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. 2018 Apr 18;9:1541. doi: 10.1038/s41467-018-04000-6

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© The Author(s) 2018

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. 4 — Persistent JNK activity causes overgrowths. a–c Discs of genotypes: a UAS-GFP;hh-Gal4 (n = 10), b UAS-RHGmiRNA/UAS-GFP; hh-Gal4/+(n = 20) and c UAS-Bsk^DN; UAS-RHGmiRNA/UAS-GFP; hh-Gal4/+(n = 11) 72 h after X-rays. EdU incorporation (red) is increased in the posterior compartment (green) of b where apoptosis is suppressed by UAS-RHGmiRNA, but when JNK activity is blocked with Bsk^DN (c), EdU incorporation is similar in both compartments, which is the normal situation (a). d Quantification of the overgrowth in UAS-RHGmiRNA/UAS-GFP;hh-Gal4/+disc (n = 32) after irradiation compared to non-irradiated discs (n = 35) (***p-value < 0.0001). Suppression of JNK by Bsk^DN (n = 14) reverses the posterior compartment to normal size (***p-value < 0.0001), indicating that JNK is responsible for the overgrowth. e, f Discs of genotypes: e UAS-GFPtubGal80^ts/+;droncⁱ²⁴/hh-Gal4droncⁱ²⁹ (n = 10) and f UAS-Bsk^DN;UAS-GFPtubGal80^ts/+;droncⁱ²⁴/hh-Gal4droncⁱ²⁹ (n = 23) 96 h after X-rays (see details of the experiment in the Methods section). Discs were stained with Mmp1 (red) to monitor JNK activity and GFP (green) to identify the P-compartment; TOPRO staining delimits the discs. The disc in e shows Mmp1 label in both compartments, but expression of the Bsk^DN blocks JNK activity (Mmp1, red) as shown in f. g Quantification of the posterior compartment volume over the total disc volume for the genotypes: UAS-GFPtubGal80^ts/+; droncⁱ²⁴/hh-Gal4 droncⁱ²⁹ (column: hh>GFPdronc⁻ X-rays, n = 79), UAS-Bsk^DN; UAS-GFPtubGal80^ts/+;droncⁱ²⁴/hh-Gal4droncⁱ²⁹ expressing Bsk^DN 24 h (column: hh>Bsk^DNdronc⁻ 24 h after X-rays, n = 43) or 48 h after irradiation (column: hh>Bsk^DNdronc⁻ 48 h after X-rays, n = 18). There is a significant size decrease when Bsk^DN is expressed 24 h and 48 h after irradiation (***p-value < 0.0001), indicating that inhibiting JNK after irradiation suppresses overgrowth. Quantification for the genotypes: UAS-GFPtubGal80^ts/UAS-SOD:UAS-Cat;droncⁱ²⁴/hh-Gal4droncⁱ²⁹ (column: hh>SOD:Cat dronc⁻ 24 h after X-rays, n = 14) inducing the expression of SOD:Cat 24 h after irradiation and UAS-GFPtubGal80^ts/UAS-molRNAi; droncⁱ²⁴/hh-Gal4droncⁱ²⁹ (column: hh>molRNAidronc⁻ 24 h after X-rays, n = 7) inducing the expression of molRNAi 24 h after irradiation. There is a significant size decrease after the expression of SOD:Cat and the inhibition of mol (**p-value < 0.001 and *p-value < 0.02, respectively), indicating that ROS levels are important to maintain JNK overgrowth. Columns in the graphs represent mean ± s.d. Statistical test used: Mann–Whitney U-test. Scale bars are 100 μm