Fig. 2.

Establishment of immortalized uterine smooth muscle cell lines overexpressing SATB2 or NRG1 genes. The expression vectors for the SATB2 and NRG1 lines contained the coding sequences of SATB2 and NRG1, respectively, plus AcGFP. The vectors were transduced into human telomerase reverse transcriptase immortalized uterine smooth muscle cells (hTERT UtSMCs). Cells transduced with only AcGFP were used as a mock control line (control). a Cell morphology of the SATB2 line, NRG1 line, control line, and parental line (parent). Scale bars: 100 μm. b Immunofluorescent staining for vimentin (red) in the SATB2 line, NRG1 line, control line, and parental line. Nuclei were stained with DAPI (violet). Scale bars: 100 μm. c Cell morphology in three-dimensional (3D) culture of the established cell lines. The 3D culture was carried out with a collagen gel-embedded culture [26, 27]. The photographs show the cell morphology of each cell line after 2 days of culture. Dotted lines outline the nodule-like aggregates of cells. Arrows indicate the aggregates of cells. Scale bars: 200 μm. d Relative expression levels of SATB2 and NRG1 mRNA to GAPDH in the established cell lines were analyzed in triplicates by real-time RT-PCR. Values are mean ± SD. e SATB2 and NRG1 protein expression in the established cell line was analyzed by Western blotting. TUBB was used as an internal control