Fab347p

Cell surface expression of DR4 and DR5 was performed using PE-conjugated antibodies (R&D Systems, FAB347P and FAB6311P). Cells were harvested at 70–80% confluence and were washed twice with PBS containing 1% FBS. Anti-DR4-PE or anti-DR5-PE was then added to a final concentration of 10 µg/mL to a 25 µL cell suspension containing 1 × 10⁵ cells. Control samples were incubated with isotype-matched PE-conjugated control antibodies (R&D Systems, IC002P and IC0041P). Antibody incubation was performed for 1 h at 4 °C in the dark. Cells were then washed twice with PBS containing 1% FBS and resuspended in 0.5 mL of the solution for flow cytometric analysis.

The expression levels of death receptors DR4, DR5, Fas, and TNFR1 were measured on cells cultured in either the monolayer or the suspension culture, for seven days. Cells were dissociated into single cell suspensions, using a non-enzymatic dissociation buffer (Cell Stripper). Cells were spun down for 5 min at 800× g and re-suspended at 5.0 × 10⁶ cells/mL, in a blocking solution (1.0% bovine serum albumin, 5.0% normal goat serum (Invitrogen), in PBS). Cells were blocked against the non-specific binding for 20 min on ice. Cells were then labeled with anti-DR4-PE (IgG₁, R&D Systems, FAB347P), anti-DR5-PE (IgG_2B, R&D Systems, FAB6311P), anti-TNFR1-PE (IgG₁, R&D Systems, FAB225P), or anti-Fas-PE (IgG_1κ, BD Pharmigen, 555674), for 45 min, in the dark, on ice, according to the manufacturer’s recommendations. Respective IgG isotype controls were used to determine the nonspecific interactions between the antibodies and the cell surface proteins. Cells were washed twice with ice-cold PBS, re-suspended in 1% BSA-PBS flow cytometry buffer, and analyzed using a BD Accuri C6 flow cytometer. Surface receptor expression was determined by calculating the median fluorescence intensity of the target protein, minus the median fluorescence intensity of the corresponding isotype control. All data are shown relative to the corresponding monolayer-cultured samples of each cell-type.

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.