Fig 1: Recombinant Shh affected neuronal PANoptosis and S100A10 protein expression after OGD/R injury in vitro.(A) The experimental procedure. (B–F) Relative expression of Shh, Ptc-1, Smo, and Gli1 in primary cortical neurons after rShh treatment, as determined by Western blotting. (G–P) Western blot analysis was used to assess the levels of PANoptosis-related proteins in the control and OGD/R groups. (Q) Neuronal viability was evaluated using the CCK-8 assay. Data are expressed as the mean ± SD. All experiments were independently repeated at least three times. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, vs. control group; #P < 0.05, ##P < 0.01, vs. Control + rShh group; ^P < 0.05, ^^P < 0.01, ^^^P < 0.001, vs. OGD/R+rShh group (two-tailed Student’s t test [C–F] or one-way analysis of variance followed by Tukey’s multiple comparison test [H‒J, L, M, O‒Q]). CASP1: Cysteinyl aspartate specific protease 1, caspase 1; CASP3: cysteinyl aspartate specific protease 3, caspase 3; CASP8: cysteinyl aspartate specific protease 8, caspase 8; CCK-8: Cell Counting Kit-8; GAPDH: glyceraldehyde phosphate dehydrogenase; Gli1: Glioma-associated oncogene 1; NLRP3: nucleotide-binding domain-like receptor protein 3; ns: not significant; OGD/R: oxygen‒glucose deprivation/reperfusion; p-MLKL: phosphorylated mixed lineage kinase domain‒like protein; Ptc-1: patched-1; rShh: recombinant Shh; S100A10: S100 calcium-binding protein A10; Shh: Sonic Hedgehog; Smo: smoothened; WB: Western blot; ZBP1: Z-DNA binding protein 1.
Fig 2: Recombinant Shh reversed the effects of S100A10 knockdown on PANoptosis after MCAO/R injury in rats.(A) The experimental procedure. (B) Immunofluorescence staining of S100A10 (green), NeuN (red), and DAPI (nuclei, blue) in the Ad-NC and Ad-shS100A10 groups. Scale bar: 100 μm. (C) Quantitative analysis of NeuN+S100A10+ cells. (D–Q) Western blot analysis of the expression of PANoptosis-related proteins in the sham and MCAO/R 3d groups (n = 3). (R, S) TTC staining was used to assess the infarct volume 3 days post-reperfusion (n = 5). While in R indicates infarct regions. (T–V) Neurological deficit assessments 3 days after MCAO/R injury (n = 10) were quantified through the Longa score (T), mNSS (U), and Bederson score (V). Data are expressed as the mean ± SD. All experiments were independently repeated at least three times. *P < 0.05, **P < 0.01, ***P < 0.001, vs. sham group; #P < 0.05, ##P < 0.01, ###P < 0.001, vs. MCAO/R + rShh group; ^P < 0.05, ^^P < 0.01, ^^^P < 0.001, vs. MCAO/R + Ad-shS100A10 + rShh group (two-tailed Student’s t-test [C] or one-way analysis of variance followed by Tukey’s multiple comparison test [E–K, M, N, P, Q, S–V]). CASP1: Cysteinyl aspartate specific protease 1, caspase 1; CASP3: cysteinyl aspartate specific protease 3, caspase 3; CASP8: cysteinyl aspartate specific protease 8, caspase 8; DAPI: 4’,6-diamidino-2-phenylindole; GAPDH: glyceraldehyde phosphate dehydrogenase; Gli1: glioma-associated oncogene 1; IF: immunofluorescence; MCAO/R: middle cerebral artery occlusion/reperfusion; NeuN: neuronal nuclei; NLRP3: nucleotide-binding domain-like receptor protein 3; ns: not significant; p-MLKL: phosphorylated mixed lineage kinase domain-like protein; Ptc-1: patched-1; rShh: recombinant Shh; S100A10: S100 calcium-binding protein A10; Shh: sonic hedgehog; Smo: smoothened; TTC: 2,3,5-triphenyl tetrazolium chloride; WB: western blot; ZBP1: Z-DNA binding protein 1.
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