XMIR-21 Knockdown of Endogenous PDCD4 Proteins Levels

A) Exosomes from HEK-293 cells transfected with a miRNA-21 XMIR oligo (XMIR-21) or an anti-miRNA-21 AXMIR oligo (AXMIR-21) were added to naïve HEK-293 cells in culture. After 24 hours, total cell lysates were taken and Western blots for PDCD4, a known miR-21 target, were performed. GAPDH protein levels detected in the Westerns were used as a loading control and reference signal for band intensity quantitation analysis. B) Quantitative analysis of band intensities from the Western blot shown in Panel A. Addition of exosomes from cells transfected with XMIR-21 resulted in down regulation of endogenous levels of PDCD4, and addition of exosomes from cells transfected with AXMIR-21 resulted in increased levels of PDCD4. These results confirm that XMIRs act on endogenous targets and that AXMIRs effectively act as miRNA inhibitors in cells when delivered via exosomes. Notably, exosomes from cells transfected with 20 nM XMIR-21 oligo caused maximum PDCD4 down regulation, whereas exosomes from cells transfected with 100 nM AXMIR-21 increased PDCD4 levels, illustrating the need to optimize conditions for each XMIR/AXMIR oligo (PDCD4 antibody obtained from Cell Signaling Technologies, catalog # 9535, and GAPDH antibody obtained from Abcam, catalog # ab9485).