Journal: bioRxiv

Article Title: Mitoxantrone inhibits and downregulates ER α through binding at the DBD-LBD interface

doi: 10.1101/2025.01.07.631371

Figure Lengend Snippet: ( A,B ) MTO (1 μM) treatment reduces ER protein levels over time in MCF7 ( A ) and T47D ( B ) cells expressing wildtype or Y537S/D538G knock-in mutant ER, quantified relative to β-actin. ( C ) In MCF7 cells expressing exogenous HA-tagged Y537S ER mutants, MTO decreases both endogenous and exogenous ER levels, with HA-tagged mutant quantified relative to β-actin. ( D ) Immunofluorescence microscopy reveals dose-dependent reduction of ER protein in MCF7 cells treated with MTO (0.1, 0.5, and 1 μM, 48 hours). ( E ) In-cell western assay shows ER levels in MCF7 cells after 18-hour treatment with E2, MTO, or fulvestrant, normalized to DNA content (DRAQ5) and expressed as percentage of DMSO control. ( F ) Time-course immunofluorescence in T47D cells shows nuclear ER reduction after 2-hour MTO treatment. ( G ) Subcellular fractionation reveals early (2-hour) ER accumulation in cytoplasm with concurrent nuclear depletion. Data represent mean ±SEM (n=4). One-way ANOVA with Dunnett’s test: * p < 0.05; ** p < 0.01; *** p < 0.005.

Article Snippet: ER protein expression was quantified using a LI-COR Odyssey imaging system and normalized to DRAQ5 DNA stain (Thermo Scientific, # 62251, 1:10,000).

Techniques: Expressing, Knock-In, Mutagenesis, Immunofluorescence, Microscopy, In-Cell ELISA, Control, Fractionation