Figure 5.

TAZ regulates Nos3 gene transcription with the KLF2 transcription factor. A) Liver sinusoidal endothelial cells (LSECs) were assessed using chromatin immunoprecipitation-quantitative polymerase chain reaction, to evaluate the recruitment of TAZ on the Nos3 promoter region. B) LSECs were immunoprecipitated with anti-TAZ antibodies and the interaction between TAZ and KLF2 was confirmed using immunoblot assay. IgG was used as an immunoprecipitation control. C) Physical interaction of FLAG-tagged TAZ and Myc-tagged KLF2 was verified in HEK293T cells, through a co-immunoprecipitation assay. D) Wild-type (WT) or deletion mutants of TAZ plasmids were introduced into HEK293T cells, along with a Myc-tagged KLF2 expression plasmid. Interaction of TAZ and KLF2 was confirmed using immunoprecipitation with Myc-tag antibodies. WCE, whole cell extracts. E) Transcriptional activity of the Nos3 promoter-containing luciferase reporter construct was analyzed using a reporter gene assay. The constructed vector was introduced into HEK293T cells, along with the TAZ- and/or KLF2-expressing plasmid. pRL-null renilla luciferase plasmid was used for normalization. F) TAZ-WT or -deletion mutant plasmids were transfected into HEK293T cells, along with pGL3-Nos3 promoter and Myc-KLF2 plasmids. Transcriptional activity was assessed via a luciferase reporter gene assay. G) Control (Con) and TAZ-knockdown (Ti) MS-1 cells were transfected with the pGL3-Nos3 promoter, along with pRL-null renilla luciferase plasmid. Transcriptional activity was assessed using a luciferase reporter gene assay. The experiment was done in triplicate (A, E, F, and G). Eight to ten-week-old mice were used for panel A and B. Data are shown as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.0005, and ****P < 0.0001, A; as assessed using two-tailed Student's t-test, E and F; as assessed using one-way ANOVA with Tukey's multiple-comparison test, G; as assessed using two-way ANOVA with Sidak's multiple-comparison test).