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Fig. 4 | Journal of Translational Medicine

Fig. 4

From: Secretion of miRNA-326-3p by senescent adipose exacerbates myocardial metabolism in diabetic mice

Fig. 4

miRNA-326-3p decreases NMVMs’ contractile and mitochondrial function by inhibiting Rictor. A Predicted upstream regulators of differentially expressed genes in AMVMs isolated from STZ mice by IPA analysis. B Western blotting analysis of Rictor, p-AKT, AKT and Gapdh in UT or NMVMs transfected with 326-3p- or negative control- (NC-) miR-mimic. (n = 3 per group). C Schematic diagram showing binding of miR-326-3p to 3′-UTR of Rictor. Complementary sequences are highlighted in red, mutated sequence in blue. D Luciferase reporter assays performed with firefly luciferase under control of the Rictor 3′ UTR (wild type (WT) or mutant (MT)), control Renilla luciferase and the miR-326-3p (n = 3 per group). E Contractile velocity of UT or NMVMs transfected with 326-3p- or NC-miR-mimic (n = 6 per group). F Representative micrographs of UT or NMVMs, transfected with 326-3p- or NC-miR-mimic, stained for mitochondrial membrane potential (TMRM), mitochondrial superoxide (MitoSox), and DAPI, mitochondrial membrane potential and mitochondrial superoxide relative intensity of fluorescence are shown (n = 5 per group). G Real-time oxygen consumption rates (OCR) were evaluated for UT or NMVMs transfected with 326-3p- or NC-miR-mimic, Basal (H) and maximal (I) respiration rates are shown (n = 8–9 per group). J Contractile velocity of NMVMs transfected with Rictor- or NC-siRNA (n = 6 per group). K Representative micrograph and fluorescence intensity quantification of NMVMs transfected with Rictor- or NC-siRNA and stained for mitochondrial membrane potential (TMRM) or mitochondrial superoxide (MitoSox) and DAPI (n = 5 per group). L Real-time oxygen consumption rates (OCR) were evaluated for NMVMs transfected with Rictor- or NC-siRNA; basal and maximal respiration rates are shown (n = 6 per group). Data are presented as the mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to UT group; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 compared to NC-miR-mimic group; $P < 0.05, $$P < 0.01, $$$P < 0.001, $$$$P < 0.0001 compared to INC-siRNA group

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