Fig 2: Generation and validation of Gpatch2-/- mice.A Gpatch2 primers and genotyping. Schematic representation of primers located in the introns flanking exon 3 of Gpatch2 for CRISPR-targeted deletion of exon 3 (blue) and genotyping (purple) to distinguish wild-type and Gpatch2 mutant alleles. B Exon 3 was deleted by CRISPR/Cas9 to cause a frameshift in Gpatch2-/- mice. C Immunoblotting for GPATCH2 in lysates from the testis of wild-type and Gpatch2-/- mice with hybridoma supernatant. D Observed numbers of wild-type, heterozygous Gpatch2+/-, and homozygous Gpatch2-/- offspring obtained from inter-crosses of Gpatch2+/- mice.

Fig 3: The absence of GPATCH2 does not affect LPS-induced Tnf expression in mice or BMDMs.A Mice were injected with 18 mg/kg LPS, and 3 h post-injection serum TNF levels were determined by ELISA (n = 4 mice per genotype). (n = 1) B Kaplan–Meier survival curves of wild-type and Gpatch2-/- mice following injection with 5 mg/kg LPS (n = 5 mice per genotype). Uptick represents mice that did not reach ethical endpoint during the LPS challenge, and were sacrificed in a healthy state 10 h post-injection. (n = 1) C Serum TNF levels in mice of the indicated genotypes before and 2 h after injection of 5 mg/kg LPS. D Fold-change in Tnf mRNA plotted relative to Hprt mRNA in BMDMs of the indicated genotypes treated with LPS for the indicated times, as determined by qPCR (n = 3 mice per genotype). (n = 1) E Levels of TNF in the culture supernatant from BMDMs of the indicated genotypes following LPS treatment for the indicated times as determined by ELISA. LPS-supplemented culture medium was replaced 120 min post-LPS treatment and replaced with medium without LPS. F BMDMs of the indicated genotypes were treated with 100 ng/mL LPS for 2 h before lysis, and extracts were subjected to immunoblotting to detect GPATCH2 with concentrated hybridoma supernatant. GPATCH2 is indicated with an arrow, and lanes show BMDMs from individual mice (n = 3 wild-type, n = 1 Gpatch2+/-, n = 2 Gpatch2-/-). (n = 1). White bars correspond to wild-type (WT) and grey bars to Gpatch2-/- BMDMs throughout. Data are presented as mean ± SEM, all data points represent individual mice. n.d. not detected. ns not significant. No statistically significant p values were obtained from comparison of wild-type and Gpatch2-/- mice (A, C–E Mann–Whitney test, B log-rank test).

Fig 4: Loss of GPATCH2 does not alter the response to subcutaneous injection of a SMAC-mimetic.A Clinical scoring of SM-induced lesions in wild-type and Gpatch2-/- mice. Box plot whiskers show minimum and maximum scoring values. B Representative images of SM-induced lesions in wild-type or Gpatch2-/- mice 1 day or 3 days post-injection. C Representative images of SM-induced skin lesion centres in wild-type or Gpatch2-/- mice stained with H&E, for CC3 or Ki67. Scale bars: 50 µm. D Cytokine levels in lysates from skin lesions in untreated, day 1 or day 3 mice of the indicated genotypes were determined by ELISA, and cytokine levels are shown in pg per mg of total protein in skin lysates. White bars correspond to wild-type (WT) and grey bars to Gpatch2-/- mice throughout. Data are presented as mean ± SEM, data points represent individual mice (n = 4 per genotype). n.d. not detected. No statistically significant p values were obtained from comparison of wild-type and Gpatch2-/- mice (Mann–Whitney test). (n = 1).