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. 2022 Jul 1;132(13):e142548. doi: 10.1172/JCI142548

Figure 4. miR-181b targets Sema3A, and TGF-β induces Twist and SMA expression.

Figure 4

(A) Representative immunoblots showing protein expression in AEECs treated with 5 ng/mL TGF-β for up to 48 hours. GAPDH was used as the loading control. (B) Representative immunoblots showing protein expression in AEECs incubated with or without TGF-β (0, 1, 2, or 5 ng/mL). (C) Representative immunoblots show protein expression in AEECs that were transfected with miR-181b antagomir (antagomir-181b) or scrambled control miRNA and then treated with or without TGF-β (5 ng/mL). (D) Confirmation of the hsa-miR-181b target site in the Sema3A 3′-UTR. Schematic representation of the Sema3A 3′-UTR indicating the predicted hsa-miR-181b binding site. AEECs were transfected with the pMIR-REPORT-Sema3A 3′-UTR (intact) or pMIR-REPORT-Sema3A 3′-UTR (mutant) luciferase reporter vector. The fold induction in relative luciferase activity was plotted (n = 3). *P < 0.05 compared with the scrambled control, by 2-tailed Student’s t test. (E) AEECs were transfected with the miR-181b TSB for 24 hours, after which AEECs were treated with or without TGF-β for 6 hours. Sema3A mRNA expression was measured by qRT-PCR (n = 3). *P < 0.05 compared with control; #P < 0.05 and ###P < 0.001 compared with TGF-β; 1-way ANOVA with Bonferroni’s post hoc test. (F) AEECs were transfected with the negative control mimic (NC mimic) or the synthetic miR-181b mimic, with or without the miR-181b TSB. Sema3A mRNA expression was measured by qRT-PCR (n = 3). **P < 0.01 compared with NC mimic; ##P < 0.01 compared with miR-181b mimic; 1-way ANOVA with Bonferroni’s post hoc test. (G) Representative quantitative data for the immunoblots showing protein expression in AEECs transfected with miR-181b TSBs for 24 hours (n = 3). ***P < 0.001 versus the NC mimic group; ###P < 0.001 versus the 181b mimic group; 1-way ANOVA with Bonferroni’s post hoc test. (H) AEECs were transfected with Sema3A siRNA or scrambled siRNA. Representative immunoblots and densitometric quantification of protein expression are shown (n = 3). **P < 0.01 and ***P < 0.001 compared with scrambled siRNA–treated group, by 2-tailed Student’s t test. t, total.