Figure 1.

YAP-SAV1 negative feedback loop maintains lung epithelial cell homeostasis. (A) Quantification of colony numbers of different lung cell lines in soft agar. (B) Intersection of genes up-regulated in BEAS2B cells with those non-significantly upregulated in HCC827/H2170 cells. (C) After overexpression of YAP, the expression of the target gene SAV1 was measured by western blot (WB) analysis. (D) Using GTEx and TCGA transcriptome data, the transcripts per million (TPM) expression value shows the positive correlation between SAV1 and YAP in normal lung samples rather than in cancer samples. (E) TCGA data shows a stronger correlation between SAV1 and YAP in normal tissue than cancer group in 20 tissues. (F) Schematic of SAV1 promoter region and the two TEAD binding sites (Wt1#, Wt2#) with two corresponding mutant sites are shown (Mut1#, Mut2#). (G) SAV1 promoter activity was assessed with the dual luciferase reporter assay in HEK-293T transfected with indicated plasmids. (H) Dual-Luciferase reporter assay in different lung cell lines. Verteporin: YAP-TEAD inhibitor. (I) CHIP-qPCR confirmed that Wt2# is the effective binding site located in the SAV1 promoter region rather than Wt1#. (J) Quantification of colony numbers of YAP-overexpressing BEAS2B cells with or without SAV1 knockdown in soft agar. Results represent means ± SD of at least two independent experiments. Statistical significance: P< 0.05 (Student's t-test).