8A). However, decreased phosphorylation of STAT3 was observed in both GSI-treated HL7702 cells and liver extracts from RBP-J KO mice after I/R injury (Fig. 5D; Supporting Fig. 7C,D). this website Because STAT3 transactivates MnSOD,19, 29 blocking Notch signaling might down-regulate the transcription of MnSOD through decreased STAT3 activation, leading to increased ROS and aggravated I/R injury. SOCS3, an inhibitor of STAT3 activation, was slightly

up-regulated in GSI-treated HL7702 hepatocytes (Supporting Fig. 8B) during I/R injury, in contrast to Notch-deficient macrophages.23 Hes proteins bind to STAT3 and facilitate phosphorylation of STAT3 by JAK2 (data not shown).30 Using immunoprecipitation, we found that during I/R injury in both mice and HL7702 cells, the absence of Notch signaling resulted in decreased Hes5-STAT3 complex (Fig. 5E,F; Supporting Fig. 9). This finding indicated that disruption of Notch signaling reduced STAT3 activation by decreasing the expression of Hes5 under I/R. HL7702 hepatocytes were transfected

with constitutively active STAT3 (STAT3C) (Supporting Fig. 10A). TUNEL staining indicated that overexpression of constitutively active STAT3 abrogated GSI-induced increase of apoptosis during I/R in vitro (Fig. 6A,B). Constitutively active STAT3 also reduced the level of ROS, which increased upon Notch signal deficiency during I/R (Fig. 6C,D). Using Western blot analysis, we found that constitutively active STAT3 up-regulated MnSOD, which was repressed by GSI during I/R injury (Fig. Protein Tyrosine Kinase inhibitor 6E; Supporting Fig. 11A). HL7702 cells were triggered by coculturing with OP9-Dll122 and were subjected to I/R injury in vitro. Compared with HL7702 cells cocultured with OP9-GFP, forced activation of Notch reduced ROS and apoptosis (Fig. 7A) after reperfusion, suggesting that Notch activation protected hepatocytes from I/R injury by reducing ROS. Hes5 is the major effector of Notch signal in

hepatocytes during I/R injury (Fig. 1A; 上海皓元 Fig. 5E,F). HL7702 hepatocytes were stably transfected with pcDNA3.1-Hes5 or pcDNA3.1 (Supporting Fig. 10B) and were subjected to I/R in vitro in the presence of DMSO or GSI. Overexpression of Hes5 ameliorated apoptosis during I/R injury, even in the presence of GSI (Fig. 7B,C). Concomitantly, increased ROS (Fig. 7D,E), decreased MnSOD, and STAT3 phosphorylation were also reversed by overexpression of Hes5 (Fig. 7F; Supporting Fig. 11B). These data further suggest that Notch signaling protected hepatocytes through the Hes5-STAT3-MnSOD-ROS pathway during I/R injury. Our results demonstrated in vitro and in vivo that Notch signaling regulates I/R injury by modulating ROS. The homeostasis of ROS is maintained by its production and scavenge.