Eight well glass chamber slide

The BT-483 and T-47D cells were seeded into eight-well glass chamber slides (Thermo Fisher Scientific). The mitochondrial transmembrane potential was measured using the MitoScreen test (Becton Dickinson, Heidelberg, Germany) according to the manufacturer’s protocol. The oxidative stress in the cytoplasm was detected using the CellROXDeepRed test (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Jurkat cells (10 × 10⁶) were induced for apoptosis with staurosporine (Sigma-Aldrich, 1 µM/10⁶ cells in 1 ml RPMI medium containing pen/strep/glutamine and 10% FBS) for 5 h. Cells were washed three times in PBS, labeled with CypHer5E (GE Healthcare; 1 µl CypHer5E/1 ml serum-free medium) for 30 min, and washed twice with PBS. Peritoneal macrophages were isolated as described, and 150 × 10³ cells were plated on an eight-well glass chamber slide (Nunc) and fed with 750 × 10³ pre-labeled apoptotic Jurkat cells for 4 h in a total of 150-µl medium with or without soluble PROS1 (25 nM; from Enzyme Research Laboratories). Next, the medium was aspirated, and bound cells were washed gently with PBS. Adherent cells were subsequently fixed in 200 µl of 4% PFA; 5% sucrose for 15 min. Cells were washed in PBS and incubated with 200 µl phalloidin (5 U/ml, CF488A conjugate, Biotium) at 4^°C overnight. Then, cells were washed three times with PBS (10 min each) and stained in 200 µl of Hoechst 3570 (20 µg/ml, Invitrogen) for 5 min, and rinsed with PBS. The chambers were removed, mounted with Fluoromount G, and visualized under a Nikon A1 confocal microscope. The number of CypHer5E⁺ engulfed ACs per macrophage was scored.

Antibodies against N-cad, Vim, ZEB1, E-cad, Smad2, Smad3, phosphorylated Smad2, and phosphorylated Smad3 were sourced from Cell Signaling Technology (Danvers, MA, USA). RPMI 1640 medium and TGF-β1 were obtained from Gibco (Grand Island, NY, USA) and PeproTech (Rocky Hill, NJ, USA), respectively. Invitrogen (Carlsbad, CA, USA) provided Alexa Fluor 488-IgG antibody for Vim, Texas Red-IgG antibody for N-cad, and 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining. The eight-well glass chamber slide was obtained from Nunc (Nalge Nunc International, Rochester, NY, USA).