Caspase 8 sirna

For caspase 8 downregulation experiments, hESC were transfected with siRNA using a Neon transfection instrument (Invitrogen) according to the manufacturer's instructions. 100 pmoles of caspase 8 siRNA (Santa Cruz Biotechnology Inc.) were added to 1 × 10⁵ cells and transfection was performed with one 1100 V pulse for 30 ms. Cells were seeded on Matrigel and cultured in CM without antibiotics. Cells were harvested at 48 and 72 hours after transfection.

His-tagged Ub and Fn14 siRNA were described in previous publications 12 (link). Human Pellino1 cDNA was amplified by PCR and subcloned into pMSCV-Flag retroviral vector (Addgene, Cambridge, MA). Pellino1 S39A and S39D mutants were constructed using QuickChange^® Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) with the primers as below: S39A sense, 5′-CGATAGAGGAAGGAGGAAAGCCAGGTTTGCTTTGTTTAAAAG-3′ and S39A antisense, 5′-CTTTTAAACAAAGCAAACCTGGCTTTCCTCCTTCCTCTATCG-3′; S39D sense, 5′-GCGATAGAGGAAGGAGGAAAGACAGGTTTGCTTTGTTTAAAAG-3′; and S39D antisense, 5′-CTTTTAAACAAAGCAAACCTGTCTTTCCTCCTTCCTCTATCGC-3′. The V5-tagged DAPK1 was constructed using pLX304 vector (Genecopoeia, Rockville, MD). The kinase-dead (KD) DAPK1 mutant was generated as described previously 26 (link). RIP1 and RIP3 siRNA were purchased from Dharmacon (Lafayette, CO). Caspase-8 siRNA and DAPK1 shRNA were ordered from Santa Cruz Biotechnology (Santa Cruz, CA). For CRISPR-Cas9 gene knockout, CRISPR guide sgRNAs targeting Pellino1 and DAPK1 were subcloned to commercial pCRISPR-SG01 vectors (HCP254204-SG01-3 and HCP270000-SG01-3) which were purchased from GeneCopoeia (Rockville, USA).