Figure 8.

The miR-200b–CITED2 circuit mediates the proapoptotic effect of high glucose through ER stress. Representative images of the TUNEL assay and quantitative data of apoptotic cell numbers in cells transfected with the control oligo or the miR-200b inhibitor under 5 mmol/L glucose or 25 mmol/L glucose conditions (A and B), in cells transfected with the blank vector or the CITED2 plasmid under 5 mmol/L glucose or 25 mmol/L glucose conditions (C and D), and in cells transfected with scramble siRNA or CITED2 siRNA in the absence or presence of 2 mmol/L 4-PBA (E and F). Apoptotic cells were labeled red, and all cell nuclei were stained by DAPI (blue). Experiments were repeated three times (n = 3). Protein levels of cleaved caspase 3 in cells under 5 mmol/L or 25 mmol/L glucose conditions with or without the miR-200b inhibitor (G), in cells under 5 mmol/L or 25 mmol/L glucose conditions with or without the transfection of the CITED2 plasmid (H), and in cells treated with CITED2 siRNA with or without 4-PBA (I). Levels of miR-200b (J), CITED2, BiP, and CHOP mRNA and proteins (K and L) in embryos cultured under 5 mmol/L glucose or 16.7 mmol/L glucose (high glucose) with or without the miR-200b inhibitor are shown. E8.5 embryos were cultured for 36 h, and the number of NTD and normally developed (Normal) embryos are shown (M). χ² test was used for statistical analyses (M). Experiments were repeated three times (n = 3), and the quantification of data is shown in the bar graph. * and # indicate significant differences (P < 0.05) compared with the other three groups.