Figure 5.

PAR5 interacts with Enhancer of Zeste Homolog 2 (EZH2) modulating E-cadherin expression. (A,B) RNA immunoprecipitation (RIP) assay was performed on extracts obtained from FRO and 8505c cells transfected with pCMV-PAR5 or the EV, using an anti-EZH2 antibody or a pre-immune (IgG) serum, as control. qRT-PCR was performed on the samples to assess fold enrichment of PAR5 precipitated by the anti-EZH2 antibody relative to IgG control, set equal to 1. (C) Western blot analysis of EZH2 expression in FRO and 8505c stably expressing PAR5 or carrying the corresponding EV. β-actin was used to normalize the amount of loaded protein. Densitometric analysis was performed by using ImageJ software to analyse protein expression compared to the EV, set equal to 1. (D) qRT-PCR analysis to evaluate the expression of EZH2 after PAR5 transfection. Data were reported as relative expression ± SD and were compared to the EV, set equal to 1. (E) Correlation scatter plot (Spearman’s Rank) between qRT-PCR levels of PAR5 and Western blot levels of EZH2 analyzed in six ATC samples (r = −0.3143; p = 0.5639). (F,G) Western blot analysis of H3K27me3 and E-cadherin expression in FRO and 8505c stably expressing PAR5 or carrying the corresponding EV. H3 and β-actin were used to normalize the amount of loaded protein. Densitometric analysis was performed by using ImageJ software to analyze protein expression compared to the EV, set equal to 1. (H) qRT-PCR analysis to evaluate the expression of E-cadherin after PAR5 transfection. Data were reported as relative expression ± SD and compared to the EV, set equal to 1.