Caspase 8

Cell lines HCT116 wild type (WT) and HCT116 Bax^−/−, androgen-dependent LNCaP cells and androgen-independent cell lines (DU145 Mock, DU145 Bax reconstituted and PC-3 cells) were either provided by Drs B. Vogelstein and Peter Daniel or purchased from American type culture collection.^{23 (link),24 (link)} All cells were cultured using their respective medium and maintained at 37 °C in a humidified atmosphere in the presence of 5% CO₂. Antibodies and sources are: IL-8 (Santa Cruz Biotechnology, Inc. Dallas, TX, USA, Cat # sc-8427); Caspase-8 (Enzo Life Sciences, Inc., Farmingdale, NY, USA, Cat # ALX-804-242-C100); Caspase-9 (Cell Signaling Technology, Danvers, MA, USA, Cat # 9502), beta actin (Santa Cruz Biotechnology, Inc., Cat # sc-47778 HRP) and Hsp60 (Millipore-Sigma, Burlington, MA, USA, Cat # MAB3514). All reagents used in this study were of highest grade of purity.

Antibodies with the following specificities were used for Western blot analysis: TRAF2, β-actin (abcam, Cambridge, UK), Caspase-3, cIAP1, cIAP2 (Cell signaling, Danvers, MA, USA), Caspase-8 (Enzo Life Sciences, Lausen, Switzerland), PARP, Cul3 (BD Pharmingen, Heidelberg, Germany), α-Tubulin (Millipore, Billerica, MA, USA) and RIP3 (Imgenex, San Diego, CA, USA). HRP-conjugated goat anti-mouse IgG1, goat anti-mouse IgG2a, goat anti-mouse IgG2b and donkey anti-rabbit antibodies were obtained from Southern Biotech (Birmingham, AL, USA) and Dianova (Hamburg, Germany). Killer-TRAIL was purchased from Alexis biosciences (Carlsbad, CA, USA). The pan-caspase-inhibitor zVAD-fmk was obtained from Bachem (Heidelberg, Germany). Necrostatin-1 was purchased from Enzo Life Sciences (Lausen, Switzerland). Fc-CD95L and Flag-TWEAK were produced as previously described.^{33 (link)} TRAF2 siRNA (HS-TRAF2_4, sequence as follows: 5′-GGACCAAGACAAGAUUGAATT-3′), alternate TRAF2 siRNA's (HS-TRAF2_5, sequence: 5′-CGAGGGCAUAUAUGAAGAATT-3′ HS TRAF2_6, sequence: 5′-GUUCGGCCUUCCCAGAUAATT-3′ HS TRAF2_7, sequence: 5′-GCUGCGGAGCAGACGUGAATT-3′) and negative control siRNA were provided by Qiagen (Hilden, Germany). siRNA transfection was performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

Antibodies and sources: CDK9 (1.1000), pCDK9 (Thr187) (1.1000), RNAPII (1.1000), phospho-RNAPII(Ser2) (1.1000), PARP (1.1000), and NUP98 (1.1000) (Cell Signaling Technology); HEXIM1 (1.1000), NELF-a (1.1000), LARP-7 (1.1000), c-Myc (1.1000), SPT5 (1.1000), and GAPDH (1.5000) (Santa Cruz Biotechnology, Dallas, TX, USA); BRD4 (1.1000) (Abcam, Waltham, MA, USA); Caspase-8 (1.1000) (Enzo Life Sciences, Farmingdale, NY, USA); β-Actin (1.10000), (Sigma-Aldrich, St. Louis, MO, USA); Cyclin T (1.1000) (Bethyl, Montgomery, TX, USA). Reagents and sources: CellTiter-Blue Cell Viability assay and Caspase-Glo 3/7 assays (Promega, Madison, WI, USA); AnnexinV and 7AAD (BD); BAY1251152, BI894999, ABBV744, Paclitaxel and Carboplatin (Selleckchem, Houston, TX, USA); BioCoat Matrigel invasion chamber (Corning, Corning, NY, USA); Migration chamber (Ibidi, Gräfelfing, Germany); RNeasy Plus kit (Qiagen, Venlo, The Netherlands). The following vectors were used: pCas9(BB)-2A-Puro (PX459) V2.0 (62988, Addgene, Watertown, MA, USA); p3xFlag-CMV-7.1 (E7533, Sigma, Ronkonkoma, NY, USA).