Fig 2: The effect of Pim-1 on cell apoptosis under HR stimulation(A) Quantitative PCR analysis of Pim-1 in MIHA cells subjected to control conditions or hypoxia–reoxygenation (HR) challenge 12 h postreperfusion (n = 3). ∗∗∗p < 0.001. Student’s t test was used.(B) Western blot analysis of Pim-1, caspase 3, cleaved caspase 3, Bax and Bcl-2 proteins in MIHA cells stably overexpressing Pim-1 and their control cells under normoxia or HR challenge 12 h postreperfusion (n = 3). β-Actin was used as a loading control. ∗∗∗p < 0.001. Student’s t test was used. The data were normalized to the expression levels of β-actin.(C) Representative images from a TUNEL assay and the proportions of apoptosis in MIHA cells stably overexpressing Pim-1 and their control cells under normoxia or hypoxia–reoxygenation challenge 12 h postreperfusion (n = 3). ∗∗∗p < 0.001 vs. control. Student’s t test was used.(D) Quantitative PCR analysis of Pim-1 in MIHA cells with Pim-1 knockdown and control cells under normoxia or HR challenge at 12 h postreperfusion (n = 3). ∗∗∗p < 0.001. Student’s t test was used.(E) Western blot analysis of Pim-1, caspase 3, cleaved caspase 3, Bax and Bcl-2 proteins in MIHA cells with Pim-1 knockdown and control cells under normoxia or hypoxia–reoxygenation challenge 12 h postreperfusion (n = 3). β-Actin was used as a loading control. ∗∗∗p < 0.001. Student’s t test was used. The data were normalized to the expression levels of β-actin.(F) Representative images of a TUNEL assay and the proportions of apoptosis in MIHA cells with Pim-1 knockdown, control cells, and ectopically overexpression of Pim-1 from Pim-1 knockdown under normoxia or hypoxia–reoxygenation challenge 12 h postreperfusion (n = 3). ∗∗∗p < 0.001 vs. control. Student’s t test was used. All data are presented as the means ± SDs. HR, hypoxia–reoxygenation.

Fig 3: Pim-1 overexpression effectively reversed the inhibitory role of miR-214 on PDGF-BB-induced cell migration in smooth muscle cells. (A) The Pim-1 protein expression level in A7r5 cells transfected with pcDNA3.1 or pcDNA-Pim-1 for 24 h or 48 h and detected by western blot. (B) The relative miR-214 expression level in smooth muscle cells stimulated by PDGF-BB after co-transfecting with miR-214 mimics or NC mimics and pcDNA3.1 or pcDNA-Pim-1 was measured by qPCR. (C) The Pim-1 protein expression level in A7r5 cells treated as in B was measured by western blot. (D) Transwell assay was used to detect the cell migration in A7r5 cells treated as in (B) with crystal violet staining; ×100 magnification. Data are presented as mean ± SD. The data are derived from one of the 3 independent experiments. *, P<0.05; **, P<0.01. PDGF-BB, platelet-derived growth factor-BB; NC, negative control; qPCR, quantitative polymerase chain reaction; SD, standard deviation.

Fig 4: miR-214 impaired the role of PDGF-BB on Pim-1 expression in smooth muscle cells. Smooth muscle cell line A7r5 with PDGF-BB treatment or miR-214 mimics transfection alone or in combined. (A) The relative miR-214 expression levels were measured by qPCR. (B) The protein expression level of Pim-1 was measured by western blot. (C) Flow cytometry was used to detect the cell cycle distribution. Data are presented as mean ± SD. The data shown are representative results of 3 independent experiments. **, P<0.01. PDGF-BB, platelet-derived growth factor-BB; qPCR, quantitative polymerase chain reaction; SD, standard deviation.

Fig 5: PDGF-BB promoted cell migration and Pim-1 expression in smooth muscle cells. Smooth muscle cell line A7r5 were treated with indicated concentrations of PDGF-BB for 24 h. (A) Transwell assay was performed to measure the cell migration potential with crystal violet staining; ×40 magnification; (B) qPCR assay was used to detect the Pim-1 mRNA expression. (C) The protein expression level of Pim-1 was detected by western blot after cells were treated with PDGF-BB. Data are presented as mean ± SD. The data shown are representative results of 3 independent experiments. **, P<0.01. PDGF-BB, platelet-derived growth factor-BB; qPCR, quantitative polymerase chain reaction; mRNA, messenger RNA; SD, standard deviation.