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Fig. 3.

Effects of autophagy via microtubule regulation and effects of caspase-3 cleavage via autophagy regulation in PDGF-BB-stimulated VSMCs.

Serum-starved VSMCs were incubated with 1 µM paclitaxel, 0.2 µM vinorelbine, 5 mM 3-MA (an autophagy inhibitor), or 0.2 µM rapamycin (an autophagy stimulator) for 24 h followed by 25 ng/ml PDGF-BB treatment for 0–48 h. Additionally, the cells were treated with 0.1 µM bafilomycin A1 for 4 h before the end of the reaction. Cell lysates applied to SDS-PAGE and subsequently immunoblotted. The band densities were normalized to those of β-actin. The gel images shown are representative of those obtained from three or five independent experiments. Data shown in graphs for relative density are expressed as means±SEM. (A) The conversion of LC3-I to LC3-II was measured as an autophagy key marker in microtubule-regulated VSMCs before and after (24–48 h) PDGF-BB treatment. Mean values of the vehicle group (0.1% DMSO) were set to 1 fold. **p<0.01 vs. vehicle. (B, C) Measurement of autophagic flux. Bafilomycin A1 (0.1 µM) was added to the vehicle (0.1% DMSO)-treated, microtubule-regulated (paclitaxel or vinorelbine), or PDGF-BB-stimulated VSMCs 4 h prior to harvest, and accumulation of LC3-II was measured by western blotting. The mean value of vehicle was set to 1 fold. *p<0.05, **p<0.01 vs. the indicated group. (D) The changes of apoptosis by autophagy regulation were tested by the measurement of caspase-3 cleavage levels. Mean values of the only rapamycin-treated group were set to 1 fold.

Korean J Physiol Pharmacol 2018;22:349-360 https://doi.org/10.4196/kjpp.2018.22.3.349
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