HSPB1 Regulates Autophagy and Apoptosis in Vascular Smooth Muscle Cells in Arteriosclerosis Obliterans

Figure 3

Tangeretin triggers autophagy in VSMCs. (a–c) VSMCs were treated with pCMV-Cmyc-HSPB1 and HSPB1-siRNA for 48 hr, followed by μg/ml ox-LDL for 24 hr. (a) The levels of LC3, p62, and β-actin were examined. (b) Levels of LC3-II relative to those of LC3-I. (c) Levels of p62 relative to those of β-actin. (d) VSMCs were treated with pCMV-Cmyc-HSPB1 for 48 hr, followed by 60 μg/ml ox-LDL for 24 hr, and then, TEM was used to observe autophagosomes. Scale bar, 5 μm (×1.2 k, left) and 1 μm (×6.0 k, right). (e, f) VSMCs were treated with pCMV-Cmyc-HSPB1 and HSPB1-siRNA for 48 hr. (e) Cell lysates were harvested to examine the protein levels of Beclin-1, HSPB1, Cmyc, and β-actin by Western blotting. (f) Densitometry was used to determine the fold change in the expression of Beclin-1 relative to the expression of β-actin. (g–i) VSMCs were treated with pCMV-Cmyc-HSPB1 and HSPB1-siRNA for 48 hr. Then, they were pretreated with 100 μM corynoxine B and 1 μM neriifolin for 2 h before being treated with 60 μg/ml ox-LDL. (g) Cell lysates were harvested to examine the protein levels of LC3, p62, and β-actin by Western blotting. (h) Levels of LC3-II relative to those of LC3-I. (i) Levels of p62 relative to those of β-actin ( for each group; , , and ).

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