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. 2021 Jun 30;22(13):7064. doi: 10.3390/ijms22137064

Figure 1.

Figure 1

Metformin treatment limits senescence of bronchial–alveolar epithelial cells and associated senescence associated secretory phenotype in whole thorax irradiated mice. (A) C57BL/6 mice were left untreated (Ctrl), received five-times a week metformin (Met) treatment, 15Gy whole thorax irradiation (WTI) or received WTI in combination five-times a week metformin treatment for first three weeks post WTI. (B) Senescence-associated beta-galactosidase activity was analyzed on frozen lung sections, 21 days post irradiation. Photomicrographs depict representative pictures (scale bar = 200 µm). (C) Senescence mediator cyclin-dependent kinase inhibitor 1 (Cdkn1a/p21) protein expressions were analyzed in whole lung protein lysates using Western Blot analyses Expression levels were quantified by densitometry and data are presented as mean values ±SEM of individual biological replicates (n = 6–9 per group). p-values indicate: ** p ≤ 0.01 by one-way ANOVA with post hoc Tukey’s multiple comparison test. (D) Cdkn1a/p21 as well as cyclin D1 (Ccnd1) mRNA expression levels were further quantified using Real-Time RT-PCR. Relative transcript levels were normalized to beta-actin (set as 1). Symbols depict individual biological replicates measured in duplicates each (0Gy and 15Gy Ctrl: n = 9 per group; 0Gy Met, 15Gy Met: n = 6 per group). Data represent mean values ±SEM, p-values indicate: * p ≤ 0.05, ** p ≤ 0.01 by one-way ANOVA with post hoc Tukey’s multiple comparison test. Additional unpaired (two-tailed) t-test analysis is depicted by # p ≤ 0.05. (E) SASP factor (Ccl2, Mmp2, Cxcl1, Il6, Ccl7, Ccl8) expression levels were analyzed (in duplicates) on mRNA level of whole lung RNA isolates (0Gy and 15Gy Ctrl: n = 9 per group; 0Gy Met, 15Gy Met: n = 6 per group). Individual biological replicates and mean values ±SEM are shown. p-values indicate: * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001 by one-way ANOVA with post hoc Tukey’s multiple comparison test and additionally by unpaired (two-tailed) t-tests as depicted by ## p ≤ 0.01, ### p ≤ 0.001.