375 tec

Cells were seeded into monolayer or suspension for 7 days, at which point the cells were isolated, using a non-enzymatic dissociation buffer (CellStripper, Invitrogen, Carlsbad, CA, USA), and re-seeded into either the tissue culture polystyrene or the Ultra-Low Attachment 96 well plates, at a concentration of 10,000 cells/well. Cells were maintained in a complete medium, overnight, prior to the TRAIL treatment. The cells were treated with the recombinant human TRAIL (TRAIL) (R&D Systems, 375-TEC), at two different doses (10 ng/mL and 100 ng/mL for MDA-MB-231 and ZR75-1 cell lines; 50 ng/mL and 500 ng/mL for MCF7 cell lines). The concentrations were based on our previous research, in which the IC50 for TRAIL treatment over 24 h was found to be 1–5 ng/mL for MDA-MB-231, 7–8 ng/mL for ZR75-1, and >500 ng/mL for MCF7 cells [37 (link)]. Plates were incubated with TRAIL over 24 h and analyzed for either viability or protein expression, at indicated timepoints.

The human breast cancer cell lines including AU565, BT474, MDA-MB-453, HCC1428, MDA-MB-361, T47D, MCF7, ZR751, HCC1500, HCC1937, HCC1954, MDA-MB-468, SKBR3, MDA-MB-157, MDA-MB-231, HCC38, HCC2157, and BT549 were purchased from American Type Culture Collection (ATCC). All cell lines were cultured per ATCC recommendation and were tested for the absence of mycoplasma contamination regularly. Recombinant human TRAIL protein, containing amino acids 114-281 of human TRAIL was produced by E. coli as homotrimers and was purchased from R&D Systems (375-TEC). Antibodies specific to human caspase-3 (8G10), caspase-8 (1C12), DR4 (D9S1R), and DR5 (D4E9) were purchased from Cell Signaling Technology. Keratin 8/18 antibodies were purchased from Cell Signaling (C51) and BioLegend (1E8). GAPDH antibody was purchased from Novus (2D4A7). Horseradish peroxidase–conjugated goat anti-rabbit IgG1 (sc-2054), goat anti-mouse IgG1 (sc-2969), and donkey anti-goat IgG (sc-2020) were purchased from Santa Cruz Biotechnology. Phycoerythrin (PE)-conjugated monoclonal antibodies to DR4 (FAB347P) and DR5 (FAB6311P) and corresponding IgG1 (IC002P) and IgG2b (IC0041P) controls were purchased from R&D Systems.