Figure 6.

Apoptosis analysis of motor neurons expressing mutant HSPB8. Rat motor neurons were transduced with pLenti-GFP, pLenti-WT-HSPB8-GFP or mutant pLenti-K141N/K141E-HSPB8-GFP constructs at DIV3 and analyzed for the activation of apoptosis at DIV7 with the TUNEL assay which detects DNA breaks in the nuclei of apoptotic cells. Confocal micrographs are shown of GFP expression (green in merge), nuclei of all cells (apoptotic and non-apoptotic, DAPI, blue in merge) and nuclei of apoptotic cells (TUNEL, red in merge). Treating the motor neuron cultures with 50 µm etoposide for 2 h was used as a positive control for cell death activation (A). Expression of the different constructs did not lead to detectable TUNEL staining (B–E). An additional internal positive control was provided by the TUNEL-positive nuclei resulting from dead cells always present in the primary cultures (arrowheads). Motor neurons expressing mutant K141E-HSPB8-GFP exhibit beaded or shortened neurites, but do not have TUNEL-positive nuclei (E). Arrowheads indicate TUNEL-positive cells and arrows indicate GFP or HSPB8-positive TUNEL-negative cells. Scale bar = 10 µm.