Acute suppression of Ezh2 induces apoptosis in pOLs.

<p>(<b>A</b>, <b>B</b>) Phase contrast images show that pOLs retract their projections when treated with Ezh2-shRNA. (<b>C</b>, <b>D</b>) Immunostaining for apoptosis marker, cleaved caspase 3 (green) shows increase in cell death in pOLs cultures treated with Ezh2-shRNA compared to control cultures (nc-shRNA). (<b>E</b>, <b>F</b>) Co-immunostaining using anti-cleaved caspase 3 and anti-RIP (an oligodendrocyte maker) shows restriction of cell extensions and increased cell death, in cultures treated with Ezh2-shRNA. (<b>G</b>) Western blot of protein samples prepared after 24 hours of transfection shows a decrease in Ezh2 and a strong increase in cleaved caspase 3 bands in pOLs treated with Ezh2-shRNA, compared to the cultures treated with nc-shRNA (control). PCNA (a proliferation marker) expression remained about the same in both conditions<b>.</b> (<b>H</b>) Quantification of apoptotic (cleaved caspase 3-positive) and differentiating (RIP-positive) cells in Ezh2-shRNA treated and control (nc-shRNA) pOLs. Bar graphs represent mean of three independent experiments ± S.D. (Calibration bar in (<b>A</b>) represents 100 micrometers, which is valid for all the photomicrographs (<b>A</b>–<b>F</b>); ** in (<b>H</b>) represents p<0.01).