Fig 2: Effect of ADRP knockdown on the ERK and Akt signaling pathways. At 48 h after siRNA transfection, VSMCs were exposed to PDGF for 0, 15, 30 and 60 min. The protein expression of ERK, p-ERK, Akt and p-Akt was subsequently examined by western blotting. Representative images are presented. Data are presented as the mean ± standard deviation. **P<0.01, as indicated by brackets. ADRP, adipose differentiate-related protein; ERK, extracellular signal-regulated kinase; siRNA, small interfering RNA; VSMCs, vascular smooth muscle cells; PDGF, platelet-derived growth factor; p-, phosphorylated; siCtrl, control siRNA; siADRP, siRNA targeting ADRP.

Fig 3: Effect of ADRP knockdown on neointima formation in apoE-/-mice. After establishing an in vivo model of atherosclerosis, the mice were injected with siCtr or siADRP twice a week. After 3 weeks of siRNA injection, aortas were obtained. (A) ADRP mRNA levels were examined by reverse transcription-quantitative polymerase chain reaction. (B) ADRP protein levels were determined by western blotting. ß-actin served as an internal control. (C) Aortas were subjected to hematoxylin and eosin staining, and lumen area, neointima area, media area and neointima/media area ratios were measured to evaluate neointima formation in mice. Scale bar, 50 µm. Data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01, as indicated by brackets. ADRP, adipose differentiation-related protein; apoE, apolipoprotein E; siRNA, small interfering RNA; siCtr, control siRNA; siADRP, siRNA targeting ADRP.

Fig 4: Analysis of gene expression in differentiated C2C12 cells. (A) RT-sqPCR and (B) densitometry analysis of ESR1, ESR2, CD36, PPARa, ACACA, PLIN2 and FASN expression in C2C12 cells. (C) Western blot and (D) densitometry analyses of CD36, PPARa, PLIN2, FASN, p-ACACA and ACACA in C2C12. (E) Western blot and (F) densitometry analyses of p-AKT, AKT, p-MAPK8 and MAPK8 in C2C12. Data are presented as the mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001 vs. CON group; #P<0.05, ##P<0.01 vs. PA + E2; &P<0.05, &&P<0.01, vs. PA + PPT; $P<0.5 vs. PA + DPN. RT-sqPCR, reverse transcription-semi-quantitative polymerase chain reaction; PLIN2, perilipin 2; CD36, CD36 molecule; ACACA, acetyl-CoA carboxylase a; FASN, fatty acid synthase; PPARa, peroxisome proliferator activated receptor a; ESR, estrogen receptor; OVX, ovariectomy; E2, 17ß-estradiol; p-, phosphorylated; MAPK, mitogen-activated protein kinase; AKT, AKT serine/threonine kinase; CON, control; PA, palmitic acid; PPT, propylpyrazoletriol; DPN, diarylpropionitrile; EtOH, ethanol; M, marker.

Fig 5: Effect of ADRP knockdown on VSMC migration induced by PDGF. (A) Cell migration was determined by wound healing assay at 12 and 24 h (magnification, ×100). (B) Cell migration ability was confirmed by Transwell migration assay (magnification, ×200). VSMCs were incubated for 24 h with PDGF at 24 h post-transfection. The protein levels and activities of MMP-2 and MMP-9 were examined by (C) western blotting and (D) gelatin zymography. Data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01, as indicated by brackets. ADRP, adipose differentiation-related protein; VSMCs, vascular smooth muscle cells; PDGF, platelet-derived growth factor; MMP, matrix metalloproteinase; siRNA, small interfering RNA: siCtr, control siRNA; siADRP, siRNA targeting ADRP.