Fig. 5

Modulation of proliferation and apoptosis of Leydig cells by mRNA targets of miR-504. Expression of miR-504 in miR-504 mimic-or miR-504 inhibitor-infected R2C cells at 24 h after culturing in normal or high glucose (HG). Data were normalised to U6 RNA, used as an internal control (A). Expression of MEK5 and MEF2C determined by RT-qPCR analysis. β-actin was used as an internal control (B, C). Representative immunoblotting (D) and cumulative quantification (E, F) of the protein levels of MEK5 and MEF2C in R2C cells transfected with miR-504 mimic, miR-504 inhibitor, mimic NC, or inhibitor NC. Media were collected and assayed for concentration of testosterone using ELISA (G). Cell proliferation was assayed using CCK8 (H). Detection of apoptotic cells by FACS analysis with FITC-labelled annexin V and PI staining (I). Bar graphs represent the percentage of apoptotic cells in each group (J). *p < 0.05, **p < 0.01, ***p < 0.001. n = 3