Ambient light drives verteporfin-induced protein cross-linking during standard laboratory sample processing.

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.