Figure 4
Maf1-mediated transcriptional repression in extracellular release of vesicle-loaded RNAGlu under oxidative stress. (a) Sanger sequencing of the Cas9/CRISPR-targeted Maf1 locus. The sequencing chromatograms of sgRNA target region indicate homozygous status of the Maf1 locus in Maf1 knockout (KO) cells. (b) Immunoblotting analysis with anti-Maf1 confirms the loss of Maf1 expression in BUMPT cells. Ponceau S staining was used to determine equal amounts of proteins loaded per lane. (c–e) Changes in abundance of intracellular (cell) and extracellular vesicle (EV) tRNAGlu in control and Maf1 knockout (KO) cells treated with 0.1 mM of H2O2 at the indicated timepoints. To collect conditioned media after oxidative stress, cells were treated with H2O2 for 1 h and thereafter exposed to serum-free, H2O2-free growth media for 23 h. Expression levels of tRNAGlu were normalized to total RNA amount, and then relative expression values of tRNAGlu of treated group normalized to control group were plotted. Because transient oxidative stress resulted in significant changes in total RNA levels and U6 snoRNA (not shown), relative expression of tRNAGlu was normalized in this manner (cell number and conditioned media volume). For extracellular vesicle samples, benzonase was treated as described in “Methods” section. (f) Relative number of extracellular vesicles from control and Maf1 knockout (KO) cells under oxidative stress. Cells were treated as in (c–e). Note that transient oxidative stress decreases release of extracellular vesicles (number) regardless of the status of Maf1 expression.
