Figure 1.
Effect of verteporfin on p62 in cells.
(A) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO, 100 nM bafilomycin A1, or 10 µM verteporfin for 8 h in the presence or absence of serum and cell lysates were immunoblotted for p62. β-tubulin was monitored as a loading control. (B) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO or 10 µM verteporfin for 4 h. Indicated amounts of each lysate were immunoprecipitated with anti-p62 antibody and analyzed by western blot. (C) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO, 10 µM verteporfin, or 100 nM bafilomycin A1 for 4 h in complete medium. The cells were fixed and stained with p62 antibody, and images were acquired by confocal microscopy. Scale bar, 10 µm. (D) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO, 100 nM bafilomycin A1, or 10 µM verteporfin for 4 h in complete medium. Cell lysates were collected, quantified, and normalized in the presence or absence of overhead laboratory light as indicated. 0.5 µg of lysate was used to examine p62 levels by western blotting. All images presented are representative of at least 3 independent experiments.
Figure 2.
Effect of verteporfin on p62 in vitro.
(A) Equal amounts of untreated BxPC-3 cell lysate were exposed to 10 µM verteporfin in the presence or absence of light at 4°C or 37°C for 30 min. (B) p62 was immunoprecipitated from untreated BxPC-3 cells. The immunoprecipitated material was then treated for 30 min in lysis buffer with 10 µM verteporfin in the presence or absence of light at 4°C or 37°C. (C) 100 ng purified GST-p62 was exposed to 10 µM verteporfin for 1 h at 37°C in the absence or presence of light. The above reactions were all immunoblotted for p62. All images presented are representative of at least 3 independent experiments.
Figure 3.
High-molecular weight p62 induced by singlet oxygen production.
(A) 50 ng GST-p62 was incubated with 0.05% DMSO, 5 µM verteporfin, or 5 µM rose bengal for 1 h at 4°C or 37°C in the dark. (B) Immunoprecipitated p62 from untreated BxPC-3 cells was exposed to 0.1% DMSO, 10 µM verteporfin, or 10 µM rose bengal for 30 min in the dark at 4°C or 37°C or to (C) 0.1% DMSO, 10 µM verteporfin, or 10 µM rose bengal in the dark at 37°C with or without 20 mM histidine. Reactions were immunoblotted for p62. (D) 26 ng purified His-p62 was incubated with 0.05% DMSO, 5 µM verteporfin, or 5 µM rose bengal for 1 h at 4°C or 37°C in the dark or (E) 50 ng purified GST-p62 was incubated with 0.1% DMSO, 10 µM verteporfin, or 10 µM rose bengal for 30 min at 37°C in the dark. Samples were derivatized with DNPH, subjected to SDS-PAGE, and immunoblotted with anti-DNP antibody solution according to the Oxyblot kit manufacturer’s instructions. All images presented are representative of at least 3 independent experiments.
Figure 4.
Effect of different ROS sources on p62 in vitro.
50 ng purified GST-p62 was exposed to different molar ratios of (A) NaOCl or (B) H2O2 for 1 h at 37°C, or to different concentrations of (C) peroxynitrite for 5 min at room temperature or (D) DEA/NONOate for 20 min at room temperature. All reactions were immunoblotted for p62. All images presented are representative of at least 3 independent experiments.
Figure 5.
Effect of rose bengal on autophagosome accumulation and p62 in cells.
MCF-7 EGFP-LC3 cells were exposed for 4 h to 0.1% DMSO, 10 µM verteporfin (VP), or different concentrations of rose bengal (RB) in the presence or absence of serum. (A) Cell lysates were immunoblotted for p62 and β-tubulin. This image is representative of 2 independent experiments. (B) Cells were fixed and stained with Hoechst 33342 and punctate EGFP-LC3 fluorescence was visualized and (C) quantified using a CellomicsVTI automated fluorescence microscope (**p<0.01, Student’s t-test) (mean±S.D. (error bars), n = 3).
Figure 6.
Effect of verteporfin-induced high-MW p62 on its association with polyubiquitinated proteins and LC3.
(A) MCF-7 EGFP-LC3 or (B) BxPC-3 cells were exposed for 4 h to 0.1% DMSO or 10 µM verteporfin in complete medium. p62 was immunoprecipitated and the bound polyubiquitinated proteins were detected using an anti-(Ub)n antibody. Immunoprecipitation was confirmed by western blot for p62. (C) Densitometry analysis was performed on the images presented in A and B using Quantity One software. (D) Using the same lysates prepared in (A) and (B), EGFP-LC3 was immunoprecipitated and bound p62 was detected in the IP fraction using an anti-GFP antibody. Immunoprecipitation was confirmed by western blot for GFP. Images presented for MCF-7 EGFP-LC3 cells are representative of at least 3 independent experiments. Densitometry was done using images from 2 of those experiments (mean ± S.D., n = 2) where the image quality was suitable for quantification. Images presented for BxPC-3 cells are representative of 2 independent experiments and densitometry was done using images from 1 experiment where the image quality was suitable for quantification.
Figure 7.
Effect of PB1 mutation on p62 crosslinking by verteporfin.
p62−/− MEF cells expressing GFP-p62 wt or GFP-p62 K7A/D69 or p62+/+ MEF cells were exposed to 0.1% DMSO or 10 µM verteporfin for 4 h in complete medium. Cell lysates were immunoblotted for p62 and β-tubulin. The image presented is representative of at least 3 independent experiments.
Figure 8.
Proposed model for verteporfin-mediated inhibition of autophagosome formation involving p62 crosslink products.
As p62 oligomers are recruited to the autophagosome membrane, they become oxidized and crosslinked to each other due to low-level singlet oxygen generation by verteporfin. This crosslinking event interferes with p62 binding to polyubiquitinated cargo, but does not affect LC3 binding. The generation of large p62 crosslink products with impaired function either physically disrupts proper autophagosome elongation and closure or it interferes with the function of other molecules necessary for completely autophagosome formation.