1. Arterioscler Thromb Vasc Biol
Two-Dimensional Fluorescence In-Gel Electrophoresis of Coronary Restenosis Tissues in Minipigs
by Lin Lu, Ya Nan Wang, Wei Hua Sun, Zhu Hui Liu, Qi Zhang, Li Jin Pu, Ke Yang, Ling Jie Wang, Zhen Bin Zhu, Hua Meng, Ping Yang, Run Du, Qiu Jing Chen, Li Shun Wang, Hong Yu, and Wei Feng Shen
Arterioscler Thromb Vasc Biol
Volume 33(3):
February 13, 2013
Copyright © American Heart Association, Inc. All rights reserved.

2. Representative 2-dimensional fluorescence in-gel electrophoresis (2D-DIGE) images of protein profiles of in-stent restenosis (ISR) intima tissue in minipigs.
Representative 2-dimensional fluorescence in-gel electrophoresis (2D-DIGE) images of protein profiles of in-stent restenosis (ISR) intima tissue in minipigs. A, The upregulated protein spots in ISR tissue than in non-ISR tissue in diabetic minipigs (≥1.5-fold increase). B, The upregulated protein spots in ISR tissue than in non-ISR tissue in nondiabetic minipigs (≥1.5-fold increase).
Lin Lu et al. Arterioscler Thromb Vasc Biol. 2013;33:
Copyright © American Heart Association, Inc. All rights reserved.

3. Assessment of adipocyte fatty acid binding protein (AFABP) protein levels in in-stent restenosis (ISR) and non-ISR intima tissue.
Assessment of adipocyte fatty acid binding protein (AFABP) protein levels in in-stent restenosis (ISR) and non-ISR intima tissue. A, The AFABP level was examined by Western blot in ISR and non-ISR tissue in both diabetic and nondiabetic minipigs. B, Quantification of data in A. C, Induction of AFABP by high-glucose and advanced glycation end products-bovine serum albumin (AGE-BSA) (25, 50, and 100 ug/mL) was examined in human arterial smooth muscle cells (hASMCs) by Western blot. D, Quantification of the data in C. *P<0.001, vs low-glucose; and #P<0.05, vs high-glucose. E, Induction of AFABP by rapamycin was analyzed in hASMCs by Western blot. F, Quantification of the data in E. *P<0.05, vs low-glucose; and #P<0.05, vs high-glucose.
Lin Lu et al. Arterioscler Thromb Vasc Biol. 2013;33:
Copyright © American Heart Association, Inc. All rights reserved.

4. Overexpression of adipocyte fatty acid binding protein (AFABP) alters phenotypes, and promotes proliferation and migration in human arterial smooth muscle cells (hASMCs).
Overexpression of adipocyte fatty acid binding protein (AFABP) alters phenotypes, and promotes proliferation and migration in human arterial smooth muscle cells (hASMCs). A, Expression of AFABP was examined in hASMCs after retroviral infection by Western blot. B, Contractile, synthetic, migratory, and inflammatory proteins were characterized by Western blot in stable hASMCs of vector, AFABP, and shRNA–AFABP overexpression. C, Stress fiber formation was examined in stable hASMCs stained by rhodamine phalloitin. D, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed in hASMCs with and without diphenyleneiodonium (DPI) (1 μmol/L). E, Wound-healing assay was performed. After overnight starvation, the cells were grown with and without DPI (1 μmol/L). The images were taken at 0 hours before and 24 hours after cell scratch. F, Quantification of the data in E. *P<0.05, vs low-glucose medium; #P<0.05, vs pLXSN-vector in low-glucose medium; &P<0.05, vs pLXSN-vector in high-glucose medium; vs pLXSN-AFABP in low-glucose and high-glucose medium, respectively. G, Boyden transwell assay was performed. After starvation, the cells were grown in low- and high-glucose medium with and without DPI (1 μmol/L). Images were taken after 4 hours. H, Quantification of the data in G. *P<0.05, vs low-glucose medium; #P<0.05, vs pLXSN-vector in low-glucose medium; &P<0.05, vs pLXSN-vector in high-glucose medium; vs pLXSN-AFABP in low- and high-glucose medium, respectively.
Lin Lu et al. Arterioscler Thromb Vasc Biol. 2013;33:
Copyright © American Heart Association, Inc. All rights reserved.

5. Overexpression of adipocyte fatty acid binding protein (AFABP) causes reactive oxygen species (ROS) production in human arterial smooth muscle cells (hASMCs).
Overexpression of adipocyte fatty acid binding protein (AFABP) causes reactive oxygen species (ROS) production in human arterial smooth muscle cells (hASMCs). A, Intracellular ROS was examined after incubation with 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate for 60 minutes. The images were taken immediately. B, After procedure in A, the cells were harvested and dichlorodihydrofluorescein (DCF) fluorescence intensity was detected using a flow cytometer. *P<0.05, vs low-glucose medium; #P<0.01, vs pLXSN-vector in low-glucose medium; &P<0.01, vs pLXSN-vector in high-glucose vs pLXSN-AFABP in low-glucose and high-glucose medium, respectively; and $P<0.05, vs pLXSN-vector. C, NADPH oxidase activities were detected in cells using a cell-free method. *P<0.05, vs low-glucose medium; !P<0.05, vs pLXSN-vector in low-glucose medium; #P<0.001, vs pLXSN-vector in low-glucose medium; &P<0.01, vs pLXSN-vector in high-glucose vs pLXSN-AFABP in low-glucose and high-glucose culture, respectively; and $P<0.05, vs pLXSN-vector.
Lin Lu et al. Arterioscler Thromb Vasc Biol. 2013;33:
Copyright © American Heart Association, Inc. All rights reserved.

6. Pathway determination for NADPH oxidase subunits (Nox1, Nox4, and P22) activation.
Pathway determination for NADPH oxidase subunits (Nox1, Nox4, and P22) activation. A through C, The human arterial smooth muscle cells (hASMCs) were infected with retroviral vector and adipocyte fatty acid binding protein (AFABP). After 12 hours, the cells were treated with pathway inhibitors SB (10 μmol/L), PD98059 (10 μmol/L), wortmannin (0.2 μmol/L), LY (10 μmol/L), Akt inhibitor IV (1 μmol/L), MyD88 inhibitor (100 μmol/L), control peptide (100 μmol/L), MyD88–siRNA-1 (10 μmol/L), and RalGDS–siRNA-1 (10 μmol/L) for 24 hours before harvest. Then, total RNA was isolated and real-time PCR was performed to determine mRNA levels of Nox1, Nox4, and P22. *P<0.05, vs pLXSN-vector. D, The protein levels of Nox1, Nox4, and P22 were evaluated in cells of procedure A by Western blot. E and F, The protein levels of RalGDS and MyD88 were detected in cells of procedure A.
Lin Lu et al. Arterioscler Thromb Vasc Biol. 2013;33:
Copyright © American Heart Association, Inc. All rights reserved.

7. Secretory adipocyte fatty acid binding protein (AFABP) promotes growth and migration of human arterial smooth muscle cells (hASMCs).
Secretory adipocyte fatty acid binding protein (AFABP) promotes growth and migration of human arterial smooth muscle cells (hASMCs). A, Stable hASMCs expressing vector, AFABP, and shRNA–AFABP were subjected to M199 medium with 1% FCS. After 24 hours, the supernatant was collected and AFABP level was examined by Western blot. B, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed in hASMCs treated by supernatant from stable hASMCs-expressing vector, AFABP, and shRNA–AFABP with and without AFABP antibody (abcam) and diphenyleneiodonium (DPI). *P<0.05, vs supernatant from vector-expressing hASMCs; and #P<0.05, vs supernatant from AFABP-expressing hASMCs. C, Wound-healing assay was performed in hASMCs treated by abovementioned supernatant with and without AFABP antibody (abcam) and DPI after starvation. The images were taken at 0 hours before and 24 hours after cell scratch. D, Quantification of the data in C. *P<0.05, vs supernatant from vector-expressing hASMCs; and #P<0.05, vs supernatant from AFABP-expressing hASMCs. E, Boyden transwell assay was done in hASMCs treated by abovementioned supernatant with and without AFABP antibody (abcam) and DPI. The images were taken after 4 hours. F, Quantification of the data in E. *P<0.05, vs supernatant from vector-expressing hASMCs; and #P<0.05, vs supernatant from AFABP-expressing hASMCs. G, Recombinant AFABP-induced viability of hASMCs in M199 medium with 1% FCS was analyzed by MTT assay. *P<0.05, vs control; and #P<0.05, vs AFABP of 10 ug/mL. H, After overnight starvation, hASMCs was induced by recombinant AFABP in M199 medium with 1% FCS, and wound-healing assay was performed. I, Quantification of the data in H. *P<0.05, vs control; and #P<0.05, vs AFABP of 10 ug/mL. J, Boyden chamber assay by recombinant AFABP was performed in hASMCs. K, Quantification of the data in J. *P<0.05, vs control; and #P<0.05, vs AFABP of 10 ug/mL. L, Western blot was done to examine the protein levels of cyclin D1, MMP-2, and MCP-1 in hASMCs treated by control, recombinant AFABP, and supernatant from stable hASMCs.
Lin Lu et al. Arterioscler Thromb Vasc Biol. 2013;33:
Copyright © American Heart Association, Inc. All rights reserved.