Hydrocortizone

MCF-10A human epithelial cells were cultured in growth medium composed of Dulbecco’s modified Eagle’s medium/Ham’s F-12 containing HEPES and L-glutamine (DMEM/F12, Invitrogen) supplemented with 5% horse serum (Invitrogen), 1% penicillin/streptomycin (Invitrogen), 10 µg/mL insulin (Sigma), 0.5 µg/mL hydrocortizone (Sigma), 20 ng/mL EGF (Peprotech) and 0.1 µg/mL cholera toxin (Sigma) and maintained under humidified conditions at 37 °C and 5% CO₂. Cells were passaged regularly by dissociating confluent monolayers with 0.05% trypsin-EDTA (Invitrogen) and suspending cells in DMEM/F12 supplemented with 20% horse serum and 1% penicillin/streptomycin. Cells were passaged at 1:4 in growth medium.
Other non-cancerous cells tested were RWPE1 (epithelial) and L929 (fibroblast); cancer cells tested were MCF-7 (epithelial), RWPE2 (prostate), PC3 (prostate), H292 (lung) and THP-1(leukemia)-derived macrophages. As for the genetically modified MCF-10A variants, MCF-10A APC−/− were purchased from Sigma Aldrich, while the MCF-10A RAS was transformed via introduction of the HrasV12 oncogene^{39 (link)}. They were all cultured in accordance with the ATCC recommended media and passage protocols. Also, blebbistatin (Sigma) or latrunculin A (Wako) dissolved in dimethylsulfoxide (DMSO) was used at 5 μM in growth media for the myosin or the actin polymerization inhibition experiments, respectively.

Human microvascular endothelial cells (HMEC-1) were purchased from the Center for Disease Control (CDC)36 (link) and cultured on gelatin-coated tissue culture dishes in growth medium composed of MCDB-131 (VWR International, USA) supplemented with 10% FBS (Fisherbrand, USA), 2 mM L-Glutamine (Invitrogen, USA), 1× antimycotic/antibiotic mixture (Life Technologies, USA), 10 ng/ml huEGF (Millipore, USA) and 1 μg/ml Hydrocortizone (Sigma Aldrich, USA). Human U937 monocytic cells were purchased from ATCC (Manassas, VA, USA) and cultured in suspension in growth medium composed of RPMI 1640 (Fisherbrand, USA) supplemented with 2 mM L-Glutamine (Invitrogen), 10 mM HEPES (Fisherbrand, USA), 10% FBS (Fisherbrand), antimycotic/antibiotic mixture (Life Technologies, USA), 1 mM sodium pyruvate (Life Technologies, USA) and 4.5 mg/ml glucose (Sigma Aldrich, USA).