Fig 2: Early lesions developed in AKT/TAZ mice display an inflammatory infiltrate. (A) Two weeks after hydrodynamic injection of AKT and TAZ, numerous transfected cells (indicated by black arrows) were encircled by an inflammatory infiltrate. The transfected cells and the inflammatory cells could be better appreciated at higher magnification (B), with the inflammatory cells characterized by small spherical nuclei and forming a fence around the transfected cells. The transfected cells displayed elevated TAZ (C) and CK19 (D) immunoreactivity. The inflammatory cells consisted of lymphocytes, characterized by immunoreactivity for CD4 and CD45 markers (E, F), and macrophages (immunoreactivity for the F4/80 marker) (G). Some small cells encircled by the inflammatory reaction showed signs of apoptosis, as revealed by positive immunoreactivity of apoptotic bodies (indicated by black arrows) for cleaved Caspase 3 (H) and cleaved PARP (I), implying their destruction by the inflammatory response. Original magnification: 100x in (A), 400x in (B-I); scale bar: 200 μm in (A), 50 μm in (B-I).

Fig 3: The inhibitor of miR-690 partially reversed the up-regulation osteogenesis and down-regulation adipogenesis induced by M2D-Exos. (A) The level of miR-690 in BMSCs after treated with M2D-Exos or PBS. *p < 0.05 compared to the BMSC group. (B) The level of miR-690 in M2 macrophages after treated with lipo3000, miR-690 inhibitor, or inhibitor control measured by qRT-PCR analysis. Control group was M2 macrophages treated with lipo3000. *p < 0.05 compared to the inhibitor control group. (C) The level of miR-690 in BMSCs after treated with M2D-Exos, miR-690 inhibited M2D-Exos, or inhibitor control M2D-Exos before cultured in osteogenic inducer were measured by qRT-PCR analysis. *p < 0.05 compared to the M2D-Exos NC inhibitor group. (D) The level of miR-690 in M2 macrophages after treated with lipo3000, miR-690 inhibitor, or inhibitor control before cultured in adipogenic inducer were measured by RT-PCR analysis. *p < 0.05 compared to the M2D-Exos NC inhibitor group. (E) IRS-1, TAZ, OCN, RUNX2 mRNA level of BMSCs after treated with M2D-Exos, miR-690 inhibited M2D-Exos, or inhibitor control M2D-Exos during osteogenesis were measured by qRT-PCR analysis. *p < 0.05 compared to the M2D-Exos NC inhibitor group. (F) IRS-1, TAZ, C/EBPß, and PPAR? mRNA level of BMSCs after treated with M2D-Exos, miR-690 inhibited M2D-Exos, or inhibitor control M2D-Exos during adipogenesis were measured by qRT-PCR analysis. *p < 0.05 compared to the M2D-Exos NC inhibitor group. (G) A-R staining was used to measure the formation of bone nodules in BMSCs following treated by M2D-Exos, miR-690 inhibited M2D-Exos, or inhibitor control M2D-Exos for 14 days. Scale bar = 50 µm, (H) The statistical data of Alizarin red-mediated calcium staining. (I) Oil red O staining was used to measure the formation of lipid droplets in BMSCs following treated by M2D-Exos, miR-690 inhibited M2D-Exos, or inhibitor control M2D-Exos for 14 days. Scale bar = 50µm. (J) The statistical data of Oil red O staining. *p < 0.05 compared to the M2D-Exos NC inhibitor group.

Fig 4: STK25 overexpression inhibits the expression of tumor stem cell markers and EMT in HCC cells promoted by miR-4800-3p through the Hippo pathway. (A) The stemness of HepB3 and LM3 cells was evaluated using a sphere formation assay. qPCR and Western blot analysis of tumor stem cell markers (CD44, CD133, and OCT4) (B, C) and EMT biomarkers (E-cadherin, ZO-1, and N-cadherin) (D, E) in HepB3 and LM3 cells. (F) Western blot was used to detect the expression of p-YAP, YAP, TAZ, and PCNA in HepB3 and LM3 cells. *p < 0.05 and **p < 0.01 vs. mimic+OE-NC.

Fig 5: Overexpression of constitutively activated TAZ (TAZS89A) rarely induces intrahepatic cholangiocarcinoma development in mice. (A) Scheme of the experiments conducted. Specifically, FVB/N mice were hydrodynamically injected with the empty vector (Vector; n=10) or an activated form of TAZ (TAZS89A; n=40). Five empty vector-injected mice, and 15 TAZS89A-injected mice were sacrificed 10 weeks post-injection, whereas 5 empty vector-injected and 25 TAZS89A-injected mice were killed 40 weeks post-injection. (B,C) Macroscopic images of empty vector- (B) and TAZS89A-injected (C) mouse livers at 10 weeks post-injection showing a normal appearance. (D) Development of a single tumor in a TAZS89A-injected mouse sacrificed 10 weeks post-injection. (E) Large tumor developed in a TAZS89A-injected mouse sacrificed 40 weeks post-injection. Tumors depicted in (D) and (E) exhibited histological features of intrahepatic cholangiocarcinomas, with cancer cells organized in tubular and pseudoglandular structures surrounded by desmoplastic stroma. In addition, the lesions displayed robust immunoreactivity for cytokeratin (CK) 7 and 19, two biliary markers, further corroborating their cholangiocellular differentiation. In addition, the same lesions were characterized by pronounced nuclear immunoreactivity for TAZ protein. Furthermore, the lesions exhibited remarkable positiveness for activated/phosphorylated (p-)Akt: Original magnification: 40x, 100x, and 200x, as indicated in the pictures. Scale bars are indicated in the pictures. Abbreviations: H&E, hematoxylin and eosin staining; w.p.i, weeks post-injection