Fig 1: Comparative pro-migratory gene expression levels in MOE and MOSE cellsPro-migratory mRNA levels were measured by qPCR in MOE and MOSE cells with p53 mutation and normalized to their respective controls. A. MOEp53R270H relative to MOEFloxed control cells. Cell lysates were probed with CDH6 and DCN antibodies. a - Tubulin is used as a loading control. B. Western blot on MOEFloxed and MOEp53R270H cells. C. Densitometry analysis of MOEp53R270H relative to MOEFloxed control cells. D. qPCR data obtained from MOEp53R273H relative to MOENeo control cells. E. Western blot on MOENeo and MOEp53R273H cells. F. Densitometry analysis of MOEp53R273H relative to MOENeo control cells. G. qPCR data obtained from MOSEp53R270H relative to MOSEFloxed control cells. H. Western blot on MOSEFloxed and MOSEp53R270H cells. Data represent mean ± SEM. Student t-test was used to determine significance, (*p < 0.05) relative to control.
Fig 2: CDH6 is repressed by mutant p53 in murine oviductsA. qPCR data on the pro-migratory genes mRNA levels from PAX8cre/+p53R270H/+ oviducts relative to Pax8cre/+ oviducts. B. Immunohistochemistry analysis of CDH6, DCN and p53 staining in Pax8cre/+ and PAX8cre/+p53R270H/+ oviducts C. Immunohistochemistry on human fallopian tube for CDH6, DCN and p53. Black arrow indicates positive staining. Scale bars = 100 µm. D. Immunohistochemistry on human ovaries for CDH6 and CK8. Black arrow indicates positive staining. Scale bars = 20 µm. E. CDH6 western blot in normal human IOSE cells. Actin is used as loading control. Data represent mean ± SEM. Student t-test was used to determine significance, (*p < 0.05) relative to control.
Fig 3: Gene expression profiles of DR, PO, and DO clusters. The violin plots show the distribution of key gene expressions in single cells from differentiation resistant MSCs (DR, blue), precursor osteoblasts (PO, green), and differentiated osteoblasts (DO, red). p-values represent differences of DR cluster compared to PO cluster and DO cluster respectively as follows: (A) BMP2 (3.8E-4; 2.9E-4), (B) RUNX2 (0.05; 0.04), (C) WNT5A. (0.04; 0.037), (D) SRC (4.8E-2; 3.2E-2). (E) YAP1 is significantly elevated in DR-MSCs compared to PO and DO (p = 0.03; p = 0.032, respectively). (F) Transcription factor OCT4 was significantly upregulated in DR-MSCs compared to PO and DO (p = 0.0168; p = 0.0161, respectively). (G) CDH6 expression was also markedly higher in the DR MSC cluster when compared to PO and DO (p = 0.0366; p = 0.0324, respectively).
Fig 4: Verteporfin significantly decreases solid tumor cell viability and inhibits YAP1. (A) MSCs were harvested on Day 12 following incubation in a differentiation medium. CDH6, YAP1, and OCT4 transcripts were all significantly down-regulated by 2 M Verteporfin in a time-dependent manner (A1) and the corresponding proteins by Western blotting (Blots are representative of three independent experiments, adjacent to the quantification of the related proteins) (A2). Verteporfin significantly downregulated (B) Transcripts for OCT4 in a dose-dependent manner (at both 2 and 5 M doses), but no significant changes were observed for YAP1 or CDH6 [Note that all gene expression levels are indicated expression levels relative to the untreated controls]. (C) Transcripts for CDH6 and OCT4 by PCR were significantly decreased in tumor cells by 2 M verteporfin compared to controls, but no significant differences were observed for YAP1. [All the above qPCR results were normalized against beta actin]. (D) HeLa cells were incubated in 2 and 5 M concentrations of verteporfin for 24 h and harvested. The trypan blue dye exclusion test was used to assess the viability of cells cultured with and without verteporfin. Cells were counted under a hemocytometer. Cell viability in both cancer cell lines was inversely correlated to increasing doses of verteporfin. Cell viability was significantly decreased (p < 0.05) with 2 and 5 M concentrations of verteporfin when compared to controls across both cell lines. (E) Effects of YAP1 inhibitor verteporfin on HeLa cells after 24 h incubation. Top panels E1 to E5: (E1) Bright-field image; (E2) NucBlue staining for live cells; (E3) YAP1 fluorescent staining; (E4) merged NucBlue (blue) and YAP1 antibody staining (red). (E5) Images with higher magnification show the location of YAP1. Lower panels of the E1 to E5 are the corresponding controls showing cells cultured with DMSO. Verteporfin administered at 2uM in cell culture substantially reduced the number of viable cells compared to DMSO controls. Verteporfin significantly reduced YAP nuclear localization, and cells cultured with DMSO showed YAP1 staining mostly localizing to the nucleus and staining overlapped with NucBlue.
Fig 5: YAP1-related signaling and associated proteins CDH6 and OCT4. YAP1-related signaling and associated proteins CDH6 and OCT4 were significantly up-regulated (p = 4.25E-8). Upon mechanical strain or cellular contact, cadherin can induce YAP1/TAZ nuclear localization, upregulate transcription of OCT4, and promote MSC stemness. Verteporfin reduces levels of YAP1, CDH6, and OCT4, inhibits YAP1 nuclear localization, induces YAP1 cytosolic sequestration, and promotes MSC differentiation.
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