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. 2023 Sep 12;13:15080. doi: 10.1038/s41598-023-41166-6

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© The Author(s) 2023

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Klotho affects FOXO-3a activity and catalase activity in astrocytes. (A) Western blot assay was performed to measure phosphorylation levels of FOXO-3a. Statistical analysis suggested that Klotho treatment decreased the FOXO-3a inhibitory phosphorylation. One way ANOVA followed by Tukey's multiple comparisons tests; [F = 23.86; P = 0.0003; R squared = 0.8413]. Below the graph is the representative western blot digital images of p-FOXO-3a, FOXO-3a, and β-actin. (B) FOXO gene reporter activity. After 48 h of plasmid transfections, treatments were performed by incubating cells for 24 h with DMEM containing high (4.5 g/L) or low (1 g/L) concentrations of glucose, with or without 1 nM Klotho. Results represent the ratio of fluorescence of firefly luciferase to renilla luciferase, normalized to the mean of the control group (DMEM high glucose). One-way ANOVA statistical analysis followed by Tukey’s multiple comparison tests. FOXO-3a activity was increased in all groups: CTL High vs. CTL Low (mean difference: − 11.35; q = 6.782), CTL High versus Klotho High (mean difference: − 25.26; q = 15.09), CTL High versus Klotho Low (mean difference: − 24.06; q = 14.38), CTL Low versus Klotho High (mean difference: − 13.91; q = 8.313), and CTL Low versus Klotho Low (mean difference: − 12.71; q = 7.598). No difference was observed between Klotho high and Klotho low glucose (mean difference: 1.196; q: 0.7148). P < 0.001, F = 50.66, and R squared = 0.9048. (C) Catalase relative activity. Statistical analysis suggested that all klotho concentrations increased catalase expression compared to that of the control group. [One-way ANOVA followed by Tukey’s multiple comparison test. F = 18.38; R squared 0.8386; P < 0.0001]. *P < 0.05; ***P < 0.0001. The original autoradiographs are available in Supplementary Figs. S4 and S5. Lysates were obtained from two or three wells of a 6-well plate to reach 2 × 10⁶ cells. Each point in the graphs represents a lysate obtained from independent cell cultures, and four independent cell cultures were analyzed in these assays.