Fig 2: A TAZ/miR-942-3p positive feedback loop modulates the oncogenic effects, glycolysis and redox state mediated by TAZ. a. Cells were transfected with a negative control vector, TAZ-specific shRNA or pre-miR-942. Cell viability was determined by a CCK-8 assay. b. MiR-942-3p promoted colony formation, as determined by a colony formation assay. c. A tube formation assay was used to verify the effect of the positive feedback loop on angiogenesis. d. To verify the effects on cell migration, a wound healing assay was performed. e. The migration and invasion of bladder cancer cells transfected with different vectors were evaluated with Transwell migration and invasion assays, respectively. f-g The expression levels of TAZ and EMT markers in T24 and EJ cells were evaluated by western blotting and qRT-PCR. h-i The uptake of glucose and production of lactate in cells under different conditions were determined to evaluate the glycolytic process. j-k The expression levels of glycolysis-related genes (PFKFB3, HK2 and GLUT1) were detected by qRT-PCR and western blotting in TAZ-deficient cells with or without supplementation with miR-942-3p. l. ROS levels were detected in cells transfected with a negative control vector, TAZ-specific shRNA or pre-miR-942. Data are presented as the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01 vs. the control group

Fig 3: TAZ and miR-942-3p enhance bladder cancer tumor growth in vivo. a. Equal numbers of different T24 cell populations (negative control, shTAZ or cotransfected with shTAZ and miR-942-3p) were used to establish subcutaneous xenograft tumors. The tumors were harvested and photographed (n = 4 each group). b. Tumor weight was determined when the mice were sacrificed. c. Tumor volume was measured and calculated after cells were injected into mice. d. MiR-942-3p levels were determined by qRT-PCR in tumors obtained from mice. e. TAZ, LATS2 and GAS1 were detected in mouse tumor tissues by western blotting. f. Immunohistochemical (IHC) detection of TAZ, LATS2 and GAS1 in tumors. Scale bars: 200 μm. g. Schematic diagram illustrating the novel positive feedback loop between TAZ and miR-942-3p that regulates GAS1 expression and modulates biological behaviors, EMT and glycometabolism in bladder cancer. Data are presented as the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01 vs. the control group

Fig 4: Human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) are less differentiated than HPV-negative HNSCC.(A) Human HNSCC tumor samples were stained for KRT1 (left). Scale bar = 100 μm. Graph displays the percentage of tumors that were KRT1+ (right). Statistical significance was determined by Fisher’s exact test. (B–C) Total RNA was purified from patient-derived xenograft samples and qRT-PCR was used to assess gene expression of (B) the differentiation markers KRT1, KRT10, and IVL and (C) the canonical yes-associated protein 1/TAZ targets CTGF and CYR61. Statistical significance was determined by Mann-Whitney nonparametric test (*p<0.05, **p<0.01).

Fig 5: TAZ is vital in cell growth, angiogenesis, migration, invasion, EMT, glycolysis and ROS homeostasis in bladder cancer cells. a. The knockdown efficiency of siRNAs specific for TAZ was determined by western blot analysis of T24 cells. b. A CCK-8 assay was performed to determine the effect of TAZ on cell viability. c. Depletion of TAZ inhibited the colony-forming abilities of T24 and EJ cells. d. The apoptotic rates of control and TAZ knockdown cells were detected by flow cytometry. e. A tube formation assay was performed to evaluate the effect of TAZ on tumor angiogenesis. f. A wound healing assay was used to evaluate the cell migration of control or TAZ-knockdown T24 and EJ cells. g. The cell migration and invasion of TAZ-deficient cells were evaluated by Transwell migration and invasion assays, respectively. h. EMT markers were detected in SV-HUC-1 cells and bladder cancer cell lines by western blotting. i-j The key role of TAZ in the EMT process was confirmed by western blotting and qRT-PCR. k. A schematic illustration of glycolysis is shown. l-m The uptake of glucose and production of lactate in TAZ-deficient and normal bladder cancer cells were determined. n. The alterations in PFKFB3, HK2 and GLUT1 at the protein level were confirmed by western blotting. o. qRT-PCR was performed to assess the expression levels of glycolysis-related genes (PFKFB3, LDHB, HK2, GLUT1, GLUT3 and GLUT4). p. Intracellular ROS levels were determined with DCFH–DA and analyzed by flow cytometry. Data are presented as the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01 vs. the control group