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. 2014 May 2;9(5):e96326. doi: 10.1371/journal.pone.0096326

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© 2014 Amândio et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A–A′ to C–C′) standard confocal sections of third instar wing imaginal discs with dorsal sides up, expressing (A–A″) UAS-cpa-IR^C10 and two copies of UAS-mCD8-GFP (green in A) or (B–B′) UAS-cpa-IR^C10, UAS-HA-cpa^ΔABD and UAS-cpb⁷ or (C–C′) UAS-cpa-IR^C10, UAS-cpb⁷ and one copy of UAS-mCD8-GFP (green in C) under sd-Gal4 control. Discs are stained with anti-activated-Caspase 3 (magenta), which monitors DRONC activation and (B–B′) Phalloidin (cyan blue in B) to underline wing disc shape. The scale bars represent 30 µm. (D) quantification of total C3 area per disc area for the genotypes sd>cpa-IR^C10, 2XGFP (lane 1); sd>cpa-IR^C10, HA-cpa^ΔABD, cpb⁷ (lane 2) and sd>cpa-IR^C10, cpb⁷, 1XGFP (lane 3). The means for lane 1 is 92.4 (n = 23); for lane 2 is 10.61 (n = 19); for lane 3 is 32.9 (n = 20). Error bars indicate s.e.m. P<0.0001 for comparison of lane 1 and 2 or 3 or lane 2 and 3. (E) quantification of total C3 area per disc area for wing discs containing T155>flp; cpa^107E mutant clones (lane 1) or T155>flp; cpa^107E mutant clones expressing UAS-cpb⁷ (lane 2). The means for lane 1 is 10.80 (n = 26); for lane 2 is 13.77 (n = 20). n.s. indicates non-significant P value. (F and G) western blots on protein extracts from wing discs expressing two copies of UAS-mCD8-GFP (lane 1) or UAS-cpa-IR^C10 and two copies of UAS-mCD8-GFP (lane 2) or UAS-cpa-IR^C10, UAS-cpb⁷ and one copy of UAS-mCD8-GFP (lane 3) under sd-Gal4 control, blotted with (F) anti-Cpa (upper panel) and anti-H3 (lower panel) or (G) anti-Cpb (upper panel) and anti-H3 (lower panel). Panels derive from the same experiment shown in Figure 5E and F and blots were processed in parallel (see Figure S2 showing the whole experiment). (H) mean intensity of the ratio of Cpa signals between posterior and anterior wing compartments of hh-Gal4 driving two copies of UAS-mCD8-GFP (lane 1) or UAS-cpb⁷ and one copy of UAS-mCD8-GFP (lane 2) or UAS-cpa-IR^C10 and two copies of UAS-mCD8-GFP (lane 3) or UAS-cpa-IR^C10 and UAS-cpb⁷ and one copy of UAS-mCD8-GFP (lane 4). The mean for lane 1 is 0.959 (n = 15), for lane 2 is 0.970 (n = 20), for lane 3 is 0.716 (n = 21), for lane 4 is 0.776 (n = 20). Error bars indicate s.e.m.. P<0.0001 for comparison of lanes 1 and 3 or 4 or P<0.01 for comparison of lanes 3 and 4. (I to L) graph of (I and J) cpa or (K and L) cpb mRNA levels measured by five independent qRT-PCR in wing imaginal discs expressing (I and K) two copies of UAS-mCD8GFP (lane 1) or UAS-cpa-IR^C10 and UAS-mCD8GFP (lane 2) or UAS-cpa-IR^C10 and UAS-cpb⁷ (lane 3) or (J and L) UAS-mCherry (lane 1) or UAS-cpb⁷ (lane 2) under sd-Gal4 control. (I) the means for lane 1 is 1.084; for lane 2 is 0.4328; for lane 3 is 1.086. P<0.0027 for comparison of lane 1 with 2 or P<0.0003 for comparison of lane 2 with 3. (J) the means for lane 1 is 1.07; for lane 2 is 0.824. n.s. indicates non-significant P value. (K) the means for lane 1 is 0.621; for lane 2 is 0.5031; for lane 3 is 3.735. P<0.0001 for comparison of lane 3 with 1 or 2. (L) the means for lane 1 is 0.292; for lane 2 is 1.961. P<0.0001 for comparison of lane 1 and 2. Error bars indicate s.e.m.