEC. Cell lysates from serum-starved HUVEC were tested at the indicated time-points for kinase activity and content of p-JNK1, p-JNK2 and p-JNK3 after immunoprecipitation with anti-p-JNK antibody. (f) JNK3 transduction reduces HUVEC viability; means±SD from 3 experiments; error bars: ±SD (g) JNK3 silencing improves HUVEC viability compromised by eB2-shRNA and B2-5Y transduction; means±SD from 3-5 experiments; error bars: ±SD (h) JNK3 is detected in p5 and p7 WT hyaloid vessels. Degenerating hyaloid vessels are marked by anti-JNK3 antibody staining (red); p-EphrinB immunostaining (green) is absent from JNK3+ hyaloid vessels; DAPI (blue). Scale bars: 200µm top panels, 50µm bottom panels. (i–k) Immunostaining reveals an inverse relationship between JNK3 and p-EphrinB fluorescence intensity in individual IB4+ cells within p5 and p7 WT hyaloid vessels. Cell distribution based on JNK3 and p-EphrinB2 mean fluorescence intensity; each dot represents 1 cell (i); the bar graphs show (j) the means (n=100–300/group) and range (±SD error bars) of p-EphrinB fluorescence intensity in JNK3+ cells and (k) the geographical distribution within hyaloid vessels of JNK3+ (mean fluorescence intensity =3.5)/p-EphrinB+ (mean fluorescence intensity =30) cells (means±SD; n=4 areas/group); fluorescence intensity values in each region are normalized by DAPI. P values from two-tailed Student t-test: N.S. not significant, *P<0.05, **P< 0.01, ***P<0.001.