FIG. 6.

Transfer and pathological impact of exo-miR-15a on retinal cells. We collected exosomes from INS-1 cells cultured under high-glucose condition. We incubated the exosomes in the culture media of rat Müller glial cells (rMC-1). (A) Live rMC-1 cell imaging was performed (Nikon Eclipse Ti-E inverted microscope). Representative 30, 60, and 90 min after INS-1-derived-labeled exosomes are shown here. RFP signals showed exo-RNAs inside rMC-1 cells (lower panels). Upper panel shows the negative control for this experiment, where we did not use any exosomes in rMC-1 media. The figures are 60X magnification. (B) qPCR data examining the miR-15a expression in rMC-1 cells, which were cultured with either no exosomes (Control) or high-glucose cultured INS-1-derived exosomes for 48 h (Exosome treated). The data were normalized with 5S rRNA expression. (C) Western blot analysis of Akt3 expression in rMC-1 cells, which were cultured with either no exosomes (Control) or with high-glucose cultured INS-1-derived exosomes for 48 h (Exosome treated). Akt3 (upper band) was normalized to α-tubulin (lower bands). Bar graphs show the quantification of protein expression. Protein lysate was prepared from the rMC-1 cell pellets. (D) Measurement of H₂O₂ production as an indicator of ROS generated from the rMC-1 cells either cultured with no exosomes (Control) or high-glucose cultured INS-1-derived exosomes for 48 h (Exosome treated). Apoptotic cell death was evaluated either by (E) cleaved caspase-3 expression normalized with caspase-3 (Cas-3) using Western blot or (F) Annexin V staining of rMC-1 cells after culturing for 48 h with no exosomes (control) or with high-glucose cultured INS-1-derived exosomes for 48 h (Exosome treated). *p > 0.05 versus Control. The original (un-edited) blot images can be found in Supplementary Western Data 2. RFP, red fluorescent protein.