Fig 2: Yap and Taz are functionally redundant during Akt/NRas-driven mouse hepatocarcinogenesis. (A) Study design. Yapflox/flox or Tazflox/flox conditional KO mice were injected hydrodynamically with Akt/NRas/pCMV (control) or Akt/NRas/pCMV-Cre plasmids. (B) Survival curves of Yapflox/flox and Tazflox/flox conditional KO mice injected with Akt and NRas. Mice number in each arm is labeled in the figure. (C and D) Comparisons of liver weight and liver-weight-to-body-weight ratios in (C) Yapflox/flox and (D) Tazflox/flox murine Akt/NRas liver tumors. (E) Western blot analysis showing the expression levels of HA-tag, Yap (upper band), Taz (lower band), and activation of Akt and Ras pathways in wild-type (WT) normal livers, Akt/NRas/pCMV tumors, and Akt/NRas/pCMV-Cre tumors. (F) Immunohistochemical staining of Yap in Yapflox/flox murine Akt/NRas liver tumors. T, tumor; ST, surrounding tissues. Scale bar: 200 μm.

Fig 3: Molecular biological analysis of Yap and Taz-depleted Akt/NRas liver tumors. (A) Western blot analysis showing the expression levels of HA-tag, Yap/Taz, and activation of Akt and Ras pathways. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as a loading control. (B) Immunohistochemical staining pattern of Yap in Yap/Tazflox/flox murine Akt/NRas liver tumors. Scale bar: 100 μm. (C) Quantification of HA-tag, p-AktSer473, and p-Erk1/2 expression in Yap/Tazflox/flox murine Akt/NRas/pCMV and Akt/NRas/pCMV-Cre liver tumors. (D) Expression of Ctgf and Cyr61 in Akt/NRas liver tumors developed in Yap/Tazflox/flox conditional knockout mice. T, tumor; ST, surrounding tissues.

Fig 4: Progressive myofibroblast YAP/TAZ activation occurs during human kidney fibrogenesis.(A) Serial biopsies collected from the same renal allograft at different times intratransplant and after transplant revealed a progressive increase in interstitial fibrosis, as evidenced by increased picrosirius red staining over time. Black arrows point to increased deposition of collagen (pathologic matrix), whereas black arrowheads identify tubular basement membrane. Black scale bar: 100 μm. (B) Kidney sections were costained with antibodies directed against α-smooth muscle actin (α-SMA, green) and YAP (red) with DAPI (blue) nuclear counterstaining. Each dot in the graph represents a single image (n = 5 images day 0, n = 13 images day 9, and n = 12 images month 2). White arrows point to α-SMA+ myofibroblasts with predominantly nuclear YAP staining. White scale bar: 25 μm. The percentage of α-SMA+ myofibroblasts with predominantly nuclear YAP staining was quantified. (C) Kidney sections were similarly analyzed for nuclear TAZ localization in α-SMA+ myofibroblasts (α-SMA: green, TAZ: red, DAPI: blue). Each dot in the graph represents a single image (n = 4 images day 0, n = 5 images day 9, and n = 11 images month 2). White scale bar: 25 μm. One-way ANOVA with post hoc Tukey’s test was used for comparisons. Data shown as mean ± SEM. *P < 0.05.

Fig 5: A proposed model for the role of PCAF in endothelial senescence. PCAF is upregulated in senescent endothelial cells by decreased coupling with DMD2 to inhibit the transportation of YAP/TAZ into nucleus, thereby promoting cellular senescence.