miR-181b regulates vascular stiffness age dependently in part by regulating TGF-β signaling - Fig 6

<p><b>A, Serum TGF-β of miR-181a1/b1^-/- (a/b KO) and control (WT) at 10 weeks of age (n = 4) and 22 weeks of age (n = 6). B, Western blot of p-SMAD2/3 compared to total SMAD2/3 in aorta of miR-181a1/b1^-/- (a/b KO, n = 4) and control (WT, n = 4) at 22 weeks. C, Immunohistochemistry showing pSMAD2/3 staining of the aorta from miR-181a1/b1^-/- (a/b KO) and control (WT). D</b>, Quantification of pSMAD2/3 staining for miR-181a1/b1^-/- (a/b KO) and control (WT). (n = 7) <b>E</b>, Westernblot showing VE-Cadherin (VEC marker) and α-actin (VSMC marker) of the cultured cells (n = 3). <b>F</b> TGF-β levels in the serum free media incubated for 24 hours in the VECs culture and VSMCs culture of miR-181a1/b1^-/- (a/b KO, n = 3) and control (WT, n = 3). <b>G</b>, TGF-β expressions in VSMCs of miR-181a1/b1^-/- (a/b KO) and control (WT), untreated and treated with angiotensin II. (n = 4–8) <b>H</b>, Using both TargetScan and DIANA-microT, we have predicted that position 71–77 of TGF-βi 3'-UTR can be a direct target of miR-181b (upper panel). We then performed mutagenesis at positions 72, 74, 76 and 77 from “A to C”, “A to C”, “G to C”, and “U to A”, respectively (lower panel). <b>I</b>, Luciferase activity of 3'-UTR of TGF-βi in rat aortic VSMCs (A7r5) co-transfected with TGF-βi or mutated TGF-βi reporter construct, along with miR-181b or scramble sequence. Values are mean ±SEM, *p<0.05, **p<0.01, ***p<0.001.