1：Experimental Design and Method Selection:

The study used IHC-PIDs for quantitative detection of ERα in vitro and developed the nearest-neighbor method to calculate extranuclear ERα. Validation included correlation with real-time qRT-PCR and flow cytometry. Clinical samples from 65 patients were analyzed using IHC-PIDs to measure total, nuclear, and extranuclear ERα PIDs scores and the ERα ENR.

2：Sample Selection and Data Sources:

Patient samples included 65 HR+/HER2- breast cancer patients who underwent surgery at Tohoku University Hospital (2001-2003). Cell lines (MCF-7, T-47D, BT-474, ZR-75-1, MDA-MB-231, HeLa) with varying ERα expression were used for in vitro validation.

3：3). Cell lines (MCF-7, T-47D, BT-474, ZR-75-1, MDA-MB-231, HeLa) with varying ERα expression were used for in vitro validation. List of Experimental Equipment and Materials:

3. List of Experimental Equipment and Materials: Equipment included fluorescence microscope (BX53, Olympus), confocal laser scanning microscope (LSM 780, Carl Zeiss), N-SIM (Nikon), flow cytometer (BD Aria II, BD Biosciences), real-time PCR system (Step One, Applied Biosystems). Materials included anti-ERα antibody (SP1, Ventana), PIDs, cell culture media (DMEM, RPMI1640, Gibco), transfection reagents (Lipofectamine LTX, Invitrogen).

4：Experimental Procedures and Operational Workflow:

Paraffin sections were stained with IHC-PIDs, imaged, and analyzed using the nearest-neighbor method. Cell lines were cultured, transfected (for ERα localization studies), and analyzed by IHC-PIDs, flow cytometry, and real-time qRT-PCR. Patient tissue sections were processed similarly, with statistical analysis of survival data.

5：Data Analysis Methods:

Statistical analysis used GraphPad Prism and JMP Pro software. Methods included Pearson correlation, χ2 tests, t-tests, ANOVA, Kaplan-Meier survival analysis, log-rank tests, and multivariate Cox regression.