Detection of Aggregation-Prone Behavior in Mutant P53 V157F Breast Cancer Cells Using Multipoint Thioflavin T Fluorescence

The tumor suppressor p53, encoded by the TP53 gene, plays a central role in maintaining genomic stability. TP53 mutations, particularly hotspot variants, are found in approximately 50% of human cancers and may lead to the loss of tumor-suppressive functions or the acquisition of gain-of-function properties, including aggregation into prion-like structures. In this study, we assessed the aggregation tendency of the p53 V157F mutation in Hs578T breast cancer cells compared to MCF7 cells expressing wild-type p53. Protein aggregation was evaluated using Thioflavin T (ThT) staining followed by fluorescence quantification in 96-well plate assays. Cells were stained with a ThT/Hoechst solution, washed, and analyzed using a microplate reader under defined excitation/emission parameters. Quantitative analysis using both single-point and four-point fluorescence readings revealed that Hs578T cells exhibited a 3.20- to 4.26-fold increase in ThT fluorescence intensity relative to MCF7, indicating significantly elevated levels of β-sheet-rich or amyloid-like aggregates. Multipoint measurements confirmed the widespread and consistent presence of protein aggregates across the well surface. These findings support the use of multipoint plate-reading to accurately detect protein aggregation in cell-based assays.