Bafilomycin A1 (Baf) reversed the protective effect of LA against HNE or VK3 in ARPE-19 cells.

<p>Cultures were pre-treated with LA and the indicated concentrations of Baf (30∼300 nM) for 18 h before 18 h exposure to HNE (15 µM) (<b>A</b>) or VK3 (20 µM) (<b>B</b>). MTS assay was used to measure cell viability (<b>A</b>, <b>B</b>) and caspase-3 activity assay to measure apoptosis (<b>C</b>) at the end of the 18 h HNE or VK3 treatment. In MTS assay, *P<0.05 <i>vs</i>. control, ** P<0.05 indicated that the three combinatorial treatment including HNE or VK3, LA, and Baf differed significantly from cultures treated by HNE plus LA or VK3 plus LA; in caspase-3 assay, ** P<0.05 <i>vs</i>. control, ***P, *P <0.05 indicated that the three combinatorial treatment including VK3, LA, and Baf differed significantly from either VK3 or VK3 plus LA treatment respectively. <b>D</b>, ARPE-19 cells were treated by Baf (300 nM), LA (1 µM), Epo (10 nM), LA plus Baf, or Epo plus Baf for 18 h, and then subjected to chymotrypsin-like proteasome activity assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103364#s2" target="_blank">Methods</a>. *P<0.05 indicated significant difference between LA and LA plus Baf or between Epo and Epo plus Baf treatment; **P<0.05 indicated significant difference between control and treatment conditions except by Baf. All the values in control cultures (<b>A</b>, <b>B</b>,<b>C</b>,<b>D</b>) were set at 100% and the values in treated cultures were normalized to the control values. All the results shown are mean (± SEM) of at least triplicate experiments in quadruplicate cultures.