Fig 2: Yap activity is increased in Bmal1 mutant tumors. (A and B) Representative images showing increased Yap+ nuclear signal in the cells of Apcmin + Bmal1-/- tumors (arrowheads show the cell nucleus in inset image ). Yap-activated nuclei are present in higher numbers (t test, P = .0186). Scale bar: 50 μm. (C) Western blots showing increased levels of total and activated Yap in Apcmin; Bmal1-/- mutant tumors. (D and E) Representative images showing increased Yap+ nuclear signal in the cells of Apcmin + Bmal1 cKO that are present in higher numbers (t test, P = .0009). Scale bar: 50 μm. (F and G) Control tumors show higher expression of Lgr5 RNA (blue) while Apcmin + Bmal1 cKO tumors show higher Tead4 RNA expression (red). The area of Tead4+ cells is significantly higher in Apcmin + Bmal1 cKO (ANOVA f(3, 36) = 9.241; P = .0001). Scale bar: 50 μm. (H and I) Apcmin + Bmal1 cKO tumors show higher levels of the Yap target gene Ereg (blue), but lower levels of the Wnt target gene, Axin2 (red), relative to controls. The area of Ereg+ cells is significantly higher (ANOVA f(3, 36) = 9.422; P < .0001). Histologic quantification shown for n = 5–10 regions per animal, and n ≥ 3 animals per condition. Scale bar: 100 μm. All error bars represent SEM.

Fig 3: SOX9 and YAP protein expression assessment on oADSCs cultured under both teno-differentiative conditions. (A) Representative Western blot images for SOX9, a-YAP, and total YAP protein expressions in oADSCs at P1 cultured in GM on a Petri dish for 14 days (CTR 14 d) and treated with teno-inductive CM for 14 days (+CM 14 d) (B) Representative Western blot images of SOX9, a-YAP, and YAP protein expression in oADSCs at P1 cultured in GM on a Petri dish for 48 h (CTR 48 h) or seeded on ha-PLGA fleeces for 48 h and 14 days (+Fleece 48 h and +Fleece 14 d, respectively). Relative densitometric values of SOX9, a-YAP and total YAP were normalized on α-TUBULIN expression. Data were expressed as means ± S.D. of three replicates/each biological sample. *, **, ***, **** Statistically significant values between the different studied groups (p < 0.05, p < 0.01, p < 0.001 and p < 0.0001, respectively).

Fig 4: Deletion of SAV1 or WWC1–3 induces YAP/TAZ activity in DPM cells(A) Schematic representation of two Hippo signaling modules. There are two modules within the Hippo signaling network: MST1/2-SAV1-WWC1–3-LATS1/2 (HPO1) and MAP4K1–7-NF2-LATS1/2 (HPO2).(B) Oncoplot depicting the distribution and alteration patterns of Hippo pathway genes in TCGA DPM datasets.(C) Expression analysis of Hippo pathway component in NCI-H2373, NCI-H2052, and NCI-H2452 mesothelioma cell lines using immunoblotting.(D and E) Enhanced YAP/TAZ activity in WWC1–3 tKO and SAV1 KO DPM cells. The pYAP and aYAP indicate phosphorylated and active YAP, respectively.See also Figure S1.

Fig 5: Downregulation of SAV1 or WWC1–3 expression promotes the tumor-forming capacity of DPM cells(A) Colony formation of DPM cell lines was enhanced upon SAV1 deletion.(B) Colony formation of DPM cell lines was enhanced upon WWC1–3 deletion.(C) Gross tumor images of tumor xenografts of control or SAV1 KO NCI-H2373 cells implanted in nude mice. Scale bar, 1 cm.(D and E) Growth curve and tumor weight analysis of control and SAV1 KO NCI-H2373 xenografts. Five mice with 10 tumors for each group were analyzed.(F) Gross tumor images of tumor xenografts of control or WWC1–3 KO NCI-H2373 cells implanted in nude mice. Scale bar, 1 cm.(G and H) Growth curve and tumor weight analysis of control and WWC1–3 KO NCI-H2373 xenografts. Five mice with 9 tumors for each group were analyzed.(I and J) Histological assessment of control and SAV1 KO NCI-H2373 tumor xenografts. H&E and immunohistochemistry (IHC) (Ki67 and YAP) staining and quantifications. Scale bar, 100 μm.(K and L) Histological assessment of control and WWC1–3 KO NCI-H2373 tumor xenografts. H&E and IHC (Ki67 and YAP) staining and quantifications. Scale bar, 100 μm.Data are presented as mean ± SEM from three independent experiments for (A) and (B). Statistical significance: ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001. The two-way ANOVA test was used for tumor growth curves (D, I), and the Student’s t test was used for other data.See also Figure S2.