Fig 2: MYBL2 inhibits Hippo signaling leading to YAP activation. (A) Analysis of the TCGA PCa protein dataset revealed that p-YAP-S127 expression was significantly downregulated in PCa samples with the highest quartiles versus those with the lowest quartiles of MYBL2 (two-tailed Student's t test). (B) Western blotting analysis of the indicated protein expression in the indicated cells. α-tubulin was used as a loading control for total extracts; P84 was used as a loading control for nuclear extracts. (C) Left panel: Expression and subcellular localization of YAP1 were analyzed in the indicated cells with altered MYBL2 expression and visualized by fluorescence and laser confocal microscopy. Scale bars: 20 μm. Right panel: Percentage of subcellular localization of YAP1 in the indicated cells (Nucleus (N); Cytoplasm (C)). (D) HOP/HIP luciferase activity was analyzed in the indicated cells. HOP-Flash luciferase assay shows YAP/TAZ-TEAD transcriptional activity; HIP-Flash is a HOP-flash mutant. P-values were determined by two-tailed Student's t test when comparing two groups and one-way ANOVA test when comparing three groups. (E) Fold change of the mRNA expression of indicated genes in qRT-PCR analysis comparing cells overexpressing MYBL2 versus vector (V) or MYBL2-Ri#1 (Ri#1) versus Ri-vector (Ri-V), generated by log2 transformation. Data were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: MYBL2 promotes castration-resistance and bone-metastasis of PCa by activating RhoA and YAP signaling. (A) For the RhoA activity assay, cells were immunoprecipitated with anti-active RhoA antibody and then immunoblotted with anti-RhoA antibody. A portion of the cell lysate (1/30) was also directly immunoblotted with anti-RhoA antibody. Western blotting analysis of the indicated protein expression in the indicated groups of LNCaP cells. α-tubulin was used as a loading control of total protein; P84 was used as a loading control of nuclear protein. Sim, Simvastatin, 5 μM, 24 h. (B) Left panel: Expression and subcellular localization of YAP1 was analyzed in the indicated groups of LNCaP cells with Simvastatin treatment and visualized by fluorescence and laser confocal microscopy. Scale bars: 20 μm. Right panel: Percentage of the subcellular localization of YAP1 in the indicated cells (Nucleus (N); Cytoplasm (C)). (C) Luciferase activity of the YAP/TAZ-TEAD promoter reporter was examined in the indicated groups of LNCaP cells. Simvastatin (5 μM, 24 h); Ver, Verteporfin (10 µM, 3 h). (D) QRT-PCR analysis of YAP1-targeted genes mRNA expression in the indicated groups of LNCaP cells. Gene expression levels were normalized to GAPDH. (E) Representative images (left panel) and quantification (right panel) of colonies formed by the indicated cells with Simvastatin or Verteporfin treatment. (F) Apoptosis assessed in the indicated cells treated in medium supplemented with 0.5% CSS for 4 days and subsequently treated with Simvastatin or Verteporfin. (G) Tumor growth curves of the indicated LNCaP xenograft tumors (n = 8/group). Mice were administered with intraperitoneal injection of Simvastatin (5 mg/kg, five days a week) or Verteporfin (100 mg/kg, every two days). (H) Quantification of xenograft tumor weight at the end of the experiment shown in (F). (I) Growth curve for bone metastasis burden as quantified by BLI in the indicated groups of MDA PCa 2b cells. (J) Numbers of metastatic lesions in bones from each group. (K) Kaplan-Meier curves of bone metastasis-free survival (BMFS) and overall survival (OS) in mice inoculated with the indicated groups of MDA PCa 2b cells (log-rank test). In (C-F, H and J), P-values were determined by one-way ANOVA test. In (G), P-values were determined by two-way ANOVA test. Data were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: Phospholamban stabilizes the actin cytoskeleton in a reversible manner. (A) Left: Volcano plot of gene microarray of triplicate experiments of esophageal SMCs expressing an inducible PLN transgene treated with vehicle or 2ug/ml of doxycycline for 72 hours. Red and blue dots represent >1.5-fold change of upregulated (n = 161) and downregulated (n = 382) differentially expressed genes with false discovery rate adjusted p < 0.05. Right: GO and Pathway analysis of differentially expressed upregulated (top, red) and downregulated (bottom, blue) genes following doxycycline induction of PLN expression in SMCs. (B) Representative image of phalloidin staining in latrunculin A (0.5uM, 30 minutes) or vehicle treated stable transgenic GFP (control) or PLN-expressing SMCs. . (C) Quantitation of nuclear YAP immunostaining (percent of cells that are nuclear YAP+ among total cells) in SMCs cultured on soft or rigid substrate at baseline (B) and after 3 weeks. Dots: different individuals, Lines: Mean ± SEM. *: p < .0.05 using analysis of variance with Tukey’s multiple comparisons. (D) Normalized fold change in PLN transcript after 14–21 days (as compared to baseline) in SMCs culture on rigid or soft substrate. Dots = different individuals, lines: Mean ± SEM.*p < 0.05. One-way analysis of variance (Kruskal-Wallis) Dunn’s multiple comparisons test. (E) Representative assay of normalized fold change in mRNA of α-smooth muscle (αSMA) and alpha cardiac muscle 1 (ACTC1) actins, actin-binding proteins caldesmon 1 (CALD1) and transgelin (TAGLN) in SMCs cultured for 21 days (3 weeks) on soft (clear bars) or rigid (solid bars) substrate. Repeated using three different donors. (F) Cell area (μm2) on soft (2 kPa) or rigid (32 kPa) substrate at baseline (B), 2, and 3 weeks of culture on soft or rigid substrate. *p < 0.05. Two-way analysis of variance with Sidak’s multiple comparisons test. Dots = mean of 3–5 high power fields ± SEM. Repeated using SMCs from four different donors. αSMA = alpha smooth muscle actin; ACTC1 = actin, alpha, cardiac muscle 1; CALD1 = caldesmon 1; FDR = false discovery rate; GFP = green fluorescent protein; GO = gene ontology; mRNA = messenger ribonucleic acid; PLN = phospholamban; SEM = standard error of the mean; SMC = smooth muscle cell; TAGLN = transgelin; YAP = yes-associated protein.

Fig 5: DCLK1 regulates stemness traits in prostate cancer cells through YAP signaling. (A) Western blot analysis confirmed the DCLK1 expression level in the indicated cells. (B) Representative images (left panel) and comparative quantification (right panel) of colonies formed by the indicated cell lines. (C) Representative light micrographs (left panel) and comparative quantification (right panel) of the sphere formation capacity of the indicated cell lines. (D) Relative mRNA expression of stemness-related markers and regulators analyzed by qRT‒PCR in the indicated cells. Ver, verteporfin (10 µM, 3 h). The data are presented as the means ± SDs from three independent experiments, and P values were determined by two-tailed Student's t test; **P < 0.001, ***P < 0.001.