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

Journal: Theranostics

Article Title: Open Porous Microenvironment-regulatory Microspheres Loaded with Curcumin@BSA NPs/BMSCs for Diabetic Wound Treatment

doi: 10.7150/thno.120285

Figure Lengend Snippet: Autophagy induction by BMSC@COPM enhances macrophage polarization and promotes migration and proliferation. (A) Transwell assays of NIH-3T3 cells were observed under a microscope, which were treated with BMSC@COPM in a high-glucose environment (Scale bar: 100 μm). (B) Representative WB blot of CD206 and CD86 in RAW 264.7 cells after were treated with LPS, rapamycin or chloroquine, respectively. (C) Quantitative analysis of CD206 and CD86 protein expression in (B). (D) Quantification of the transwell assay results. (E) Representative images of LC3-II and P62 protein blots in NIH-3T3 cells treated with HG and then treated with CQ (10 μM) or BMSC@COPM. (F) Statistical analysis for P62 and the LC3-II/LC3-I ratio in (E). (G) Representative images of LC3-I/II and P62 protein blots in RAW 264.7 cells, which were induced with LPS and then treated with 10 μM rapamycin (an autophagy inducer, RAPA), 10 μM CQ, or BMSC@COPM. (H) Statistical analysis protein expression related to autophagyin. vs the control group, ** P < 0.01 and *** P < 0.001; vs the LPS group and HG group, $ P < 0.05, $$ P < 0.01 and $$$ P < 0.001; vs the BMSC@COPM group, # P < 0.05, ## P < 0.01, ### P < 0.001. The data shown in the figure are presented as the means ± SDs (n = 3).

Article Snippet: The mouse embryonic fibroblast line NIH-3T3 and the macrophage-like cell line RAW 264.7 were donated by the medical research center of Mudanjiang Medical University.

Techniques: Migration, Microscopy, Expressing, Transwell Assay, Control