D9s1r

Human NSCLC tissues were stained with IHC using the EnVision™ + Dual Link System-HRP Kit (Dako; Carpinteria, CA). The rabbit monoclonal antibody against DR4 (D9S1R; #42533) was purchased from Cell Signaling Technology (Danvers, MA 01923) and used at 1:100 dilutions overnight at 4^oC. The specificity of the antibody was determined with matched IgG isotype antibody as a negative control in IHC. Moreover, a single band of correct molecular weight in Western blotting was assured. Both the percentage of positive staining in tumor cells and intensity of staining were scored. The intensity of IHC staining was measured by using a numerical scale (0 = no expression, 1 = weak expression, 2 = moderate expression and 3 = strong expression). The staining data were finally quantified as the weighted index (WI) (WI = % positive staining in tumor x intensity score) as previously described 54 (link). DR4 staining was scored as negative (≤ 10 WI) and positive staining (> 10 WI), respectively. The WI was determined by 2 individuals, and the final values were the average of the two readings. cPARP in xenograft tissues were stained with cPARP antibody purchased from Cell Signaling Technology (#5625) at 1:50 dilution.

Immunoblotting analysis was performed, as previously described [24 (link),37 (link)]. Cells lines were cultured in either monolayer or suspension growth conditions, and were harvested at specified time-points. Cells were washed with PBS and lysed using a radioimmunoprecipitation assay (RIPA) lysis buffer, with a protease inhibitor. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA). Equal amounts of lysis (20 μg) were resolved by electrophoresis, using 4–12% NuPAGE Bis-Tris gels and transferred to the PVDF membranes (Life Technologies, Carlsbad, CA, USA). Primary antibodies were used at the recommended manufacturer’s indications (1:500 to 1:1000). Membranes were stripped using the Restore Western Blot Stripping Buffer (Pierce) and re-probed with appropriate antibodies. Immunocomplexes were visualized with chemiluminescence using the Immobilon Western Chemiluminescent HRP Substrate. Antibodies specific to human DR4 (D9S1R), DR5 (D4E9), TNFR1 (C25C1), Caspase 3 (8G10), Caspase 8 (1C12), PARP (9542), and p62 (D5E2), were purchased from Cell Signaling Technologies (Danvers, MA, USA). Fas (C-20) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). GAPDH (2D4A7) and LC3B/MAP1LC3B (NB100) were purchased from Novus Biologicals (Littleton, CO, USA).

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.