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. 2019 Feb 15;10:771. doi: 10.1038/s41467-019-08686-0

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 9 — The ASPP complex promotes PP1β9C and PP1α96A AJ localization. a–h Confocal X–Y sections of pupal retinas at 40 h APF stained with anti-E-cad antibodies to mark cell outlines. Scale bar = 10 µm. a, b ccdc85 (b) mutant flies have defects in eye development compared to wild-type flies (a) (13.30 ± 1.40 vs 12.2 ± 0.4). c Mutation of ASPP in ccdc85 mutant flies worsens the phenotype (16.7 ± 2.10). d–g Expression of ASPP under the ubiquitin 63E (ubi) promoter in ASPP, ccdc85 mutants rescues the phenotype (d), but overexpression of ASPP^FA (e), ASPP^KVK (f), ASPP ^WK (g) or ASPP^FA-WK (h) do not (13.16 ± 1.058 vs 16.22 ± 1.44, 14.64 ± 1.38, 15.64 ± 1.98 and 15.60 ± 1.49) (see Supplementary table 1 for full genotypes). i Quantification of inter-ommatidial cells (IOCs) in retinas of the indicated genotypes at 40 h after puparium formation (APF). Error bars represent the standard deviation. A one-way ANOVA test was carried out to determine if the differences among means were significantly different from each other. p-values were adjusted using a Bonferroni correction. Significant differences are marked. *** indicates p < 0.0001, ** indicates p < 0.01 and * indicates p < 0.05 (n = 25 hexagons from four retinas). j Quantification of bristle misplacement. (n = 25 from four retinas). k–s Confocal X–Y sections of pupal retinas at 26 h APF showing endogenous YFP-tagged PP1β9C and stained with E-Cad. Scale bar = 10 µm. k–m PP1β9C localises at the cell–cell junctions (67.31% ± 7.45). n–p ASPP loss does not change the localisation of PP1-β9C (69.23% ± 6.29). q–s Loss of ccdc85 in an ASPP mutant background mislocalises PP1β9C from the cell–cell junctions (60.92% ± 5.51). t Quantification of percentage of junctional PP1β9C of the indicated genotypes at 26 h APF. Error bars represent the standard deviation. For quantifications of PP1β9C intensity, two regions of interest were drawn surrounding the junctions of a cell (ROI_TOTAL and ROI_CYTOPLASM). Junctional fraction was quantified by (ROI_TOTAL – ROI_CYTOPLASM). Values were normalised to 100% for PP1β9C. One-way ANOVA test was carried out to determine if the differences among means were significantly different from each other. p-values were adjusted using a Bonferroni correction. Significant differences are marked. *** indicates p < 0.0001 (n = 50 cells from four retinas)