Fig. 2

Validation of the selected EMT markers at the mRNA and protein levels. a RT-qPCR validation of downregulated (CDH1, CTNNB, ESRP1, PERP, and EPCAM) and upregulated (ZEB1) EMT genes. RNA expression of the down- and upregulated genes in AIPpos tumors (n = 6) compared to normal pituitaries (n = 5) and sporadic GH tumors (n = 3) show confirmation of the gene expression profile data. b Immunohistochemical analysis of downregulated (CDH1, CTNNB, ESRP1, and PERP) and upregulated (ZEB1) EMT genes. Protein expression in graphical form and with representative images in AIPpos tumors compared to normal pituitaries and sporadic GH tumors. E-cadherin: Normal pituitary cells are showing uniform strong to moderate membranous staining. AIPpos GH tumor displays weak diffuse cytoplasmic positivity without any membranous staining. Sporadic GH adenoma shows membranous and granular cytoplasmic positivity. Beta-catenin: Normal pituitary cells are showing strong to moderate membranous immunoreactivity. AIPpos GH tumor displays discontinuous cytoplasmic expression. Sporadic GH shows membranous and granular cytoplasmic positivity. ESRP1: Normal pituitary cells are showing strong cytoplasmic immunoreactivity. AIPpos GH tumor shows weak cytoplasmic expression. Sporadic GH tumor shows universal cytoplasmic and moderate to strong nuclear positivity. PERP: Normal pituitary cells are showing strong granular cytoplasmic positivity. AIPpos GH tumor shows cytoplasmic expression with the nuclear atypia. Sporadic GH tumor shows granular cytoplasmic and nuclear positivity of variable strength. ZEB1: Normal pituitary cells are completely negative. AIPpos GH tumor exhibits uniform moderate to strong positive nuclear staining. Sporadic GH tumor shows weak to moderate nuclear staining in the majority of the tumor cells. Overall P value for multiple comparison is shown in the left upper corner of each graph, while significance between groups are marked with *, <0.05, **, <0.01, ***, <0.001 (Kruskal–Wallis test followed by Conover–Inman test). All images are ×400 magnification and scale bar = 50 µm