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. 2022 Oct 13;13:6045. doi: 10.1038/s41467-022-33773-0

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© The Author(s) 2022

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. 2 — a Representative immunoblots and densitometry graphs demonstrating the diabetes-induced increases of (b) ICAM-1, (c) iNOS, and (d) the ratio of pIκB/IκB were inhibited in the retina of RPE-specific Akt2 cKO diabetic mice. e Representative images and (f) quantification of attached leukocytes in the retina. Arrows indicate leukocytes adherent to the retinal blood vessels. Diabetes increased the number of adherent leukocytes in the Akt2^fl/fl diabetic retina compared to nondiabetic Akt2^fl/fl mice; this number was markedly reduced in Akt2 cKO diabetic mice, Scale bar: 100 µm. g Retinal superoxide was measured using lucigenin; diabetes increased retinal production of superoxide in Akt2^fl/fl mice. These levels were attenuated in Akt2 cKO diabetic mice. h Representative images and (i) quantification of DHE-stained (red) retinal sections showing the levels of ROS in each group. The DHE stain was primarily localized in the nuclear layers, Scale bar: 100 µm. The intensity of red fluorescence was quantified at the INL and ONL as they represent the majority of red staining in the retina. The diabetes-induced increase of ROS in Akt2^fl/fl retina was not observed in diabetic Akt2 cKO mice. j Representative images and (k) quantification of DCF-stained retinal sections showing ROS accumulated in the inner and outer segments of photoreceptors. The blue stain is DAPI. The diabetic Akt2 cKO mice displayed significantly low retinal ROS levels compared to the diabetic Akt2^fl/fl mice. Scale bar: 100 µm. Data are expressed as mean ± SD. **p < 0.01; ***p < 0.001; ****p < 0.0001 show changes versus Akt2^fl/fl nondiabetic (N) controls. ^††p < 0.01, and ^††††p < 0.0001 show changes versus Akt2^fl/fl diabetic (D) mice. Statistical test used in (b, c, d, f, g, i, k) is One-way ANOVA followed by a Tukey’s post hoc test. n = 6 mice for each group. Exact p values are: b p = 0.0024 (Akt2 ^fl/fl D vs. Akt2 ^fl/fl N), p = 0.0081 (Akt2 cKO D vs. Akt2 ^fl/fl D). c p = 0.0004 (Akt2 ^fl/fl D vs. Akt2 ^fl/fl N), p = 0.0024 (Akt2 cKO D vs. Akt2 ^fl/fl D). d p = 0.0001 (Akt2 ^fl/fl D vs. Akt2 ^fl/fl N), p = 0.0036 (Akt2 cKO D vs. Akt2 ^fl/fl D). f p = 0.0001 (Akt2 ^fl/fl D vs. Akt2 ^fl/fl N), p = 0.0019 (Akt2 cKO D vs. Akt2 ^fl/fl D). g p = 0.0003 (Akt2 ^fl/fl D vs. Akt2 ^fl/fl N), p = 0.0043 (Akt2 cKO D vs. Akt2 ^fl/fl D). i p = 0.0001 (Akt2 ^fl/fl D vs. Akt2 ^fl/fl N), p < 0.0001 (Akt2 cKO D vs. Akt2 ^fl/fl D). k p < 0.0001 (Akt2 ^fl/fl D vs. Akt2 ^fl/fl N, Akt2 cKO D vs. Akt2 ^fl/fl D). cKO conditional knockout, GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer, RIS/ROS rod inner/outer segment, DHE dihydroethidium, DCF dichlorofluorescein. Source Data is provided in the Source Data file.