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. 2020 Apr 15;15:26. doi: 10.1186/s13024-020-00372-w

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 3. — NLRP12 collaborates with NLRP3 and NLRC4 to elicit pyroptosis and promote IL-1β production in ischemic injury through CASP1-dependent GSDMD cleavage a-h NLRP12/NLRP3/NLRC4 promoted IL-1β release and induced pyroptosis by CASP1-dependent GSDMD cleavage: a Knockdown of NLRP12/NLRP3/NLRC4 reduced CASP1 and GSDMD cleavage in extracts from BV2 microglia after OGDR injury, as determined by western blot analysis (n = 6). The protein levels were normalized to β-actin levels. b LDH release (n = 6). c IL-1β secretion (n = 6). d-e LDH release (d) and IL-1β production (e) of BV2 microglia treated with the CASP1 inh, YVAD (200 µM) and subjected to OGDR injury (n = 6). f Representative SEM images showed pyroptotic cell death and other morphological changes in BV2 microglia subjected to OGDR combined with or without different additional treatments (n = 5): (i): control; (ii): OGDR; (iii): OGDR plus CASP1 inh (YVAD, 200 µM); (iv): OGDR plus NLRP3 siRNA (si); (v): OGDR plus NLRP12 si; (vi): OGDR plus NLRC4 si; (vii): CASP8 CRISPR plus OGDR; (viii): OGDR plus HIF-1α si ; (ix): OGDR plus IL-1β neutralizing antibody. Scale bar: 20 µm. g-h Immunoblotting analysis for detection of pyroptotic proteins in the retinas of NLRP12-/- or WT mice with or without NLRP3/NLRC4 knockdown under RIR conditions (n = 6). The protein levels were normalized to β-actin levels. i-n Mutual regulatory relationships among NLRP12, NLRP3 and NLRC4: i-j Protein expression and mRNA levels of the indicated molecules, as determined by western blotting and qRT-PCR detection in BV2 microglia exposed to OGDR with or without NLRP3/NLRP12/NLRC4 knockdown (n = 6). The mRNA and protein levels were normalized to β-actin levels. k-n Western blotting and qRT-PCR analyses of NLRP3, NLRP12, and NLRC4 in retinas from WT mice and NLRP12 KO mice that were sacrificed 7 days after elevated IOP injury, combined with or without NLRP3/NLRC4 knockdown (n = 6). The mRNA and protein levels were normalized to β-actin levels. The data shown are representative of at least three independent experiments. The data are represented as the mean ± SD. *P < 0.05, **P < 0.01, one-way ANOVA, two-way ANOVA and two-tailed unpaired t-test.