Journal: Molecular Neurodegeneration

Article Title: NLRP12 collaborates with NLRP3 and NLRC4 to promote pyroptosis inducing ganglion cell death of acute glaucoma

doi: 10.1186/s13024-020-00372-w

Figure Lengend Snippet: 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.

Article Snippet: The BV2 microglial cell line was purchased from Procell Co., Ltd. (#CL-0493, Wuhan, China) and has been authenticated by Microread Genetics Co., Ltd. (Beijing, China) using PCR amplification.

Techniques: Western Blot, CRISPR, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: Molecular Neurodegeneration

Article Title: NLRP12 collaborates with NLRP3 and NLRC4 to promote pyroptosis inducing ganglion cell death of acute glaucoma

doi: 10.1186/s13024-020-00372-w

Figure Lengend Snippet: CASP8-mediated HIF-1α signaling is involved in retinal ischemic injury and RGCs loss. a HE staining and quantitative evaluation of total retinal thickness in retinal tissue subjected to high IOP followed by HIF-1α knockdown (20 μΜ, n = 6). Scale bar: 50 μm. b Retrograde FG labeling and quantitative measurement of RGCs survival from mice subjected to RIR injury in the absence or presence of HIF-1α interference (20 μΜ, n = 6). Scale bar: 200 μm. c Representative immunofluorescence images of RGCs in the retina from mice treated with or without HIF-1α blockage. Primary antibody against RBPMS was used to label RGCs ( n = 6). Scale bar: 100 μm. d CRISPR-CAS9 design to knock out CASP8 in BV2 cell line. Targeted vector was designed based on the exon 3 to exon 5 in WT allele. e-f RNA level and protein levels of HIF-1α in WT and CASP8 KO BV2 cell line exposed to OGDR insult ( n = 6, both). The protein and mRNA levels were normalized to β-actin levels. g BV2 microglia with the indicated genotypes were subjected to OGDR and stained with antibodies against cleaved-CASP8 and HIF-1α ( n = 6). Scale bar: 100 μm. h CASP8 activity ( n = 5). i Representative images of immunofluorescence staining targeting phospho-NF-kB P65 translocation in WT BV2 microglia and CASP8-specific KO cell line exposed to OGDR ( n = 6). Scale bar: 20 μm. j The protein levels of HIF-1α were assayed by immunoblots in BV2 microglia treated with the NF-kB P65 inhibitor, JSH-23 (40 μM, n = 6). The protein expression was normalized to β-actin expression. RIR: retinal ischemia-reperfusion; GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; HIF-1α si: HIF-1α siRNA. All of the data are representative of at least three independent experiments. Data are represented as the mean ± SD. * P < 0.05, ** P < 0.01, one-way ANOVA and two-tailed unpaired t-test.

Article Snippet: The BV2 microglial cell line was purchased from Procell Co., Ltd. (#CL-0493, Wuhan, China) and has been authenticated by Microread Genetics Co., Ltd. (Beijing, China) using PCR amplification.

Techniques: Staining, Labeling, Immunofluorescence, CRISPR, Knock-Out, Plasmid Preparation, Activity Assay, Translocation Assay, Western Blot, Expressing, Two Tailed Test

Journal: Molecular Neurodegeneration

Article Title: NLRP12 collaborates with NLRP3 and NLRC4 to promote pyroptosis inducing ganglion cell death of acute glaucoma

doi: 10.1186/s13024-020-00372-w

Figure Lengend Snippet: CASP8 promotes NLRP12/NLRP3/NLRC4 and CASP1 activation upon HIF-1α signaling. a-b The protein and mRNA levels of NLRP12/NLRP3/NLRC4 and CASP1 was detected in retinas from WT mice with or without CASP8 knockdown (20 μΜ) that were harvested at the seventh day after reperfusion ( n = 6). The protein and mRNA levels were normalized to β-actin levels. c-d CASP8 elimination diminished the activation of NLRP12/NLRP3/NLRC4 and CASP1 in BV2 microglia exposed to OGDR ( n = 6). The protein and mRNA levels were normalized to β-actin levels. e-h Immunoblot and qRT-PCR analyses of targeting NLRP12/NLRP3/NLRC4 and CASP1 in vivo and in vitro (n = 6) with or without HIF-1α knockdown. The protein and mRNA levels were normalized to β-actin levels. i-j Protein and mRNA levels of HIF-1α upon NLRP12/NLRP3/NLRC4 suppression in vitro ( n = 6). The mRNA and protein levels were normalized to β-actin levels. k-l Knockdown of CASP1 suppressed the production of HIF-1α protein and mRNA in vitro ( n = 6). 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

Article Snippet: The BV2 microglial cell line was purchased from Procell Co., Ltd. (#CL-0493, Wuhan, China) and has been authenticated by Microread Genetics Co., Ltd. (Beijing, China) using PCR amplification.

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, In Vivo, In Vitro

Journal: Molecular Neurodegeneration

Article Title: NLRP12 collaborates with NLRP3 and NLRC4 to promote pyroptosis inducing ganglion cell death of acute glaucoma

doi: 10.1186/s13024-020-00372-w

Figure Lengend Snippet: Activation of the CASP8-HIF-1α pathway elicits pyroptosis and promotes IL-1β production which in turn magnifies inflammatory cascades via the CASP8-HIF-1α-NLR axis. a The protein levels of cleaved GSDMD were detected in retinas from WT mice with or without CASP8 knockdown (20 μΜ) that were harvested at the seventh day after reperfusion ( n = 6). The protein levels were normalized to β-actin levels. b Western blot analysis of cleaved CASP1 and GSDMD in extracts from WT BV2 microglia and CASP8-specific KO cell line after OGDR injury ( n = 6). The protein levels were normalized to β-actin levels. c-d Cytotoxicity c and IL-1β production d in BV2 microglia under OGDR injury ( n = 6). e-f The protein levels of cleaved GSDMD were detected in vivo and in vitro with or without HIF-1α knockdown. The protein levels were normalized to β-actin levels. g-h Cytotoxicity g and IL-1β processing h were measured in the presence or absence of HIF-1α siRNA treatment in BV2 microglia ( n = 6, both). i Western blotting detection of the indicated proteins in BV2 microglia subjected to OGDR and OGDR concomitant with IL-1β neutralizing antibody treatment ( n = 6). The protein levels were normalized to β-actin levels. j CASP8 activity ( n = 6). The data shown are representative of at least three independent experiments. Data are represented as the mean ± SD. * P < 0.05, ** P < 0.01, experiments were assessed by one-way ANOVA, two-way ANOVA or two-tailed unpaired t-test

Article Snippet: The BV2 microglial cell line was purchased from Procell Co., Ltd. (#CL-0493, Wuhan, China) and has been authenticated by Microread Genetics Co., Ltd. (Beijing, China) using PCR amplification.

Techniques: Activation Assay, Western Blot, In Vivo, In Vitro, Activity Assay, Two Tailed Test