Fig 1: Characterization of pulmonary arterial smooth muscle cells (PASMCs) and analysis of the expressions of miR-223-3p and ITGB3 the induction of after hypoxia. A, Photographs of PASMC were screened, magnification, ×200; scale bar: 50 µm. B, PASMCs were characterized by a-smooth muscle actin (a-SMA) immunofluorescence, magnification, ×400; scale bar: 50 µm. C and D, QRT-PCR was utilized to evaluate the expression levels of miR-223-3p and ITGB3 in the hypoxic cells compared with the normal cells. E, ITGB3 protein expression was determined in normal and hypoxic cells by Western blot. All experiments were performed in triplicate. **P < 0.01
Fig 2: MiR-223-3p and ITGB3 expression level after transfection. A, ITGB3 expression after transfection with three specific siRNAs was determined. B, MiR-223-3p expression level after transfection with miR-223-3p mimics or inhibitor was confirmed via qRT-PCR. C, The ITGB3 mRNA level after transfection with pcDNA-ITGB3 or si-ITGB3 transfection was compared with matched control groups via qRT-PCR. D, The protein expression of ITGB3 was measured and compared with the matched control group after transfection with pcDNA-ITGB3 and si-ITGB3 or co-transfection with pcDNA-ITGB3 and miR-223-3p mimics. E, Design of ITGB3 3'UTR (wild type and mutant) was the target sequence. F, The dual luciferase reporter assay was performed, and decreased luciferase activity was observed in miR-223-3p mimics + pGL3-ITGB3 wt group. All experiments were performed in triplicate. **P < 0.01 compared with control siRNA, control mimics, pcDNA Vector or pGL3-NC group; ## P < 0.01 compared with control inhibitor or control siRNA
Fig 3: MiR-223-3p and ITGB3 are differentially expressed in IPAH patients. A, According to GSE33463, differentially expressed genes included ITGB3 in IPAH. B, After analysing the data of GSE67597, differentially expressed miRNAs were detected, including miR-223-3p in IPAH
Fig 4: miR-223 regulates the apoptosis of pulmonary arterial smooth muscle cell (PASMCs) after transfection under normal or hypoxic condition. Flow cytometric analysis was used to evaluate the effects of miR-223-3p mimics or miR-223-3p inhibitor or si-ITGB3 or pcDNA-ITGB3 transfection at 48 h on apoptosis of PASMCs. A, The x-axis represents Annexin V-FITC staining, and y-axis represents PI staining. Quadrant Q4 is Annexin V-FITC-and PI negative, representing viable cells. Quadrant Q3 is Annexin V-FITC positive and PI negative, representing cells in early-stage apoptosis viable cells. Quadrant Q1 is Annexin V-FITC negative and PI positive, representing dead cells. Quadrant Q2 is Annexin V-FITC and PI positive, representing cells in late-stage apoptosis. B, The percentage of early- and late-stage apoptotic cells is represented in the histogram. Data were representative of three independent experiments. **P < 0.01 compared with the blank control in the normoxia treatment group; ## P < 0.01 compared with the blank control in the hypoxia group
Fig 5: The proliferation of pulmonary arterial smooth muscle cell (PASMC) after transfection under normal or hypoxic conditions. Cell proliferation of PASMCs was measured, after transfection with miR-223-3p mimics or miR-223-3p inhibitor or si-ITGB3 or pcDNA-ITGB3 at 48 h. All experiments were performed in triplicate. **P < 0.01 compared with the blank control in the normoxia treatment group; ## P < 0.01 compared with the blank control in hypoxia group
Supplier Page from Abcam for Anti-Integrin beta 3 antibody [ITGB3/1713]