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. 2017 Jul 18;8:16074. doi: 10.1038/ncomms16074

Figure 3. Notch3 induces endothelial cell death in vitro.

Figure 3

(a) Sub-G1 analysis of HUVEC electroporated with a control plasmid (Control), or a plasmid expressing the full-length version of Notch3 for 24 h. Quantification was made for three independent experiments and a paired-ratio t-test was applied. (b) AnnexinV/Propidium iodide was performed on HUVEC cells electroporated with a control plasmid or a plasmid expressing Notch3. Quantification was made on three independent experiments and a paired ratio student t-test was applied. (c) Sub-G1 quantification was made after electroporation of HUVEC cells with a control plasmid or a plasmid expressing Notch3 after 48 h of treatment with DMSO or z-LEHD-fmk or z-VAD-fmk pan-caspase inhibitors. Quantification was made on three independent experiments and a paired ratio student t-test was applied. (d) HUVEC were electroporated with a control siRNA (control) or a siRNA targeting caspase-9 (C9). 48 h later, cells were electroporated with a control plasmid or a plasmid expressing Notch3 and 24 h later, sub-G1 analysis was made. Quantification was made on three independent experiments and a paired ratio student t-test was applied. (e) Immunoprecipitation of Myc-tagged S1-Cter Notch1 (S1-CterN1), S1-Cter Notch2 (S1-CterN2) and S1-Cter Notch3 (S1-CterN3) constructs in HEK293t cells together with a control plasmid (−) or a plasmid expressing an HA-tagged dominant-negative version of caspase-9 (C9). (f) Lysates form HEK293t cells carrying Doxycycline (DOX)-inducible HA-tagged Notch3 (piN3) or DOX-inducible HA-tagged N3ICD (piN3ICD) plasmids were immoprecipitated for endogenous caspase-9 and western blot were done to analyse Notch3 (HA) or caspase-9 in total lysates or IP. (g) Proximity Ligation Assay was performed for endogenous caspase-9 or endogenous caspase-8 and doxycycline-induced Notch3 in HEK293 cells. Quantification was made on five images containing 100–150 cells each. (h) Caspase-9 activity was measured from Notch3 immunoprecipitated lysates from HEK293t cells carrying inducible Notch3 (piN3) or inducible N3ICD (piN3ICD) plasmids.