Live-Cell Imaging of MCF10A Cells Treated individually or in combination with EGF, OSM, or TGFB

MCF10A cell culture and experimental procedures were conducted based on established methodologies (10.1038/s42003-022-03975-9). For routine maintenance and passaging, cells were cultured in a growth medium composed of DMEM/F12 (Invitrogen, #11330-032) supplemented with 5% horse serum (Sigma, #H1138), 20 ng/ml EGF (R&D Systems, #236-EG), 0.5 µg/ml hydrocortisone (Sigma, #H-4001), 100 ng/ml cholera toxin (Sigma, #C8052), 10 µg/ml insulin (Sigma, #I9278), and 1% Penicillin/Streptomycin (Invitrogen, #15070-063). For experiments involving EGF perturbation, a growth factor-free medium was prepared using DMEM/F12, 5% horse serum, 0.5 µg/ml hydrocortisone, 100 ng/ml cholera toxin, and 1% Pen/Strep. Cells were cultured to 50–80% confluency before being detached with 0.05% trypsin-EDTA (Thermo Fisher Scientific, #25300-054). Subsequently, 20,000 cells were seeded into 24-well plates (Thermo Fisher Scientific, #267062) coated with collagen-1 (Cultrex, #3442-050-01) in growth medium.After an 18-hour incubation in the new media, cells were treated with single ligand or combinations of ligands in fresh growth factor-free media: 10 ng/ml EGF (R&D Systems #236-EG), 10 ng/ml OSM (R&D Systems #8475-OM), and 10 ng/ml TGFβ (R&D Systems #240-B). Phenotypic responses to individual and combination treatment with EGF, OSM, and TGFB treatment were assessed through live-cell imaging using the Incucyte S3 microscope (Essen BioScience, #4647), which captured images every 30 minutes over a 24-hour period. The dataset includes an Excel spreadsheet that documents the experimental conditions for each imaged well. Companion RNAseq can be accessed from the Gene Expression Omnibus: GSE282654.MCF10A细胞的培养与实验流程均基于已发表的成熟方法（DOI: 10.1038/s42003-022-03975-9）。 常规培养与传代时，细胞采用含血清生长培养基培养，该培养基由DMEM/F12（Invitrogen，货号#11330-032）添加以下组分配制：5%马血清（Sigma，货号#H1138）、20 ng/ml表皮生长因子（EGF，R&D Systems，货号#236-EG）、0.5 µg/ml氢化可的松（Sigma，货号#H-4001）、100 ng/ml霍乱毒素（Sigma，货号#C8052）、10 µg/ml胰岛素（Sigma，货号#I9278）以及1%青霉素-链霉素混合液（Invitrogen，货号#15070-063）。 若开展涉及表皮生长因子干预的实验，则需配制无生长因子的基础培养基，其组成为DMEM/F12、5%马血清、0.5 µg/ml氢化可的松、100 ng/ml霍乱毒素以及1%青霉素-链霉素（Pen/Strep）。 实验前将细胞培养至汇合度50%~80%，随后使用0.05%胰蛋白酶-EDTA（Thermo Fisher Scientific，货号#25300-054）进行消化。 取20,000个细胞接种于包被有Ⅰ型胶原蛋白（Cultrex，货号#3442-050-01）的24孔板（Thermo Fisher Scientific，货号#267062）中，使用上述含血清生长培养基培养。 待细胞在新培养基中孵育18小时后，更换为无生长因子的新鲜培养基，并加入单一配体或配体组合进行干预：10 ng/ml EGF（R&D Systems，货号#236-EG）、10 ng/ml制瘤素M（OSM，R&D Systems，货号#8475-OM）以及10 ng/ml转化生长因子β（TGFβ，R&D Systems，货号#240-B）。 采用Incucyte S3活细胞成像系统（Essen BioScience，货号#4647）对EGF、OSM及TGFβ单一或联合处理后的细胞表型响应进行评估，该系统每30分钟采集一次图像，连续采集时长为24小时。 本数据集包含一份Excel表格，用于记录每个成像孔对应的实验条件。 配套的RNA测序（RNAseq）数据可从基因表达综合数据库（Gene Expression Omnibus，GEO）获取，编号为GSE282654。