Fig. 5.

Verteporfin inhibits Yap protein expression and alters ccn2a and evx1 expression. (A) Immunoblotting demonstrates reduced Yap protein expression in zebrafish fin lysates treated with 20 µM Verteporfin (VP) compared to DMSO-treated controls. Protein lysates were extracted from five pooled fins per treatment group. Tubulin was used as a loading control to normalize protein levels. (B) Quantitative analysis of Yap protein expression in zebrafish fins treated with 20 µM Verteporfin (VP) compared to DMSO-treated controls. Band intensity was measured using ImageJ and normalized to Tubulin as a loading control. Data represent the average Yap expression from four independent western blots, each performed using protein lysates from five pooled fins per treatment group. Statistical analysis (two tailed, unpaired Student's t-test P value=0.02) indicates a significant reduction in normalized Yap expression in VP-treated samples compared to controls. (C) Fins were amputated at 50% and treated with 20 µM Verteporfin at 72 hpa via injection. Fins were harvested 24 h later at 96 hpa. Amputation planes are denoted by white dotted line. In situ hybridization was performed using antisense digoxygenin-labeled probe against ccn2a to measure relative gene expression (n=5 for each treatment, with three biological replicates). There is less expression in treated fins when compared to DMSO shown with white arrows. (D) Reduction of ccn2a gene expression was quantified through qPCR in both DMSO and Verteporfin treated fins. Graph shows a mean of three biological replicates fold difference and standard deviation (n=5 fins per replicate). A fold difference of 1 means no change from wild-type expression. The Student's t-test (two tailed, unpaired) was used to assess significance with a P-value of 0.01. (E) In situ hybridization was performed using antisense digoxygenin-labeled probe against evx1 to measure relative gene expression. There is an increase in positive fins rays for evx1 with verteporfin treated fins (n=5 for each treatment with three biological replicates). Increase in evx1 gene expression was quantified through qPCR in both DMSO and Verteporfin treated fins. (F) Graph shows a mean of three biological replicates fold difference and standard deviation (n=5 fins per replicate). A fold difference of 1 means no change from wild-type expression. The Student's t-test (two tailed, unpaired) was used to assess significance with a P-value of 0.02. Scale bar represents 100 µm.