Effect of treating HRMVECs with nutlin-3 and/or norrin
To identify transcripts controlled by elevated P53 in HRMVECs we inhibited the MDM2/P53 interaction using nutlin-3. To identify cross-talk of P53 and beta-catenin dependent signaling, the experiment was performed in the presence or absence of the frizzled4 ligand norrin. Expression of beta-catenin-dependent signaling response genes induced by norrin, e.g., AXIN2 and APCDD1, were inhibited by nutlin-3. Nutlin-3 furthermore altered the expression of several gene sets, e.g., known p53 regulated genes (e.g., CDKN1A, MDM2), genes that potentially regulate beta-catenin-dependent signaling (including NCAPH, NEURL2, DRAXIN, DKK1), mediators of blood-retina barrier function (including SOX18, KIF11, ABCG2, MFSD2A), and mediators of inflammatory signaling  and leukocyte extravasation (including ICAM1, CCL2, SELE, and CXCL1). The gene expression changes mediating inflammatory signaling and leuokocyte interactions substantially overlap with those expression changes observed in endothelial cells under high glucose conditions and diabetes mellitus. Overall design: HRMVECs were treated for 24 hours: unstimulated, stimulated with norrin only, stimulated with nutlin-3 only, and stimulated with both norrin and nutlin3. Nutlin-3 was used at 10 µM, norrin (R+D Systems) was used at 200 ng/ml. Nutlin-3 was solved in DMSO. DMSO was added as necessary to keep the DMSO concentration at 0.1% v/v in all groups. Quadruplicate samples of four groups were subjected to paired-end RNA-seq of ribodepleted RNA. Ca. 20 mio reads per sample were obtained.