1：Experimental Design and Method Selection:

The study integrates multiple Gaussian-functions analysis into FRET two-hybrid assays to determine stoichiometric ratios of hetero-oligomeric complexes. It uses E-FRET and 33-FRET methods for measuring donor- and acceptor-centric FRET efficiencies (ED and EA). Gaussian functions are fitted to E-count histograms of ED and EA images to obtain peak values and stoichiometric ratios.

2：Sample Selection and Data Sources:

Human cervical cancer (Hela) cells were used, transfected with various FRET tandem plasmids (e.g., CVC, VCV, C32V, CFP-Bcl-XL, Bad-YFP). Cells were cultured in DMEM with 10% fetal calf serum and transfected using Turbofect? reagent.

3：List of Experimental Equipment and Materials:

Zeiss LSM 880 inverted laser scanning confocal microscope system with a X63/1.4 oil-immersion objective and two PMT detectors; ZEN 2.0 software; plasmids from Addgene and provided by other labs; DMEM medium; fetal calf serum; penicillin/streptomycin; Turbofect? transfection reagent.

4：4 oil-immersion objective and two PMT detectors; ZEN 0 software; plasmids from Addgene and provided by other labs; DMEM medium; fetal calf serum; penicillin/streptomycin; Turbofect? transfection reagent. Experimental Procedures and Operational Workflow:

4. Experimental Procedures and Operational Workflow: Cells were transfected with plasmids, imaged using confocal microscopy in donor, FRET, and acceptor channels. ED and EA were calculated using E-FRET and 33-FRET methods. Gaussian functions were fitted to E-count histograms to determine peak values and stoichiometric ratios.

5：Data Analysis Methods:

Gaussian peak fitting to E-count histograms; statistical analysis of ED, EA, and R values from multiple cells; use of IAA/IDD ratio to indicate co-expression levels.