Additional file 4: Table S1. of Glyceollins trigger anti-proliferative effects through estradiol-dependent and independent pathways in breast cancer cells

All differentially expressed genes. The ID of all genes are indicated with their functions and expression levels. Cell cycle analysis. MCF-7 cells (100,000 cells/well) were plated in 6-well plates. After 72 h of serum and steroid deprivation, the cells were treated for 72 h with solvent as control, 10−9 M E2, 10−5 M glyceollin I or II, or a combination of E2 and glyceollin I or II. Then, the cells were trypsinized and fixed in 70% ethanol before staining with propidium iodide. The percentage of cells in each cell cycle phase was assessed by flow cytometry with a FACS Calibur (BD Biosciences). Measurement of apoptosis MCF-7 cells (4000 cells/well) were plated in 96-well plates. After 72 h of serum and steroid deprivation, the cells were treated for 72 h with solvent as control, 10−9 M E2, 10−5 M glyceollin I or II, or a combination of E2 and glyceollin I or II. TUNEL staining was assessed with an In Situ Cell Death Detection Kit, Fluorescein (Roche) according to the manufacturer’s instructions. The fluorescence and percentage of TUNEL-positive cells were determined with an Array Scan VTI (Thermo Fisher Scientific) on the ImPACcell platform (Rennes, France). Chromatin Immunoprecipitation (ChIP) MCF-7 cells (2,000,000 cells /dishes) were plated in 10 cm dishes and then deprived of steroids and serum for 72 h. The cells were treated for 1 h with 10−9 M E2, with 10−5 M GI or GII with or without 10−9 M E2. Then, cells were cross-linked for 10 min with 1.5% of formaldehyde (Sigma). Cells were lysed in lysis buffer (50 mM Tris-HCl, pH 8.1, 10 m M EDTA, 0.5% Empigen BB and 1% SDS). Chromatin was sonicated 10 min (15 s on/off cycles) on Bioruptor (Diagenode) at highest intensity. Soluble chromatin was diluted in IP buffer (20 mM Tris-HCl, pH 8.1, 2 mM EDTA, 0.1% Triton X-100) with 2 μg of ERα antibody (E115, Abcam) and yeast RNA as non-specific competitor and incubated overnight at 4 °C on rocking platform. Then, protein G coupled sepharose beads were added to the samples and were incubated 4 h à 4 °C. Immune complexes were washed one time in washing buffer 1 (20 mM Tris-HCl, pH 8.1, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100 and 0.1% SDS), one time in washing buffer 2 (20 mM Tris-HCl, pH 8.1, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100 and 0.1% SDS), one time in washing buffer 3 (10 mM Tris-HCl, pH 8.1, 1 mM EDTA, 250 mM LiCl, 1% Deoxycholate and 1% NP-40) and finally two times in washing buffer 4 (10 mM Tris-HCl, pH 8.1, 1 mM EDTA). After washing, immune complexes were extracted with 100 μl of extraction buffer (0.1 M NaHCO3 and 1% SDS). Cross-linking was reverse by incubation of samples overnight at 65 °C and DNA was purified using the Nucleospin Gel and PCR cleanup kit (Macherey Nagel). Enrichment analysis on the ERE proximal of GREB1 (Fwd: CACTTTGAGCAAAAGCCACA and Rev.: GACCCAGTTGCCACACTTTT) and on an enhancer 1 of PgR described in [58] was normalized using an irrelevant region on the chromosome 10 (Fwd: AGGTGACAAGCCAAGTGTCC and Rev.: GCCTGGTGGCATACTAAAGG). Analysis was performed by real time PCR on a CFX 384 apparatus (BioRad) on 2 μL of immunoprecipitation or 0.2 μL of input with 500 nM of primers and iTaq Universal SYBR Green Supermix (BioRad). (XLSX 590 kb)

所有差异表达基因。所有基因的标识符均标注了其功能与表达水平。

细胞周期分析：将MCF-7细胞（100,000个细胞/孔）接种于6孔板中，经血清与类固醇剥夺培养72小时后，分别以溶剂（对照组）、10⁻⁹ M雌二醇（E2）、10⁻⁵ M大豆素I（glyceollin I）或大豆素II（glyceollin II），或雌二醇与大豆素I/II联合处理72小时。随后用胰酶消化细胞，以70%乙醇固定，再用碘化丙啶进行染色。采用BD FACS Calibur流式细胞仪（BD Biosciences）检测各细胞周期时相的细胞占比。

细胞凋亡检测：将MCF-7细胞（4000个细胞/孔）接种于96孔板中，经血清与类固醇剥夺培养72小时后，以同样的试剂处理72小时。采用罗氏荧光素原位细胞死亡检测试剂盒（In Situ Cell Death Detection Kit, Fluorescein, Roche）按照说明书进行TUNEL染色，通过赛默飞世尔科技的Array Scan VTI成像系统在法国雷恩的ImPACcell平台上检测荧光信号及TUNEL阳性细胞占比。

染色质免疫共沉淀（Chromatin Immunoprecipitation, ChIP）：将MCF-7细胞（2,000,000个细胞/培养皿）接种于10 cm培养皿中，经血清与类固醇剥夺培养72小时后，分别以10⁻⁹ M E2、10⁻⁵ M大豆素I或大豆素II，或联合10⁻⁹ M E2处理1小时。随后用1.5%甲醛（Sigma）交联细胞10分钟。用裂解缓冲液（50 mM Tris-HCl，pH 8.1，10 mM EDTA，0.5% Empigen BB及1% SDS）裂解细胞。将染色质置于Bioruptor超声破碎仪（Diagenode）中以最高强度超声10分钟（15秒开/15秒关循环）。将可溶性染色质用免疫沉淀缓冲液（IP缓冲液，20 mM Tris-HCl，pH 8.1，2 mM EDTA，0.1% Triton X-100）稀释，加入2 μg 雌激素受体α（ERα）抗体（E115, Abcam）及酵母RNA作为非特异性竞争性抑制剂，于4℃摇床孵育过夜。随后向样本中加入蛋白G耦联琼脂糖珠，于4℃孵育4小时。免疫复合物依次用洗涤缓冲液1（20 mM Tris-HCl，pH 8.1，2 mM EDTA，150 mM NaCl，1% Triton X-100及0.1% SDS）洗涤1次、洗涤缓冲液2（20 mM Tris-HCl，pH 8.1，2 mM EDTA，500 mM NaCl，1% Triton X-100及0.1% SDS）洗涤1次、洗涤缓冲液3（10 mM Tris-HCl，pH 8.1，1 mM EDTA，250 mM LiCl，1%脱氧胆酸钠及1% NP-40）洗涤1次，最后用洗涤缓冲液4（10 mM Tris-HCl，pH 8.1，1 mM EDTA）洗涤2次。洗涤完成后，用100 μL洗脱缓冲液（0.1 M NaHCO₃及1% SDS）洗脱免疫复合物。将样本置于65℃孵育过夜以逆转交联，随后用Nucleospin凝胶与PCR纯化试剂盒（Macherey Nagel）纯化DNA。

针对GREB1基因雌激素反应元件（ERE）近端区域（正向引物：CACTTTGAGCAAAAGCCACA，反向引物：GACCCAGTTGCCACACTTTT）及文献[58]中报道的孕激素受体（PgR）增强子1区域的富集分析，以10号染色体上的无关区域（正向引物：AGGTGACAAGCCAAGTGTCC，反向引物：GCCTGGTGGCATACTAAAGG）作为内参进行标准化。采用CFX 384实时荧光定量PCR仪（BioRad）进行分析，反应体系为2 μL免疫沉淀产物或0.2 μL input样本，加入500 nM引物及iTaq Universal SYBR Green Supermix（BioRad）。（XLSX格式，文件大小590 KB）