Vectashield mounting medium h 1500

The U937 macrophages were incubated in the presence of 10 μg/mL Dil-ox-LDL in RPMI 1640 medium supplemented with 10% fetal bovine serum including 100 μg/mL streptomycin and 100 U/mL penicillin at 37 °C in 5% CO₂ for 18 h [21 (link),26 (link)]. After gentle washing with PBS three times, the adherent cells were mounted in Vectashield mounting medium (H-1500, Vector Laboratories, Burlingame, CA, USA) and were imaged with BZ-X710 microscope/software (Keyence, Osaka, Japan). The fluorescent intensity of red color area per cells was quantified as described previously [21 (link),26 (link)].

Cells were fixed in 4% formaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA), permeabilized in 0.2% Triton X-100 and incubated in 50 mM NH₄Cl/0.1% glycine. Primary antibodies (E-cadherin, BD Biosciences, #610182; cleaved caspase-3, Cell Signaling Technology, #9661; vimentin, Dako, #M0725) were incubated over night at 4°C and secondary antibodies for 4 hours at room temperature. Samples were mounted using Vectashield mounting medium (H-1500, Vector Laboratories, Burlingame, CA, USA) containing 1.5 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) and analyzed in a Leica DMIRE2 microscope equipped with a TCS SP2 confocal unit (Leica Microsystems, Heerbrugg, Switzerland).

SMTP-44D [40 (link),41 (link)] was produced by TMS Co., Ltd. (Tokyo, Japan). Dil-ox-LDL was purchased from Highland Technology Center (Frederick, MD, USA). Anti-F4/80 antibody (Alexa Fluor^® 647, Ab204467) was from Abcam (Cambridge, UK). Roswell Park Memorial Institute (RPMI) 1640 medium from Sigma Aldrich (St. Louis, MO, USA), and vectashield mounting medium (H-1500) was purchased from Vector Laboratories (Burlingame, CA, USA).