Fig 2: Loss of Klotho heterozygosity leads to changes in vascular structure and abnormalities of the YAP signaling pathway in vascular endothelial cells. (A) H&E staining showed the abnormal structure of blood vessels and capillaries in the kidney tissue of Klotho+/- mice. Magnification 400×. scale bar = 30μm. (B) The measurement results of vascular lumen area and vascular elastic lamina area on renal vascular and blood capillary. **P<0.01 vs WT group, *P<0.05 vs WT group, t test. (C) Immunofluorescence staining showed that the expression of Klotho on vascular endothelial cells (CD31+) in kidney tissue of Klotho+/- mice decreased significantly. Magnification 400×. scale bar = 30μm. (D) Western blot showed that the expression of the YAPsignaling pathway in vascular endothelial cells of kidney tissue of Klotho+/- mice was abnormal.

Fig 3: Detailed expression analysis of β-catenin and YAP/TAZ signaling pathways.(A) A 23-gene signature as reported by Abitbol et al. [12], was utilized to explore β-catenin signaling in liver cancers, including canonical, liver-specific, and negatively regulated β-catenin target genes. The red/green color coding for the repaired clones, was created by determining the fold-change in expression relative to the corresponding average of parental samples. This color coding is only depicted for exploratory purposes and does not indicate that expression levels are also significantly different from the parental cells. (B) Similarly, the gene expression level was analyzed using RNA-seq data of YAP/TAZ target genes and components of the Hippo signaling pathway. (C) Western blotting for Hippo pathway components, with β-actin used as loading control. All protein density levels are normalized to β-actin in the same blot, and are presented in S10 Fig.

Fig 4: Smoking triggers the methylation of SAV1 and MB1/2 could be used in lung cancer diagnosis. (A) SAV1 gene expression data (normalized TPM) for smoking and non-smoking patients were obtained from the TCGA LUAD/LUSC samples. (B) The relative SAV1 mRNA expression level was determined by RT-qPCR in lung cancer tissue and adjacent healthy tissue samples from smoking or non-smoking patients with LUAD/LUSC. (C, D) Western blot (WB) showing expression levels of SAV1 in lung cancer tissue and adjacent healthy tissue samples from smoking or non-smoking patients with LUAD/LUSC. (E) Quantification of WB band intensity from (C,D). (F) Kaplan-Meier patient survival curves for overall survival (OS) rates based on SAV1 expression in smoking or non-smoking patients from TCGA database. (G, H) The DNA methylation level of SAV1 promoter region was analyzed by Bisulfite sequencing PCR (BSP) in lung cancer tissue and adjacent healthy tissue samples from smoking or non-smoking patients. (I, J) Two methylation blocks (MB1, MB2), CpG clustering sites locating at SAV1 promoter specifically related to lung cancer, were found by BSP. Results represent means ± SD of at least two independent experiments. Statistical significance: p< 0.05 (Student's t-test).

Fig 5: 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).