Culturing Patient-Derived IBC Cell Line

The patient‐derived IBC cell line SUM‐149 (BioIVT, Westbury, NY, USA) was cultured in Ham’s F12 medium (Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS), as previously described [54 (link), 55 (link), 56 (link)]. SUM‐149 IBC cells were seeded 1 : 3 in 10‐cm plates. Once they reached 70% confluency, 3 mL of culture media was collected every 72 h from three plates, while regular media served as control. For total protein extraction, the cells were harvested with a cell scraper and lysed on ice using NP‐40 lysis buffer containing one protease inhibitor tablet (Roche Diagnostics Corporation, Indianapolis, IN, USA). Cells were incubated for 10 min on ice and vortexed intermittently. Supernatants were collected after centrifugation [21,256 g (RCF), 4 °C, 10 min], and protein concentration was quantified using the Precision Red protein assay kit (Cytoskeleton, Inc., Denver, CO, USA), as described previously [54 (link), 56 (link), 57 ].