Fig 1: Silencing of RUNX2 or inhibition of the PTEN/Akt signaling pathway neutralizes the protective effect of miR-149 inhibitor. The PTEN signaling pathway inhibitor SF1670 was added to miR-149-overexpressing cells, and RUNX2 was further inhibited in BEWO cells that interfered with miR-149. (a) The expression of RUNX2, PTEN and Akt1 as well as the extent of Akt1 phosphorylation verified by western blot analysis and RT-qPCR. (b) Detection of cell proliferation by EdU staining. (c) Cells were stained with CFSE, and cells were tested for cell viability by flow cytometry. (d) Detection of cell apoptosis by flow cytometry. (e and f) Transwell assay detected the migration and invasion of HTR-8 cells and BEWO cells. Two-way ANOVA (panel a) or one-way ANOVA (panel b–f) was adopted for comparisons among multiple groups, followed by Tukey's multiple comparisons test.
Fig 2: Plasma ED-EVs prevent fibroblast senescence and accelerate skin wound healing in diabetic mice by promoting YAP nuclear translocation and activating the PI3K/Akt/mTOR pathway.
Fig 3: miR-582 activates the PI3K/Akt/Snail pathway in GC cells. (A) The protein expression and phosphorylation level of PI3K/Akt/Snail signaling pathway measured by immunoblotting. Then, PI3K specific agonist 740Y-P was added into miR-582 inhibitor-transfected cells. (B) EdU staining of GC cell viability. (C) immunofluorescence of E-cadherin. (D) Migration ability of GC cells assessed by Transwell assays. (E) Invasion ability of GC cells evaluated by Transwell assays. The data are displayed as the mean ± SD of three independent experiments. One-way ANOVA and Tukey’s multiple comparison test was applied to determine statistical significance. *p < 0.05.
Fig 4: miR-149 disrupts the PTEN/Akt signaling pathway by targeting RUNX2. (a) TargetScan, StarBase, miRDB and miRTarBase predicted possible targeting mRNAs of miR-149. (b) StarBase predicted the targeted binding sequence of miR-149 and RUNX2. (c) Luciferase activity experiment verified the binding relationship between miR-149 and RUNX2. (d) The mRNA expression of RUNX2 in placental chorionic tissues of 21 RM patients and 13 controls was detected by RT-qPCR. (e) Pearson's correlation test analyzed the relationship between miR-149 and RUNX2 in RM patients. (f and g). The mRNA and protein expression of RUNX2 in HTR-8 and BEWO cells determined by RT-qPCR and western blot analysis. (h) The protein expression of PTEN and Akt1 as well as the extent of Akt1 phosphorylation determined by western blot analysis. Comparisons between the two groups were performed using an unpaired t test (panel d). Two-way ANOVA (panel c and h) or one-way ANOVA (panel f and g) was adopted for comparisons among multiple groups, followed by Tukey's multiple comparisons test.
Fig 5: Plasma ED-EVs inhibit DSF premature senescence through the PI3K/Akt/mTOR pathway. (A and C) Western blot analysis detected levels of PI3K/Akt/mTOR pathway-and senescence-related proteins. (B) SA-ß-gal assay detected DSF senescence. (D) ELISA measured SASP level (MMP-3, IL-6 and IL-1ß) in DSF. (E) H2DCFDA probe measured ROS level in DSF. Compared with the blank group, *p < 0.05, **p < 0.01, ***p < 0.001; compared to the ED-EVs group, #p < 0.05, ##p < 0.01, ###p < 0.001. Data in panels (A and D) were analyzed by two-way ANOVA, and data in panels (B, C and E) were analyzed by one-way ANOVA, followed by Tukey's multiple comparisons test. Repetitions = 3.
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