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Fig. 5 | Molecular Neurodegeneration

Fig. 5

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

Fig. 5

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.

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