Fig 2: Compensatory decrease of YAP stability and activation of the Hippo kinase cascade in the YapS112A mice. (A) Faster turnover of endogenous YAPS112A protein compared with endogenous wild-type (WT) YAP protein. Confluent cultures of wild-type and YapS112A MEFs were treated with 50 µg/mL CHX for different periods of time and probed with the indicated antibodies. A representative blot is shown. YAP protein levels were quantified by the LI-COR Odyssey imaging system from three parallel experiments, normalized to actin, and arbitrarily set as 1 at time 0 in the graph shown at the right. Note that twice as much YapS112A cell lysates were loaded in each lane to adjust for the lower levels of YAP protein in the YapS112A cells (*). (B) Western blot analysis of liver protein lysates from wild-type and YapS112A mice. The graphs show quantification of YAP, TAZ, NF2, Mst1, Mst2, Lats1, Lats2, p-Lats, TEAD1-4, and CTGF levels in mutant livers relative to wild-type livers. Data are mean ± SD from three animals for each genotype. (*) P < 0.05, t-test.

Fig 3: Reduced expression of stretch-induced contractile markers and decreased myogenic response in Y/T KO (YAP/TAZ [yes-associated protein 1/WW domain containing transcription regulator 1] knockout) mice. The portal veins (n=3 in all groups and targets) were kept in organ culture for 24 h either stretched with gold weight (loaded) or not stretched (unloaded). Reverse transcription-quantitative polymerase chain reaction analysis of selected smooth muscle markers in portal veins from Y/T KO and Ctrl (control) mice: (A) Tagln, (B) Cnn1, (C) Des, and (D) Acta2. E–J, Mesenteric arteries were mounted in a pressure myograph. E, Intraluminal pressure was increased systematically, and active vessel diameter was recorded in Ca2+ containing HEPES-buffered Krebs solution. F, Passive vessel diameter was measured in Ca2+-free solutions. G, The relative change in passive diameter was used as an indicator of vascular distensibility. H, Myogenic response was calculated as the relative difference between active and passive diameter. I, A single dose of 100 nmol/L Ang II (angiotensin II) was added to the preparations followed by gradual increase in intraluminal pressure. Active diameter was monitored, and myogenic response was calculated (E–I; Ctrl, n=6; Y/T KO, n=6). J, Peak transient contraction relative to baseline after 100 nmol/L Ang II stimulation (Ctrl, n=5; Y/T KO, n=5). All data are presented as mean±SEM. FC indicates fold change.

Fig 4: SFTA1P is transcriptionally regulated by YAP/TAZ/TEAD in NSCLC cell lines.A A schematic illustration of the lncRNA profiling in MKN28 gastric cancer cells. SFTA1P was identified as a candidate target of YAP/TAZ. Briefly, engineered YAP/TAZ-knockout cells (YKO/TKO) were transduced with empty vector (TKO-EV or TKO) or active form of YAP (TKO-YAP or YOE)/TAZ (YKO-TAZ or TOE). RNA sequencing (n = 2 biologically independent samples) was performed to identify the differentially expressed lncRNAs with the criteria indicated. Venn diagram shows the lncRNAs that were up/downregulated upon overexpression of YAP/TAZ. Bar chart plots the relative expression of SFTA1P using Fragments Per Kilobase of transcript per Million mapped reads (FPKM). The heatmap demonstrates the relative expression of differentially expressed lncRNAs upon YAP/TAZ overexpression. The LOG2 fold-change of each lncRNA upon YAP/TAZ overexpression in two replicates were calculated and plotted. The red arrow pinpoints SFTA1P. B The expression of SFTA1P in lung cell lines was evaluated by RT-qPCR. The relative expression of SFTA1P was calculated by normalizing the SFTA1P expression in the indicated cell lines against that in the BEAS-2B cell line. Mean ± SD, n = 3 (biological replicates). C Western blot analysis of pYAP (S127), YAP, pTAZ (S89), TAZ, CYR61, and GAPDH in the indicated lung cell lines. D–G Knockdown of YAP (D–E), TAZ (D–E), and TEADs (F–G) in H1299 cells. The knockdown efficiencies were evaluated by western blot analysis in (E, G). The RNA expression of SFTA1P, ANKRD1, CTGF, and CYR61 were measured by RT-qPCR following the knockdowns of YAP and TAZ (D) or TEAD1 and TEAD4 (F), Mean ± SD, n = 4 (biological replicates). H The transcriptional activity of the regulatory sequence in the vicinity of SFTA1P TSS was assessed by dual-luciferase assay upon co-transfection of the reporter plasmids and siRNAs against YAP, TAZ, or TEADs in H1299 cells. A schematic illustration of the promoter sequence cloned into a pGL3-Basic vector was shown in the top panel: the 910 bp upstream and 2520 bp downstream sequence of the SFTA1P’s TSS (overlapping with the 1st exon, 1st intron, and the partial sequence of the 2nd exon) was examined. Mean ± SD, n = 3 (biological replicates). I Subcellular localization of SFTA1P was determined by RT-qPCR following subcellular fractionation in H1299 cells. The expression of GAPDH (cytoplasmic marker), U2 snRNA (nuclear marker), and SFTA1P were each quantified and normalized to their corresponding expression levels in the whole-cell lysate. The percentage of the expression in cytoplasmic fraction or nuclear fraction versus the summation of them were calculated and plotted. Mean ± SD, n = 3 (biological replicates). J The presence of Poly(A) tail in SFTA1P transcript was measured by RT-qPCR following reverse transcription using either random hexamer or oligo-dT. The relative expression of each gene was obtained by normalizing against the expression of beta-actin. The comparisons were calculated by normalizing the relative expression in the cDNA library synthesized using random hexamer. Mean ± SD, n = 3 (biological replicates). Statistical significance was determined using Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: Hepatic Let-7a pathways in the liver of the offspring of control and obese dams after partial hepatectomy. Hepatic Let-7a-5p (A) and Prkaa2 (B) gene expression (qPCR), AMPKa2 and pAMPKa2 immunoblotting (C), YAP1 (D) gene expression (qPCR), and YAP/TAZ immunoblotting (E) from the offspring of control (CT-O) and obese (HF-O) dams, before (baseline) or 4 h (PHx 4 h) or 48 h (PHx 48 h) after partial hepatectomy surgery. Data are plotted as individual values (dot plots), means and SEM, represented by vertical bars. n = 5–10/group. *p = 0.05 and **p = 0.005 after Student’s t-test (CT-O versus HF-O). Representative image and histogram of cell cycle distribution (n = 2) by flow cytometry in AML12 cells transfected with negative control, anti-Let-7a, or Let-7a mimic (F).