Research Paper Volume 17, Issue 3 pp 757—777

Figure 1. Increase in senescence makers in irradiated and aged flies and meltrin knockdown in the enterocytes reduces intestinal permeability and senescence makers in flies following irradiation. (A) Representative immunohistochemistry images of the gut show increased SA-β gal and γ-H2Ax positive cells in X-ray irradiated flies (100Gy) compared to control flies (0Gy). Fluorescence intensity was measured using ImageJ; Scale bar = 20 μm. (A-i). Quantification of fluorescence intensity of SA-β gal (arbitrary units, A.U.) between 0 Gy and 100 Gy. Mean fluorescence intensity was measured using ImageJ and averaged across the gut of 15–16 flies. Data represent mean ± SEM for each group. (A-ii) Quantification of γ-H2AX positive cells expressed as a percentage of total cells in both control and 100 Gy X-ray exposed groups. Data are presented as mean ± SEM from the gut of 15–16 flies. Exposure to 100 Gy of X-ray radiation significantly increased the number of γ-H2AX positive cells compared to the control group, indicating elevated DNA double-strand breaks in response to radiation. (B) Representative immunohistochemistry images of the gut show increased SA-β gal and γ-H2Ax positive cells in X-ray irradiated flies (20 days) compared to control flies (7 days). Fluorescence intensity was measured using ImageJ; Scale bar = 20 μm. (B-i) Quantification of fluorescence intensity of SA-β gal (arbitrary units, A.U.) between 7-day and 20-day-old flies. Mean fluorescence intensity was measured using ImageJ and averaged across the gut of 13–16 flies. Data represent mean ± SEM for each group. (B-ii) Quantification of γ-H2AX positive cells expressed as a percentage of total cells in both 7-day-old and 20-day-old flies. Data are presented as mean ± SEM from the gut of 13-16 flies. The gut of 20 days old flies significantly increased the number of γ-H2AX positive cells compared to the 7-day-old, indicating elevated DNA double-strand breaks in response to radiation. (C-i, C-ii) Smurf assay for assessing gut permeability was performed with 5966-GS>UAS-Meltrin RNAi flies on day ten after irradiation. Control 100Gy R (−) was maintained on 1.5 YE food without RU486, whereas meltrin expression was knocked down in ECs in 100Gy R (+). Results plotted as the mean proportion of Smurf to non-Smurf flies of 4 vials in each group with 25 flies in each vial. Error bars indicate SEM. (D) Immunohistochemistry images of gut showing an increase in SA-β gal and γ-H2Ax positive cells in non-irradiated and X-Ray irradiated in control (RU-) flies and meltrin RNAi and flies (RU+). (D-i, D-ii) Quantification of SA-β gal intensity and γ-H2Ax positive cells in irradiated-non irradiated flies in the gut of RU- and RU+ flies. (A-i, A-ii, B-i, B-ii, C, C-i, D-i and D-ii) Error bars indicate SEM. The significance level was determined by ANOVA or Student’s t-test, denoted by asterisks: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, and ^****p < 0.0001.