Drosophila Muscleblind Is Involved in troponin T Alternative Splicing and Apoptosis
Figure 3
mblC overexpression activates apoptosis in vivo, but not significantly in cell culture.
Confocal micrographs of third instar wing imaginal discs from en-Gal4 mblC/+ (A,C,E) and en-Gal4/+ controls (B,D) stained with an anti-Mbl (A), anti mammalian Caspase-3 antibody (B,C), or TUNEL assay (D,E). Wing imaginal discs of en-Gal4 UAS-mblC (C) flies show activation of executioner caspase-3 in cells over-expressing MblC (A) in the posterior compartment where the en-Gal4 driver is active. Despite the fact that MblC overexpression levels are similar in posterior pouch (A, bent arrow) and notum cells (A, arrowhead), caspase-3 is not detected activated in prospective notum cells (C). A TUNEL assay to detect DNA fragmentation that results from apoptosis signalling cascades reproduced the same pattern of apoptotic cells (D,E) detected by caspase-3 activation. (F) Bar graph representing the average number (from quadruplicates) of live cells 48 h after transfection of plasmids expressing the indicated Muscleblind protein isoforms. Overexpression of Muscleblind isoforms did not significantly reduce Drosophila S2 cell viability in cell culture conditions. Error bars are standard deviations. (G) Western blot of protein extracts from S2 cells transfected as in (F) with the indicated Muscleblind proteins and detected with an anti-Muscleblind antibody [47]. Lower molecular weight bands in lanes MblA and MblB are degradation products. MblD could not be detected by western blotting. Predicted molecular weights are: MblA, 22.65 kDa; MblB, 34.46 kDa; MblC 26.91 kDa.