Fig. 3

Functional regulation of MIR9-2 by NANOG. (A) Cropped western blotting showing exogenous expression of NANOG (pSIN-NANOG) in HEK293 cells compared to that in the empty vector (biological triplicates with expression of ACTB used as a loading control. Full-length blots are presented in Additional file 8: Fig. S3). NANOG expressing cells were subjected to a luciferase reporter assay using MIR9-2 reporter vectors (empty control, MIR9-2, and MIR9-2 Δ deletion, n = 3; *P < 0.05 compared to the control; Student’s t-test). (B) NANOG expression in TCGT cell lines and normal testes. TCam-2 cells are derived from SEM and NT2/D1, and Tera-1 cells are derived from EC (n = 5; *P < 0.05 compared to normal testis; Two-way ANOVA followed by Tukey’s multiple comparisons test). (C) qRT-PCR data showing downregulated expression of NANOG after stable knockdown (left) and concomitant upregulated expression of MIR9-2-5p and MIR9-2-3p (right) (n = 3); *P < 0.05 compared to the scrambled control; One-way ANOVA and Two-way ANOVA, respectively, followed by Tukey’s multiple comparisons test). Cropped western blotting showing the expression of NANOG in TCam-2 cells transduced with either scrambled or shRNA-NANOG (shA, biological triplicates with expression of ACTB used as a loading control. Full-length blots are presented in Additional file 8: Fig. S3)