Fig. 3

HES1 status alters invasive behavior in thyroid cells and affects expression of Notch1 and Dtx1. a Validation of HES1 knockdown in stable NThy.scr and HES1 overexpression in NThy.mir-182 by shHES1 and HES1 wild type (HES1 wt), respectively, on mRNA and protein level. Empty pcDNA3.1 and shControl (shctrl) plasmids were used as controls. Actin shows equal loading of protein. b-c Invasion and migration of thyroid cells after knockdown of HES1 (b) or HES1 overexpression (c). Values are relative fold compared to related controls of three individual experiments with representative images from fluorescence microscopy for Boyden chamber and bright field microscopy for wound healing assays; ** p < 0.01, *** p < 0.001. d NThy.miR-182 (top panel) and NThy.scr cells (bottom) were transfected with plasmids encoding HES1 wild type or shHES1. After one day specific amounts of cells were seeded and counted every 24 h to determine proliferation. e Detection of Notch1, Dtx1, and HES1 transcript levels in indicated thyroid cell lines after specific treatment: HES1 up- and downregulation (top), depletion of miR-182 (center, bottom right) and blockade of RET-associated signaling (bottom left). Cells were transfected with corresponding control plasmids and RNA was isolated 24 h later. Actin was used for equal loading. f Immunofluorescence staining of HES1, Notch1, and Dtx1 in miR-182 expressing NThy-ori 3.1 cells compared to control cells with miR-scr. Nuclei were counter-stained with DAPI and fluorescence was visualized by confocal laser scanning microscopy. Immunoblot indicates cleaved Notch1 and actin in NThy.miR-182 cells (bottom, right)