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. Author manuscript; available in PMC: 2017 Feb 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2015 Dec 3;36(2):350–360. doi: 10.1161/ATVBAHA.115.306713

Figure 5. LRP1 knockdown increases PARP-1 activity-dependent phosphorylation of Rb and CDK2 in HRECs.

Figure 5

A–C, LRP1 knockdown increases CDK2 and retinoblastoma (Rb) phosphorylation. HRECs were transfected with LRP1 or control siRNA. Two days later, cells were incubated under normoxia (21% O2) or hypoxia (2% O2) for 2 hours. Cell lysates were immunoblotted with indicated antibodies. The ratios of the phosphorylated CDK2 and Rb to their total respective protein amounts were quantified by Image J (B and C). D, PARP-1 activity increased in LRP1 knockdown HRECs in response to hypoxia at 2% O2. ELISA enzymatic assay for PARP-1 was performed with PAR as a substrate of PARP-1 coated in the plate. Quantitative data were presented as a ratio of relative light unit to protein amount in cell lysates. E–G, PARP-1 inhibition decreases CDK2 and retinoblastoma (Rb) phosphorylation. HRECs were incubated with PARP-1 inhibitor PJ-34 at 3 μM and under normoxia (21% O2) or hypoxia (2% O2) for 2 hours. Cell lysates were immunoblotted with indicated antibodies. The ratios of the phosphorylated CDK2 and Rb to their total respective protein amounts were quantified by Image J (F and G). H–J, PJ-34 blocks the increases in CDK2 and retinoblastoma (Rb) phosphorylation following LRP1 knockdown. HRECs were transfected with LRP1 or control siRNA. Two days later, cells were treated with PARP-1 inhibitor PJ-34 at 3 μM and incubated under normoxia (21% O2) or hypoxia (2% O2) for 2 hours. Cell lysates were immunoblotted with indicated antibodies. The ratios of the phosphorylated CDK2 and Rb to their total respective protein amounts were quantified by Image J (I and J). K, Schematic illustration to shown how LRP1 regulates cell cycle progression, endothelial proliferation and retinal angiogenesis induced by hypoxia. *, P<0.05, compared to same cells under normoxia. #, P<0.05, compared to control HRECs exposed to same oxygen level. **, P<0.05, compared to LRP1 siRNA-transfected cells without treatment of PJ-34 inhibitors. n=3. All data were analyzed with two-way ANOVA analysis followed by the Fisher’s LSD or Turkey multiple comparison test.