Extended Data Fig. 1: Generation of Oasl1-/- mouse using the CRISPR-Cas9 system and the function of Oasl1 in virus-induced necroptosis.
a, Schematic of the genomic target in OASL1 gene and OASL1 exon 2 sgRNA. The sgRNA sequence (designed in http://chopchop.cbu.uib.no) is marked in green and the protospacer adjacent motif (PAM) sequence is marked in red. Blue arrow points the predicted cleavage site. b, Mouse Oasl1 genomic sequences from Oasl1+/+ and Oasl1-/- littermate mice. PAM sequence is marked in red. 5 base pairs deletion was shown in Oasl1-/- mice. c,d, Immunoblot analysis of OASL1 expression in Oasl1+/+ and Oasl1-/- primary fibroblasts infected with MCMV (c) or treated with IFN-β (d) for the indicated times. e, Quantification of necrotic cell death by measuring the intracellular ATP levels in Oasl1+/+ and Oasl1-/- primary fibroblasts upon TNF-α (50 ng/ml) and/or zVAD (20 mM) treatment. f, Measurement of lactate dehydrogenase (LDH) release to the medium in Oasl1+/+ or Oasl1-/- primary fibroblasts infected with MCMV-WT (m.o.i. = 5) for 8 h. g, Immunoblot analysis of RIPK3 and MLKL phosphorylation in Oasl1+/+ or Oasl1-/- primary fibroblasts infected with MCMV-WT (m.o.i. = 5) for the indicated hours. h, MCMV-WT titer in Oasl1+/+ and Oasl1-/- primary fibroblasts were determined by plaque assays by titrating culture medium at the indicating time points (n = 4 for Oasl1+/+, n = 2 for Oasl1-/-). i, (Left) Immunoblot analysis of CRISPR Cas9-mediated knockout of human OASL in A549 cells upon mock or Sendai virus (SeV) infection (Asterisk, non-specific band). Quantification of necrotic cell death by measuring the intracellular ATP levels in WT and hOASL KO A549 cells infected with (middle) HSV-GFP (m.o.i. = 10) upon IFN-β stimulation (n = 3) or (right) VACV (m.o.i. = 5) (n = 2). c,d,g, Data are representative of two independent experiments. e,f, Data are presented as mean ± SEM from three independent experiments. Statistical analyses were performed using a two-tailed unpaired t-test (f) or two-way analysis of variance (ANOVA) (e,h,i). ns, not significant.
