Overexpressing HA-cpa in wing discs knocked-down for cpb restores cpb mRNA and protein levels and suppresses apoptosis.(A–A′ and B–B′) standard confocal sections of third instar wing imaginal discs with dorsal sides up, expressing (A–A″) UAS-cpb-IR45668 and one copy of UAS-mCD8-GFP (green in A) or (B–B′) UAS-cpb-IR45668 and UAS-HA-cpa89E under sd-Gal4 control. Discs are stained with anti-activated-Caspase 3 (magenta), which monitors DRONC activation and (B–B′) anti-HA (green in B), reflecting HA-cpa89E expression. The scale bars represent 30 µm. (C) quantification of total C3 area per disc area for the two genotypes shown in A–A′ and B–B′. The means for sd>cpb-IR45668, GFP is 62.19 (n = 22); for sd>cpb-IR4566, HA-cpa89E is 26.67 (n = 32). Error bars indicate s.e.m. P<0.0001 for comparison of between both genotypes. (D and E) western blots on protein extracts from wing discs expressing two copies of UAS-mCD8-GFP (lane 1) or UAS-cpb-IR45668 and two copies of UAS-mCD8-GFP (lane 2) or UAS-cpb-IR45668 and UAS-HA-cpa89E and one copy of UAS-mCD8-GFP (lane 3) under sd-Gal4 control, blotted with (D) anti-Cpa (upper panel) and anti-H3 (lower panel) or (E) anti-Cpb (upper panel) and anti-H3 (lower panel). (F to I) graphs of (F and G) cpa or (H and I) cpb mRNA levels measured by (F and H) three or (G and I) five independent qRT-PCR in wing imaginal discs expressing (F and H) two copies of UAS-mCD8GFP (lane 1) or UAS-cpb-IR45668 and UAS-mCD8GFP (lane 2) or UAS-cpb-IR45668 and UAS-HA-cpa89E (lane 3) or (G and I) UAS-mCherry (lane 1) or UAS-HA-cpa89E under sd-Gal4 control. (F) the means for lane 1 is 0.90; for lane 2 is 1.04; for lane 3 is 9.8. P<0.033 for comparison of lane 1 with 3. (G) the means for lane is 1.07; for lane 2 is 1.88. P<0.0001 for comparison of lane 1 and 2. (H) the means for lane 1 is 0.59; for lane 2 is 0.25; for lane 3 is 0.4319. P<0.0018 for comparison of lane 1 and 2 or P<0.048 for comparison of lane 2 and 3. (I) the means for lane 1 is 0.29; for lane 2 is 0.31. n.s. indicates non-significant P value. Error bars indicate s.e.m.
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