Extended Data Fig. 8: APT2 mediates depalmitoylation of GSDMD and promotes pyroptosis.
From: A palmitoylation–depalmitoylation relay spatiotemporally controls GSDMD activation in pyroptosis
a, b, HEK293T cells were transfected with HA tagged-ATP1 or APT2 and Flag tagged-GSDMD or GSDMD-NT for 24 hours. APT1/2 binding was revealed by immunoprecipitation and immunoblotting. c–e, WT (d and c) or Dox-inducible GSDMD-NT-reconstituted (e) BMDMs were transfected with 50 nM siRNA targeting Apt2 for 48 hours followed by LPS (100 ng/ml) treatment for 3 hours (c), or treated with LPS (100 ng/ml) for 3 hours followed by ML349 (50 μM) treatment for 1 hour (d), or incubated with Dox (2 μg/ml) in the presence of ML349 (50 μM) for 12 hours (e). Palmitoylation of GSDMD (c and d) or GSDMD-NT (e) was detected using IP–ABE assay. f–i, BMDMs were transfected with 50 nM siRNA targeting Apt2 for 48 hours and stimulated with LPS (100 ng/ml) for 3 hours followed by Nig (10 μM) (f and g) or ATP (2 mM) (h and i) treatment for indicated times (f and h) or 2 hours (g and i). Cell death was measured by SytoxGreen positivity assay (f and h). The resulting supernatants were quantified for LDH release (g and i). j, k, Primary Apt2+/+ and Apt2-/- BMDMs were pre-treated with 5z7 (200 nM) for 0.5 hours followed by treatment with LPS (40 ng/ml) (j), or treated with LFn-Rod (100 ng/ml) plus protective antigen (1 µg/ml) (k) for the indicated times. Cell death was measured by SytoxGreen positivity assay. l, BMDMs were transfected with 50 nM siRNA targeting the indicated APTs for 48 hours and then stimulated with LPS (100 ng/ml) for 3 hours followed by treatment with Nig (10 μM) for indicated times. Cell death measured by SytoxGreen positivity assay was shown on the left. mRNA expression of the indicated APTs was shown on the right. Data are presented as mean ± s.d. of n = 4 (f and h), 6 (g, i and l, left), 8 (j), 5 (k) or 3 (l, right) independent wells of one representative experiment. Unpaired two-tailed Student’s t-test (g and i).
