---
title: "Ambient light during sample processing causes verteporfin-driven protein cross-linking"
id: "plos-one-17-ambient-light-drives-verteporfin-induced-protein-cross-linking-during-standard"
canonical_url: "https://medichelpline.com/clinical-feed/plos-one-17-ambient-light-drives-verteporfin-induced-protein-cross-linking-during-standard"
content_type: "clinical_feed_article"
specialty: "Pharmacology"
source_name: "PLOS ONE (Medicine)"
source_url: "https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0358654"
published_at: "2026-09-21T14:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# Ambient light during sample processing causes verteporfin-driven protein cross-linking
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/plos-one-17-ambient-light-drives-verteporfin-induced-protein-cross-linking-during-standard
- **Specialty:** [Pharmacology](https://medichelpline.com/clinical-feed/pharmacology.md)
- **Primary Source:** PLOS ONE (Medicine)
- **Source URL:** [Original Journal Publication](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0358654)
- **Published At:** 2026-09-21T14:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- Verteporfin (VP), a photosensitizing benzoporphyrin used clinically and as a purported light-independent **YAP**-TEAD inhibitor, can drive formation of **high-molecular-weight complexes (HMWCs)** during routine laboratory processing. - The study tested MEL270, HEK293, and MCF-7 cells treated with therapeutic and supra-therapeutic VP under four systematically varied light/dark conditions. - HMWC formation for **p62**, DIAP1, ROCK1, **YAP**, and phospho-YAP was driven principally by ambient-light exposure during post-lysis sample processing rather than light during cellular treatment. - VP-induced cross-linking occurs in cell-free lysates: lysates spiked with VP formed HMWCs within 1 hour of ambient-light exposure and accumulated over 6 hours. - Vehicle controls processed under identical ambient-light workflows showed no HMWC formation, indicating VP is required for the light-dependent effect. - Pre-treatment of lysates with the radical scavenger **N-acetylcysteine (NAC)** or the singlet oxygen quencher **L-histidine** markedly attenuated HMWC formation, implicating both radical-mediated (Type I) and **singlet oxygen**–mediated (Type II) photochemistry. - Anti-methionine sulfoxide immunoblots demonstrated light-dependent methionine oxidation that paralleled HMWC formation, consistent with singlet-oxygen attack on susceptible residues. - **YAP** and phospho-YAP were highly susceptible to photo-cross-linking, while **TEF1/TEAD1** was comparatively resistant, a pattern that can mimic selective YAP-TEAD inhibition on standard immunoblots. - Light exposure during lysis and processing contributed more to HMWC formation than light during the treatment phase, though some treatment-phase contribution was detected. - A single-experiment CCK-8 assay reported lower viability under light than darkness at 24 hours, consistent with immunoblot findings; the source notes this observation requires independent replication. - The findings support a light-artifact hypothesis for many reported VP effects in cell culture and have implications for interpretation of preclinical VP data and the design of clinical investigations. - Data availability, methods, and some viability details were provided in source repositories, but portions of the viability results are incomplete or truncated in the provided source text.
## Clinical Analysis & Structured Key Points
Ambient light drives verteporfin-induced protein cross-linking during standard laboratory sample processing | PLOS One Browse Subject Areas ? Click through the PLOS taxonomy to find articles in your field. For more information about PLOS Subject Areas, click here . Article Authors Metrics Comments Media Coverage Reader Comments Figures Figures Abstract Verteporfin (VP) is widely used as a light-independent pharmacologic inhibitor of the YAP-TEAD transcriptional complex, despite its potent photosensitizing properties. Here we show that VP-associated high–molecular–weight complexes and apparent depletion of key proteins on immunoblots can be substantially driven by ambient-light exposure during post-lysis handling, more than by light during cellular treatment. In this study, the hypothesis is tested independently using MEL270, HEK293, and MCF-7 cells treated with therapeutic and supra-therapeutic concentrations of VP under four systematically varied light/dark conditions. The results demonstrate that high-molecular-weight complex (HMWC) formation for p62, DIAP1, ROCK1, YAP, and phospho-YAP is driven principally by light exposure during sample processing rather than during cell treatment, occurs in cell-free lysates within 1 hour of ambient-light exposure, and is markedly attenuated by both N-acetylcysteine and L-histidine, consistent with contributions from both radical-mediated and singlet oxygen-mediated photochemistry. Notably, YAP and phospho-YAP are highly susceptible to photo-cross-linking, whereas transcription enhancer factor 1 (TEF1)/TEAD1 remains comparatively resistant, a pattern that mimics selective YAP-TEAD inhibition on standard immunoblots. In a single-experiment CCK-8 assay, viability was markedly lower under light than in darkness at 24 hours, an observation consistent with the immunoblot findings but requiring independent replication. These findings support the light-artifact hypothesis and may have implications for how preclinical data on VP are interpreted in the design of clinical investigations. Citation: Baroutis KG, Corrêa VSMC, Ntentakis D, Narimatsu T, Gragoudas ES, Miller JW, et al. (2026) Ambient light drives verteporfin-induced protein cross-linking during standard laboratory sample processing. PLoS One 21(9): e0358654. https://doi.org/10.1371/journal.pone.0358654 Editor: Sripathi M. Sureban, Regeneron Pharmaceuticals Inc, UNITED STATES OF AMERICA Received: March 10, 2026; Accepted: September 3, 2026; Published: September 21, 2026 Copyright: © 2026 Baroutis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All data files are available from the Zenodo repository ( https://doi.org/10.5281/zenodo.18913106) . The repository includes publication-ready figures, uncropped western blot images, raw CCK-8 plate reader data, compiled datasets, ANOVA analysis code and output, coomassie staining data, and a membrane-to-figure mapping guide. Funding: The author(s) received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. Introduction Verteporfin (VP) was approved by the U.S. Food and Drug Administration in 2000 for the treatment of subfoveal choroidal neovascularization associated with age-related macular degeneration (AMD) [ 1 , 2 ]. In the first step of photodynamic therapy (PDT), liposomal VP (Visudyne) is administered intravenously and, after a quarter of an hour, accumulates in the retinal vasculature and is activated by a semiconductor red-light diode laser at 689 nm [ 2 ], generating reactive oxygen species [ 1 ], which cause thrombotic occlusion of the targeted neo-vessels [ 3 ]. Although intravitreal injections targeting vascular endothelial growth factor have become the first-line treatment of neovascular AMD, PDT with VP remains an important treatment for polypoidal choroidal vasculopathy, chronic central serous chorioretinopathy, and other chorioretinal conditions [ 3 , 4 ]. Moreover, VP is a small-molecule inhibitor of the Yes-associated protein (YAP)-TEA domain (TEAD) transcriptional interaction, thereby extending its utility beyond PDT [ 5 ]. YAP is a central effector of the Salvador-Warts-Hippo signaling pathway, also known as the Hippo pathway [ 6 ], which controls organ size, tissue homeostasis, and cell proliferation and, when dysregulated, drives the transcription of pro-proliferative and anti-apoptotic genes in many cancers [ 7 ]. Initial studies claimed that VP directly binds to YAP and inhibits the YAP-TEAD interaction, independently of light activation [ 8 ]. Subsequent studies have indicated further mechanisms, including sequestration of YAP in the cytoplasm via 14-3-3σ [ 9 , 10 ]. This has led to extensive preclinical studies of VP as the prototypical pharmacological YAP inhibitor in a variety of cancers, including glioblastoma, breast, bladder, and pancreatic cancers [ 11 – 14 ]. Here, we tested the hypothesis that ambient light exposure during routine sample processing is a primary determinant of VP-associated protein cross-linking and apparent YAP depletion. VP was also identified as an autophagy inhibitor. The selective autophagy receptor p62/SQSTM1 is covalently cross-linked into high-molecular-weight complexes (HMWCs) [ 15 , 16 ]. This protein aggregation extends well beyond p62: Zhang et al. reported broad-spectrum proteotoxicity independent of YAP1 expression [ 17 ], and Condurat et al. showed that VP-induced proteotoxicity was not abolished in CRISPR-mediated YAP/TAZ-knockout cells [ 18 ]. VP was classified as a covalent protein polymerizer with non-specific cross-linking activity [ 19 ]. In vivo data on VP-induced antiproliferative effects were equivocal [ 20 , 21 ]. This major discrepancy between high in vitro cytotoxic activity and weak in vivo effects of VP raises a critical mechanistic question: is the effect attributed to VP in cell culture confounded by an uncontrolled experimental variable? The photochemistry of porphyrin derivatives provides a well-established explanation for this discrepancy. Benzoporphyrins generate singlet oxygen upon exposure to visible light. Singlet oxygen oxidizes susceptible amino acids such as methionine, histidine, tryptophan, cysteine, and tyrosine, resulting in covalent cross-links between polypeptides [ 22 , 23 ]. Fancy and Kodadek took advantage of this photochemistry to map protein-protein interactions and showed that even a short exposure to light in the presence of porphyrin-treated samples leads to extensive covalent oligomerization [ 24 , 25 ]. In a recent study, Jiang et al. showed that singlet oxygen also oxidizes disulfide bonds, resulting in the formation of thiosulfinate intermediates that cross-link polypeptides [ 26 ]. Together, these data suggest that the high-molecular-weight complexes, apparent YAP depletion, and cytotoxic effects observed in VP-treated cells may be photochemical artifacts rather than a true pharmacological effect of VP. This hypothesis was tested directly in a study, which reported that VP-induced formation of cross-linked oligomers and high-molecular-weight complexes is mediated by light and causes cellular toxicity. Its retraction [ 27 ] removed from the peer-reviewed literature the primary experimental support for the light-artifact hypothesis, leaving this hypothesis resting only on the general photochemistry of porphyrin derivatives, thus requiring independent re-evaluation [ 22 – 26 ]. We do not rely on any of its data here. Since VP is now being tested in clinical trials as a systemic anticancer drug [ 28 ], and the next-generation TEAD-specific inhibitors are also being tested in clinical trials [ 29 ], it has become urgent to clarify whether the reported effects of VP on YAP and other targets are real pharmacological responses or merely photochemical artifacts. In the present study, we directly tested this hypothesis by treating three cell lines with VP at therapeutic and supra-therapeutic concentrations under four systematically varied light/dark conditions. The results of this study, which were obtained independently, provide new primary evidence for a hypothesis that currently lacks such evidence and may have an impact on how preclinical VP data are generated and interpreted, including the design of ongoing clinical investigations. Results Ambient light during sample processing drives VP-dependent HMWC formation To determine whether ambient light mediates VP-induced protein cross-linking, MEL270, HEK293, and MCF-7 cells were treated with vehicle, low-dose (LD; 1.25 µg/mL), or high-dose (HD; 7.5 µg/mL) VP under four systematically varied light/dark conditions. Western blot analysis of p62, DIAP1, and ROCK1 revealed that HMWC formation was determined principally by light exposure during the sample-processing phase. Cells treated in darkness and subsequently lysed under ambient light (condition ii: dark treatment/light lysis) exhibited prominent HMWC bands for all three proteins, whereas matched samples processed entirely in darkness (condition iv: complete dark) displayed only monomeric bands at the expected molecular weights ( Fig 1 ). The p62 immunoblots showed the most conspicuous cross-linked species, migrating above 120 kDa, with a concomitant reduction in the monomeric 62-kDa band. DIAP1 and ROCK1 similarly exhibited HMWC bands above 150 kDa exclusively in samples processed under ambient light. This pattern was consistent across all three cell lines. β-actin loading controls confirmed equivalent protein loading across all conditions. Vehicle-treated samples carried through the identical ambient-light workflow showed no HMWC formation and preserved monomeric bands for every protein examined, indicating that ambient light alone, in the absence of VP, does not produce cross-linking. Download: PNG larger image TIFF original image Fig 1. Ambient light during sample processing drives VP-dependent HMWC formation. Western blots of (A) p62, (B) DIAP1, and (C) ROCK1 in MEL270, HEK293, and MCF-7 cells treated with vehicle, low-dose (LD; 1.25 µg/mL), or high-dose (HD; 7.5 µg/mL) VP for 0 or 6 h. Samples underwent dark treatment/light processing (condition ii) or darkness throughout (condition iv). High-molecular-weight complexes (HMWCs) appeared in light-processed VP samples but were absent under darkness. Vehicle lanes are VP-free light controls. β-actin is the loading control. https://doi.org/10.1371/journal.pone.0358654.g001 VP-induced cross-linking occurs in cell-free lysates and accumulates over hours To establish that VP-induced cross-linking does not require intact cellular machinery, lysates from untreated MEL270, HEK293, and MCF-7 cells were spiked with HD VP (7.5 µg/mL) and incubated on ice. After 6 hours under ambient light, prominent HMWC bands were observed for p62, DIAP1, and ROCK1 in all three cell lines ( Fig 2 ). Matched lysates incubated in near-darkness for the same duration showed no HMWC formation, with only monomeric bands detected. In a time course of light-exposed lysates, cross-linked species were absent immediately after VP addition (0 hours), first detectable at 1 hour, and increased in intensity through 6 hours ( Fig 3A ). Vehicle-treated samples from all three cell lines, carried through the same ambient-light workflow with no VP added at any stage, showed no HMWC formation and preserved monomeric p62, DIAP1, and ROCK1 ( Fig 3B ), confirming that ambient light in the absence of VP does not generate cross-linked species. The same complexes form when intact cells are treated with VP before lysis and the lysates are subsequently processed under ambient light ( Fig 1 ). These findings demonstrate that VP-induced protein cross-linking is a photochemical reaction that occurs in the post-lysis protein milieu and does not require active cellular processes. Download: PNG larger image TIFF original image Fig 2. VP induces protein cross-linking in cell-free lysates. Lysates from untreated MEL270, HEK293, and MCF-7 cells were spiked with high-dose (HD; 7.5 µg/mL) VP and incubated for 6 h under ambient light or near-darkness. High-molecular-weight complexes of p62, DIAP1, and ROCK1 formed under light but not in darkness, demonstrating that cross-linking can occur after lysis without intact cellular machinery. β-actin is the loading control. https://doi.org/10.1371/journal.pone.0358654.g002 Download: PNG larger image TIFF original image Fig 3. Cross-linking in cell-free lysates accumulates over time and requires VP. (A) Lysates from HEK293, MEL270, and MCF-7 cells were spiked with high-dose (HD; 7.5 µg/mL) VP and exposed to ambient light for 0, 1, 3, or 6 h. High-molecular-weight complexes of p62, DIAP1, and ROCK1 first appeared at 1 h and increased thereafter. (B) Vehicle-treated lysates processed under identical light conditions showed no complexes and preserved monomeric bands, serving as VP-free light controls. β-actin is the loading control. https://doi.org/10.1371/journal.pone.0358654.g003 Singlet oxygen and free radical scavengers attenuate VP-induced cross-linking To identify the reactive species responsible for cross-linking, cell homogenates were pre-incubated with the radical scavenger N-acetylcysteine (NAC; 100 mM) or the singlet oxygen quencher L-histidine (7.5 mM) for 30 minutes before addition of HD VP (7.5 µg/mL) and incubation under ambient light for 6 hours. NAC pre-treatment markedly reduced p62 HMWC formation in MEL270, HEK293, and MCF-7 lysates, leaving only faint residual high-molecular-weight signal with a preserved monomeric 62-kDa band ( Fig 4 , upper panels). L-histidine pre-treatment also reduced HMWC formation, although residual high-molecular-weight signal remained in all three cell lines and the reduction was less complete than with NAC ( Fig 4 , lower panels). In both cases, the monomeric p62 signal was maintained, indicating that the scavengers limited cross-linking rather than promoting non-specific protein degradation. These results are consistent with the involvement of both Type II (singlet oxygen-mediated) and Type I (radical-mediated) photochemical pathways in VP-induced cross-linking. Download: PNG larger image TIFF original image Fig 4. NAC and L-histidine attenuate VP-induced p62 cross-linking. MEL270, HEK293, and MCF-7 homogenates were pre-incubated for 30 min with N-acetylcysteine (NAC; 100 mM; upper panels) or L-histidine (7.5 mM; lower panels), followed by high-dose (HD; 7.5 µg/mL) VP and 6 h of ambient-light exposure. Both scavengers reduced high-molecular-weight complex formation and preserved monomeric p62; attenuation was greater with NAC. VP-negative lanes underwent the same light workflow. β-actin is the loading control. https://doi.org/10.1371/journal.pone.0358654.g004 Methionine oxidation parallels light-dependent cross-linking Immunoblotting with an anti-methionine sulfoxide antibody revealed light-dependent methionine oxidation across all three cell lines ( Fig 5 ). Under ambient light, immunoreactive bands in the 120–140-kDa range increased in intensity with VP dose and treatment duration. These signals were more prominent in VP-treated than in vehicle-treated samples, although light-exposed vehicle samples also displayed detectable methionine sulfoxide immunoreactivity. Samples processed in near-darkness manifested minimal methionine sulfoxide signal regardless of VP treatment. The concordance between methionine oxidation and HMWC formation indicates that singlet oxygen was generated under these conditions. Methionine is one of several residues attacked by singlet oxygen, alongside tryptophan, tyrosine, histidine and cysteine, and we did not determine which residues carry the covalent linkages responsible for HMWC formation. Download: PNG larger image TIFF original image Fig 5. Light-dependent methionine oxidation parallels VP-induced cross-linking. Anti-methionine sulfoxide immunoblots of MEL270, HEK293, and MCF-7 cells treated with vehicle, low-dose (LD; 1.25 µg/mL), or high-dose (HD; 7.5 µg/mL) VP for 0 or 6 h under ambient light throughout (condition i) or darkness throughout (condition iv). Immunoreactivity at approximately 120–140 kDa generally increased with VP dose and duration under light and was lower in dark-processed samples. Vehicle lanes are VP-free light controls. β-actin is the loading control. https://doi.org/10.1371/journal.pone.0358654.g005 YAP and phospho-YAP undergo light-dependent cross-linking; TEF1 is comparatively resistant Given the widespread use of VP as a purported YAP-TEAD inhibitor, we examined whether YAP, phospho-YAP (Ser127), and TEF1/TEAD1 are susceptible to light-dependent cross-linking. Under dark treatment/light lysis conditions, YAP exhibited HMWC bands and reduced monomeric band intensity in a dose- and time-dependent manner across MEL270, HEK293, and MCF-7 cells ( Fig 6 ). Phospho-YAP (Ser127) displayed a parallel pattern of HMWC formation with diminished monomeric signal under light exposure. Under near-darkness, both YAP and phospho-YAP monomeric bands were preserved at levels comparable to vehicle-treated controls, with no detectable HMWC formation. Download: PNG larger image TIFF original image Fig 6. YAP and phospho-YAP undergo light-dependent cross-linking, whereas TEF1/TEAD1 is comparatively resistant. Western blots of YAP, phospho-YAP (Ser127), and TEF1/TEAD1 in (A) MEL270, (B) HEK293, and (C) MCF-7 cells treated with vehicle, low-dose (LD; 1.25 µg/mL), or high-dose (HD; 7.5 µg/mL) VP for 0 or 6 h. Samples underwent dark treatment/light processing (condition ii) or darkness throughout (condition iv). Light-processed VP samples showed YAP and phospho-YAP high-molecular-weight complexes with reduced monomeric bands, whereas TEF1/TEAD1 remained largely stable. β-actin is the loading control. https://doi.org/10.1371/journal.pone.0358654.g006 TEF1/TEAD1 exhibited substantially less HMWC formation than either YAP or phospho-YAP. The monomeric TEF1 band remained largely stable across doses and time points, with reduced monomeric signal and a discrete higher-molecular-weight species evident only at the high dose at 6 hours under light ( Fig 6 ). This differential susceptibility suggests that structural determinants governing cross-linking vulnerability vary among proteins, with TEF1 being comparatively, though not entirely, resistant to VP-induced photochemical modification. Light during treatment contributes less to HMWC formation than light during processing When cells were treated with VP under ambient light but lysed and processed in near-darkness (condition iii: light treatment/dark lysis), HMWC formation for DIAP1, ROCK1, and p62 was attenuated relative to conditions in which lysis occurred under light ( Fig 7 ). This result indicates that, although some cross-linking may occur during the treatment phase, light exposure during the lysis and sample-processing steps is the primary determinant of HMWC formation. Download: PNG larger image TIFF original image Fig 7. Light during treatment contributes less to HMWC formation than light during processing. Western blots of DIAP1, ROCK1, and p62 in MEL270, HEK293, and MCF-7 cells treated with vehicle, low-dose (LD; 1.25 µg/mL), or high-dose (HD; 7.5 µg/mL) VP for 0 or 6 h under light treatment/dark processing (condition iii). High-molecular-weight complex formation was attenuated relative to dark treatment/light processing (condition ii; Fig 1 ), indicating that post-lysis illumination is the stronger determinant of cross-linking. β-actin is the loading control. https://doi.org/10.1371/journal.pone.0358654.g007 VP combined with ambient light reduces cell viability in a dose- and time-dependent manner Cell viability was quantified by CCK-8 assay in MEL270, HEK293, and MCF-7 cells treated with vehicle, LD, or HD VP under ambient light or dark conditions for
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