Effect of verteporfin-induced high-MW p62 on its association with polyubiquitinated proteins and LC3.

<p>(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.</p

Effect of verteporfin on p62 <i>in vitro</i>.

<p>(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.</p

Effect of different ROS sources on p62 <i>in vitro</i>.

<p>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.</p

Effect of verteporfin on p62 in cells.

<p>(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.</p

Effect of PB1 mutation on p62 crosslinking by verteporfin.

<p>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.</p

Effect of rose bengal on autophagosome accumulation and p62 in cells.

<p>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 Cellomics^VTI automated fluorescence microscope (**p<0.01, Student’s t-test) (mean±S.D. (error bars), n = 3).</p

Proposed model for verteporfin-mediated inhibition of autophagosome formation involving p62 crosslink products.

<p>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.</p

Hortonones A to C, Hydroazulenones from the Genus <i>Hortonia</i>

The new hexahydroazulenones hortonones A (<b>1</b>) to C (<b>3</b>) were isolated from the leaves of three representative species of the endemic Sri Lankan genus <i>Hortonia</i> that belongs to the family Monimiaceae. Hortonones A (<b>1</b>) and B (<b>2</b>) have the unprecedented rearranged hortonane sesquiterpenoid carbon skeleton, and hortonone C (<b>3</b>) has the unprecedented rearranged and degraded 13-norhortonane skeleton. Hortonone C (<b>3</b>) exhibited in vitro cytotoxicity against human breast cancer MCF-7 cells at 5 μg/mL

Time-course and reversibility of mTORC1 inhibition and EGFP-LC3 processing by NTZ and TIZ.

<p>(A) Cells were incubated with 10 µM NTZ or 10 µM TIZ for the indicated times. (B) Cells were incubated with 10 µM NTZ, 10 µM TIZ or 30 nM rapamycin for 4 h. The drugs were then washed away and cells were incubated in drug-free medium for the indicated times post-washout. Cell lystates were immunoblotted as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002691#ppat-1002691-g002" target="_blank">Figure 2</a>.</p

Increased EGFP-LC3 processing and inhibition of mTORC1 signaling by NTZ and TIZ.

<p>Cells were treated with the indicated concentrations of NTZ, TIZ or rapamycin for 4 h (A) or 24 h (B). In panel C, cells were treated with 10 µM NTZ, 10 µM TIZ, 30 nM rapamycin, or DMSO without or with 0.1 µM bafilomycin A1 for 4 h. EGFP-LC3 processing was examined by immunoblotting with antibodies against GFP, mTORC1 activity using antisera against phospho-S6K Thr³⁸⁹ and total S6K, and mTORC2 activity with antisera against phospho-AKT Ser⁴⁷³ and total AKT. Total AKT was also used as a loading control.</p