Ambient light drives verteporfin-induced protein cross-linking during standard laboratory sample processing
- 1Department of Ophthalmology, Retina Service, Ines and Frederick Yeatts Lab in Retina Research, MassachusettsEye and Ear, Harvard Medical School, Boston, Massachusetts, United States of America
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.
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BibTeX
@article{baroutis2026ambient,
title = {{Ambient light drives verteporfin-induced protein cross-linking during standard laboratory sample processing}},
author = {Konstantinos G. Baroutis and Victor San Martin C. Corrêa and Dimitrios Ntentakis and Toshio Narimatsu and Evangelos S. Gragoudas and Joan W. Miller and Lucy H. Young and Demetrios G. Vavvas},
journal = {PLOS One},
year = {2026},
month = sep,
doi = {10.1371/journal.pone.0358654},
url = {https://doi.org/10.1371/journal.pone.0358654},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Ambient light drives verteporfin-induced protein cross-linking during standard laboratory sample processing
AU - Konstantinos G. Baroutis
AU - Victor San Martin C. Corrêa
AU - Dimitrios Ntentakis
AU - Toshio Narimatsu
AU - Evangelos S. Gragoudas
AU - Joan W. Miller
AU - Lucy H. Young
AU - Demetrios G. Vavvas
PY - 2026
DA - 2026/09/21
JO - PLOS One
DO - 10.1371/journal.pone.0358654
UR - https://doi.org/10.1371/journal.pone.0358654
AB - 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.
SN - 1932-6203
ER -