Figure 1.
LA or Epo increased protein levels of ATG5, ATG7, and the conversion of LC3-I to LC3-II.
A, ARPE-19 cells were treated with Epo (0.3∼10 nM, left panel) or LA (100-1000 nM, right panel) for 18-24 h, and proteins were harvested and subjected to immunoblotting for ATG5, ATG7, LC3 and GAPDH. The blots shown were typical of at least triplicate experiments. The ratios of ATG5/GAPDH, ATG7/GAPHH, and LC3-II/GAPDH for Epo (B) or LA treatment (C) were mean (+ SEM) of at least triplicate experiments. The ratios for control were set as 100% and the values from treatment conditions were normalized to the control values. P<0.05 vs control.
Figure 2.
LA or Epo induced autophagic flux.
A, ARPE-19 cells were treated with DMSO, Epo (10 nM), or LA (1 µM) for 18 h and Baf (400 nM) was added to the cultures for the final 4 h. Proteins were harvested and subjected to immunoblotting against LC3. The blots shown are typical of at least triplicate experiments. The optic densities were averaged and quantified in B, the values in control were set as 100% and the values in treated conditions were normalized to the control values. * P<0.05 vs control; **P<0.05 indicated that Epo or LA plus Baf differed significantly from Epo or LA treatments. C, ARPE-19 cells were treated with DMSO, Epo (10 nM), or LA (1 µM) for 18 h, and labeled with fluorescence as described in Methods, and imaged by confocal laser microscope. The images shown were typical of the images from five non-contiguous fields in each dish from triplicate experiments. Scale bar, 20 µM. The LC3-positive puncta overlaying labeled lysosome for Epo, or LA treatment, and sham condition were averaged from 20 cells and quantified in D. * P<0.05 vs control. Blue, DAPI-labeled nuclei; Red, lyso tracker-labeled lysosome; Green, FITC-labeled LC3. The merged images were shown in the most right column and the orange-stained cells indicated LC3, co-localized with lysosome.
Figure 3.
LA or Epo decreased phospho-AKT and phospho-mTOR protein levels.
ARPE-19 cells were treated with Epo (0.3∼10 nM) (A) or LA (100–1000 nM) (B) for 18–24 h, and proteins were harvested and subjected to immunoblotting for mTOR, phospho-mTOR(p-mTOR), AKT, phospho-AKT(p-AKT), p62. The optical density ratios (mTOR/GAPDH, AKT/GAPDH, p-mTOR/GAPDH, p-AKT/GAPDH, p62/GAPDH) for Epo (C) or LA treatment (D) were averaged from at least triplicate experiments and the ratios for the mTOR/GAPDH, AKT/GAPDH were not shown. The values for control were set as 100%; the values for treatment condition were normalized to the control values. *P <0.05 vs control.
Figure 4.
Bafilomycin A1 (Baf) reversed the protective effect of LA against HNE or VK3 in ARPE-19 cells.
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) (A) or VK3 (20 µM) (B). MTS assay was used to measure cell viability (A, B) and caspase-3 activity assay to measure apoptosis (C) at the end of the 18 h HNE or VK3 treatment. In MTS assay, *P<0.05 vs. 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 vs. 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. D, 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 Methods. *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 (A, B,C,D) 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.
Figure 5.
Bafilomycin A1 (Baf) reversed the protection of Epo against HNE or VK3.
Cultures were pre-treated with the indicated concentrations of Epo for 18 h before 18 h exposure to HNE (15 µM) (A) or VK3 (20 µM) (B); or the cultures were pre-treated with Epo (10 nM) and the indicated concentrations of Baf (30∼300 nM) for 18 h before 18 h exposure to HNE (15 µM) (C) or VK3 (20 µM) (D). MTS assay was used to measure cell viability at the end of the 18 h HNE or VK3 treatment. The results shown in A, B, C, and D are mean (± SEM) of at least three independent experiments in quadruplicate cultures. The values in the control cultures were set at 100% and the survivals in treated cultures were normalized to the control values. * P<0.05 vs. control, ** P<0.05 indicated that the three combinatorial treatment including HNE or VK3, Epo, Baf differed from either Epo plus VK3 or Epo plus HNE treatment.
Figure 6.
Knockdown of Atg7 attenuated the protective effect of LA or Epo.
ARPE-19 cells were transfected with scramble siRNA (SCR), or Atg7-specific siRNA (SiATG7), continued to be cultured for 24 h, followed by pre-treatments with Epo (10 nM, C) or LA (1 µM, D), or sham treatment for 18–24 h, and then subjected to VK3 (20 µM) treatment for 18 h. After the 18 h VK3 treatment, the cultures were subjected to western blot analyses (A) or MTS assay (C, D). The knockdown effects by siRNA were quantified in B, *P<0.05 indicated significant difference between the knockdown effect of SiATG7 and SCR. In C, D, *P<0.05 indicated significant differences between LA or Epo treatment and LA or Epo plus VK3 treatments; ** P<0.05 indicated significant differences between the protective effects of LA or Epo treatment in SCR group and those in SiATG7 group. All the results were averaged from at least triplicate experiments and the values in control were set as 100% and the values in treated conditions were normalized to the control values.