Fig 1: Cell migration and proliferation were also regulated by IL-2/sorafenib co-treatment through the JNK-TAZ pathways. a–c Immunofluorescence assay for cell proliferation-related factors. IL-2/sorafenib co-treatment elevated the expression of CDK4 and cyclin D1, which was repressed by SP600125, an inhibitor of the JNK-TAZ pathways. d, e An EdU assay was performed to quantify the cell proliferation. The number of EdU-positive cells was recorded. f–h Cell migration factors such as CXCR4 and CXCR7 were measured via western blotting. *P < 0.05 vs. control group; @P < 0.05 vs. IL-2+ sorafenib group. Cont control
Fig 2: Sjögren's syndrome occurrence is accompanied by a downregulation of the Hippo signaling pathway. Western blotting analysis showed that the protein expression levels of E-cadherin, a-catenin, p-TAZ and TAZ were higher than those of SMGs in 17-week-old NOD mice. *P<0.05 vs. Control. E-cad, E-cadherin; NOD, non-obese diabetic; p-, phosphorylated; TAZ, transcriptional coactivator with PDZ-binding motif.
Fig 3: Knocking down the expression of TAZ reduces the postponing effect of ADSCs on SS. (A) shRNA lentivirus vector targeting TAZ was injected into tail vein of NOD mice (with ADSCs treated). (B) The expression of TAZ decreased after lentivirus transfection in SMGs in mice. *P<0.05, #P<0.05 vs. sh-NC. (C) Knocking down TAZ reduced the delaying effect of ADSCs on SS, but this effect is still stronger than the PBS-treated Control group. *P<0.05; #P<0.05. ADSCs, adipose-derived stem cells; NOD, non-obese diabetic; sh, short hairpin RNA; SMG, submandibular gland; SS, Sjögren's syndrome; TAZ, transcriptional coactivator with PDZ-binding motif.
Fig 4: ADSCs delay the progression of Sjögren's syndrome by upregulating the Hippo signaling pathway. (A) Western blotting analysis showed that the protein expression levels of TAZ in the ADSC group and Control group decreased gradually between 13 and 17 weeks, and that the downward trend of TAZ in the ADSC group was slower compared with the Control group. (B) Immunofluorescence staining showed that the expression of E-cad (green) in SMGs from mice in the ADSC group was significantly higher compared with that in the Control group. DAPI (blue) was used to stain the nucleus. (C) Immunofluorescence showed that the expression of TAZ in SMGs of ADSC group was significantly higher than that of the Control group. DAPI (blue) was used to stain the nucleus. *P<0.05, **P<0.01. ADSCs, adipose-derived stem cells; E-cad, E-cadherin; SMG, submandibular gland; TAZ, transcriptional coactivator with PDZ-binding motif.
Fig 5: YAP/TAZ nuclear localization mediates stiff environment-induced cancer proliferation and metastasis. a) Immunofluorescence imaging of YAP/TAZ in cells grown on different substrates (scale bar, 20 µm, 48 h). Stiff substrate regulation of YAP/TAZ nuclear localization is heterogeneous in cell lines derived from different metastatic sites. Stiff substrate induces YAP nuclear localization in PC3 cells, but not in LNCaP cells. b) Higher YAP/TAZ nuclear localization ratio was observed in PC3 cells grown on stiff substrates compared with PC3 cells grown on soft substrates (n = 3, p = 8.1573 × 10-6). c) Phos-Tag sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (P-tag) demonstrates YAP1 dephosphorylation levels in PC3 and LNCaP cells grown on stiff substrates (46.7 KPa). The stiff substrate induces YAP1 dephosphorylation failure in LNCaP cells. d) TAZ and YAP expression in cells transfected with empty vector (PC3-Con) and YAP1/TAZ-interfering RNA (PC3-Y/Ti). e) The proliferative capacity of PC3-Con cells and PC3-Y/Ti cells grown on different substrates was evaluated by EdU staining (n = 3, p = 0.0149). f) The migration ability of PC3-Con and PC3-Y/Ti cells growing on different substrates was assessed using Transwell assays (n = 3, p = 0.0160). g) Tumor diameter in nude mice. PC3-Con cells and PC3-Y/Ti cells were grown on different substrates for 7 days and injected into subcutaneous tumors in nude mice; the tumor diameter was measured 4 weeks after injection (n = 5, P = 5.6823 × 10-5). h) The expression of the YAP1 mechanical induction nuclear localization-related proteins talin, integrin-b1, and p-FAK (phosphorylated FAK [Tyr397]) in PC3 and LNCaP cells. i) Immunofluorescence imaging of pFAK in PC3 and LNCaP cells (scale bar, 2.5 µm). The difference in focal adhesion morphology between PC3 and LNCaP cells was revealed by p-FAK; PC3 cells had longer (n = 53, p = 1.6069 × 10-13) and wider (n = 53, p = 1.7690 × 10-23) focal adhesions than LNCaP. j) Schematic showing that the response heterogeneity of different metastasis site-derived cells to stiff substrates is due to differences in YAP/TAZ localization. *p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001, t-tests.
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