Fig. 5

pSMAD1/5 phosphorylation level in blastocysts after IVC with or without miRNA-148b mimic. (A) Blastocyst analyzed for localization and abundance of phosphorylated (active) form of SMAD1/5 (pSMAD1/5) transcription factor. pSMAD1/5 Immunostaining (green) counterstained with Hoechst 33,342 for nuclear staining (blue). Embryos cultured from presumptive zygotes to blastocyst in SOF + 0.3% BSA (Control), supplemented with 1 µM control mimics (CMimic), or supplemented with 1 µM of miR-148b (miR148b) or embryos cultured from presumptive zygotes to 16- cell stage in SOF + 0.3% BSA supplemented with 1 µM of miR-148b (miR148b-OV), or embryos cultured from 16-cell stage to blastocyst in SOF + 0.3% BSA supplemented with 1 µM of miR-148b (miR148b-UT). (B and C) Quantification of phosphorylation levels and representative WB images of pSMAD1/5 signal from each group. WB data were normalized relative to the abundance of β-actin as a loading control and expressed as mean ± SEM. Values with different superscripts indicate statistically significant differences at (P ≤ 0.05)