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Experiment design. (A) In Experiment 1, the effect of rtPA on the ICH mouse model was investigated. (B) In Experiment 2, the effect and the possible mechanisms of rtPA in the ICH model using primary cortical neurons in vitro were investigated. (C) In Experiment 3, the mechanism of rtPA’s effect on the ICH model in neurons in vitro was examined using the PI3K pathway inhibitor. (D) In Experiment 4, the protein domain that mediates rtPA’s neuroprotective effect in the ICH model in neurons in vitro was investigated. DMSO: Dimethyl sulfoxide; ER: <t>endoplasmic</t> reticulum; H&E staining: hematoxylin & eosin staining; ICH: intracerebral hemorrhage; rtPA: recombinant tissue plasminogen activator; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling.
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SPH regulates NLRP3 inflammasome, ER stress, oxidative stress, and apoptosis/survival pathways in the hearts of hypertensive mice. (A, B) Representative Western blots and densitometric analysis for ER stress <t>(GRP78),</t> NLRP3 inflammasome components (NLRP3, ASC, C-Caspase-1, C-IL-1β, C-GSDMD), and oxidative stress (SOD1). (C–H) Relative mRNA expression of Nlrp3, Asc, Casp1, Gsdmd, IL1b, and IL18 by qRT-PCR. (I, J) Representative Western blots and densitometric analysis for apoptosis markers (BAX, BCL2, C-Caspase-3) and PI3K/AKT pathway proteins (p-PI3K, PI3K, p-AKT, AKT). GAPDH or total proteins were used for normalization. Data are mean ± SEM (n = 3 per group). Statistical analysis was by one-way ANOVA with Tukey's post-hoc test.
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Experiment design. (A) In Experiment 1, the effect of rtPA on the ICH mouse model was investigated. (B) In Experiment 2, the effect and the possible mechanisms of rtPA in the ICH model using primary cortical neurons in vitro were investigated. (C) In Experiment 3, the mechanism of rtPA’s effect on the ICH model in neurons in vitro was examined using the PI3K pathway inhibitor. (D) In Experiment 4, the protein domain that mediates rtPA’s neuroprotective effect in the ICH model in neurons in vitro was investigated. DMSO: Dimethyl sulfoxide; ER: endoplasmic reticulum; H&E staining: hematoxylin & eosin staining; ICH: intracerebral hemorrhage; rtPA: recombinant tissue plasminogen activator; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling.

Journal: Neural Regeneration Research

Article Title: Recombinant tissue plasminogen activator protects neurons after intracerebral hemorrhage through activating the PI3K/AKT/mTOR pathway

doi: 10.4103/NRR.NRR-D-23-01953

Figure Lengend Snippet: Experiment design. (A) In Experiment 1, the effect of rtPA on the ICH mouse model was investigated. (B) In Experiment 2, the effect and the possible mechanisms of rtPA in the ICH model using primary cortical neurons in vitro were investigated. (C) In Experiment 3, the mechanism of rtPA’s effect on the ICH model in neurons in vitro was examined using the PI3K pathway inhibitor. (D) In Experiment 4, the protein domain that mediates rtPA’s neuroprotective effect in the ICH model in neurons in vitro was investigated. DMSO: Dimethyl sulfoxide; ER: endoplasmic reticulum; H&E staining: hematoxylin & eosin staining; ICH: intracerebral hemorrhage; rtPA: recombinant tissue plasminogen activator; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling.

Article Snippet: The following primary antibodies were used for analysis: bcl2 (rabbit, 1:1000, Proteintech, Cat# 12789-1-AP, RRID: AB_2227948), bax (rabbit, 1:1000, Proteintech, Cat# 50599-2-Ig, RRID: AB_2061561), coiled-coil myosin-like bcl2-interacting protein (beclin1; rabbit, 1:1000, Proteintech, Cat# 11306-1-AP, RRID: AB_2259061), sequestosome-1/ubiquitin-binding protein p62 (SQSTM1/p62; rabbit, 1:1000, Abclonal, Cat# A11250, RRID: AB_2758477), microtubule-associated proteins 1A/1B light chain 3B (LC3; rabbit, 1:1000, Abcam, Cat# ab48394, RRID: AB_881433), endoplasmic reticulum chaperone BiP (Grp78/BIP; mouse, 1:1000, Proteintech, Cat# 66574-1-Ig, RRID: AB_2881934), cyclic AMP-dependent transcription factor ATF-6 alpha (ATF6; rabbit, 1:1000, Proteintech, Cat# 24169-1-AP, RRID: AB_2876891), PRKR-like endoplasmic reticulum kinase (PERK; rabbit, 1:1000, Cell Signaling Technology, Cat# 3192S, RRID: AB_2095847), phospho-PERK (rabbit, 1:1000, Cell Signaling Technology, Cat# 3179S, RRID: AB_2095853), eukaryotic translation initiation factor 2 subunit alpha (eIF2α; rabbit, 1:1000, Cell Signaling Technology, Cat# 9722S, RRID: AB_2230924), phospho-eIF2α (rabbit, 1:1000, Cell Signaling Technology, 9721S, RRID: AB_330951), phosphatidylinositol 3-kinase regulatory subunit alpha (PI3 kinase p85; rabbit, 1:1000, Cell Signaling Technology, Cat# 4257S, RRID: AB_659889), RAC-alpha serine/threonine-protein kinase (AKT; rabbit, 1:1000, Cell Signaling Technology, Cat# 4691S, RRID: AB_915783), phospho-AKT (rabbit, 1:1000, Cell Signaling Technology, Cat# 4060S, RRID: AB_2315049), mammalian target of rapamycin (mTOR; rabbit, 1:1000, Cell Signaling Technology, Cat# 2983S, RRID: AB_2105622), phospho-mTOR (rabbit, 1:1000, Cell Signaling Technology, Cat# 2971S, RRID: AB_330970), and β-actin (mouse, 1:1000, Proteintech, Cat# 66009-1-Ig, RRID: AB_2687938).

Techniques: In Vitro, Staining, Recombinant, TUNEL Assay

rtPA attenuates neurological behavior impairment and apoptosis after ICH. (A–C) Left forelimb placement experiment, corner turn experiment, and modified Garcia score testing were conducted at 1 hour before surgery and 6, 24, and 72 hours after surgery ( n = 14 per group). (D) H&E staining (top) and Nissl staining (bottom) of peri-hematoma tissue at 72 hours after ICH and rtPA treatments ( n = 3 per group). Scale bars: 100 µm. (E) Representative picture of TUNEL staining of peri-hematoma tissue conducted at 72 hours after ICH and rtPA treatments ( n = 3–6 per group). Scale bars: 100 µm. (F) The proportion of TUNEL-positive cells to all nucleated cells surrounding the hematoma ( n = 3–6 per group). (G–J) Analysis of apoptosis-associated proteins at 24 and 72 hours after treatment ( n = 3). Data are represented as mean ± SEM. * P < 0.05, **** P < 0.0001, vs. sham group; &P < 0.05, && P < 0.01, &&&& P < 0.0001, vs . ICH group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs . ICH + vehicle group (two-way analysis of variance followed by Bonferroni post hoc test (A–C) or one-way analysis of variance followed by Tukey’s post hoc test (F, I, J). bax: Apoptosis regulator bax; bcl2: apoptosis regulator bcl2; DAPI: 4′,6-diamidino-2-phenylindole; ER: endoplasmic reticulum; H&E staining: hematoxylin & eosin staining; ICH: intracerebral hemorrhage; rtPA: recombinant tissue plasminogen activator; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling.

Journal: Neural Regeneration Research

Article Title: Recombinant tissue plasminogen activator protects neurons after intracerebral hemorrhage through activating the PI3K/AKT/mTOR pathway

doi: 10.4103/NRR.NRR-D-23-01953

Figure Lengend Snippet: rtPA attenuates neurological behavior impairment and apoptosis after ICH. (A–C) Left forelimb placement experiment, corner turn experiment, and modified Garcia score testing were conducted at 1 hour before surgery and 6, 24, and 72 hours after surgery ( n = 14 per group). (D) H&E staining (top) and Nissl staining (bottom) of peri-hematoma tissue at 72 hours after ICH and rtPA treatments ( n = 3 per group). Scale bars: 100 µm. (E) Representative picture of TUNEL staining of peri-hematoma tissue conducted at 72 hours after ICH and rtPA treatments ( n = 3–6 per group). Scale bars: 100 µm. (F) The proportion of TUNEL-positive cells to all nucleated cells surrounding the hematoma ( n = 3–6 per group). (G–J) Analysis of apoptosis-associated proteins at 24 and 72 hours after treatment ( n = 3). Data are represented as mean ± SEM. * P < 0.05, **** P < 0.0001, vs. sham group; &P < 0.05, && P < 0.01, &&&& P < 0.0001, vs . ICH group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs . ICH + vehicle group (two-way analysis of variance followed by Bonferroni post hoc test (A–C) or one-way analysis of variance followed by Tukey’s post hoc test (F, I, J). bax: Apoptosis regulator bax; bcl2: apoptosis regulator bcl2; DAPI: 4′,6-diamidino-2-phenylindole; ER: endoplasmic reticulum; H&E staining: hematoxylin & eosin staining; ICH: intracerebral hemorrhage; rtPA: recombinant tissue plasminogen activator; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling.

Article Snippet: The following primary antibodies were used for analysis: bcl2 (rabbit, 1:1000, Proteintech, Cat# 12789-1-AP, RRID: AB_2227948), bax (rabbit, 1:1000, Proteintech, Cat# 50599-2-Ig, RRID: AB_2061561), coiled-coil myosin-like bcl2-interacting protein (beclin1; rabbit, 1:1000, Proteintech, Cat# 11306-1-AP, RRID: AB_2259061), sequestosome-1/ubiquitin-binding protein p62 (SQSTM1/p62; rabbit, 1:1000, Abclonal, Cat# A11250, RRID: AB_2758477), microtubule-associated proteins 1A/1B light chain 3B (LC3; rabbit, 1:1000, Abcam, Cat# ab48394, RRID: AB_881433), endoplasmic reticulum chaperone BiP (Grp78/BIP; mouse, 1:1000, Proteintech, Cat# 66574-1-Ig, RRID: AB_2881934), cyclic AMP-dependent transcription factor ATF-6 alpha (ATF6; rabbit, 1:1000, Proteintech, Cat# 24169-1-AP, RRID: AB_2876891), PRKR-like endoplasmic reticulum kinase (PERK; rabbit, 1:1000, Cell Signaling Technology, Cat# 3192S, RRID: AB_2095847), phospho-PERK (rabbit, 1:1000, Cell Signaling Technology, Cat# 3179S, RRID: AB_2095853), eukaryotic translation initiation factor 2 subunit alpha (eIF2α; rabbit, 1:1000, Cell Signaling Technology, Cat# 9722S, RRID: AB_2230924), phospho-eIF2α (rabbit, 1:1000, Cell Signaling Technology, 9721S, RRID: AB_330951), phosphatidylinositol 3-kinase regulatory subunit alpha (PI3 kinase p85; rabbit, 1:1000, Cell Signaling Technology, Cat# 4257S, RRID: AB_659889), RAC-alpha serine/threonine-protein kinase (AKT; rabbit, 1:1000, Cell Signaling Technology, Cat# 4691S, RRID: AB_915783), phospho-AKT (rabbit, 1:1000, Cell Signaling Technology, Cat# 4060S, RRID: AB_2315049), mammalian target of rapamycin (mTOR; rabbit, 1:1000, Cell Signaling Technology, Cat# 2983S, RRID: AB_2105622), phospho-mTOR (rabbit, 1:1000, Cell Signaling Technology, Cat# 2971S, RRID: AB_330970), and β-actin (mouse, 1:1000, Proteintech, Cat# 66009-1-Ig, RRID: AB_2687938).

Techniques: Modification, Staining, TUNEL Assay, Recombinant

rtPA attenuates neuron apoptosis and autophagy after experimental ICH in vitro . (A–C) The DEGs between control group and hemin group associated with autophagy animals (KEGG: mmu04140), positive regulation of neuron apoptotic process (GO: 0043525), and positive regulation of response to endoplasmic reticulum stress (GO: 1905898) were screened, and the transcriptional levels of DEGs in each group are presented as heatmaps. (D, E) Analysis of apoptosis-associated proteins. (F) Transmission electron microscopy images of neurons after hemin and rtPA treatment. Red asterisk indicates the autophagosome, black arrows indicate the endoplasmic reticulum, and N means nucleus. Scale bars: 1 µm. (G–J) Analysis of autophagy-associated proteins. Data are shown as mean ± SEM ( n = 3–4). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . control group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs. hemin group (one-way analysis of variance followed by Tukey’s post hoc test). bax: Apoptosis regulator bax; bcl2: apoptosis regulator bcl2; beclin1: coiled-coil myosin-like bcl2-interacting protein; DEGs: differential expression genes; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; LC3: microtubule-associated proteins 1A/1B light chain 3B; p62: sequestosome-1/ubiquitin-binding protein p62; rtPA: recombinant tissue plasminogen activator.

Journal: Neural Regeneration Research

Article Title: Recombinant tissue plasminogen activator protects neurons after intracerebral hemorrhage through activating the PI3K/AKT/mTOR pathway

doi: 10.4103/NRR.NRR-D-23-01953

Figure Lengend Snippet: rtPA attenuates neuron apoptosis and autophagy after experimental ICH in vitro . (A–C) The DEGs between control group and hemin group associated with autophagy animals (KEGG: mmu04140), positive regulation of neuron apoptotic process (GO: 0043525), and positive regulation of response to endoplasmic reticulum stress (GO: 1905898) were screened, and the transcriptional levels of DEGs in each group are presented as heatmaps. (D, E) Analysis of apoptosis-associated proteins. (F) Transmission electron microscopy images of neurons after hemin and rtPA treatment. Red asterisk indicates the autophagosome, black arrows indicate the endoplasmic reticulum, and N means nucleus. Scale bars: 1 µm. (G–J) Analysis of autophagy-associated proteins. Data are shown as mean ± SEM ( n = 3–4). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . control group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs. hemin group (one-way analysis of variance followed by Tukey’s post hoc test). bax: Apoptosis regulator bax; bcl2: apoptosis regulator bcl2; beclin1: coiled-coil myosin-like bcl2-interacting protein; DEGs: differential expression genes; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; LC3: microtubule-associated proteins 1A/1B light chain 3B; p62: sequestosome-1/ubiquitin-binding protein p62; rtPA: recombinant tissue plasminogen activator.

Article Snippet: The following primary antibodies were used for analysis: bcl2 (rabbit, 1:1000, Proteintech, Cat# 12789-1-AP, RRID: AB_2227948), bax (rabbit, 1:1000, Proteintech, Cat# 50599-2-Ig, RRID: AB_2061561), coiled-coil myosin-like bcl2-interacting protein (beclin1; rabbit, 1:1000, Proteintech, Cat# 11306-1-AP, RRID: AB_2259061), sequestosome-1/ubiquitin-binding protein p62 (SQSTM1/p62; rabbit, 1:1000, Abclonal, Cat# A11250, RRID: AB_2758477), microtubule-associated proteins 1A/1B light chain 3B (LC3; rabbit, 1:1000, Abcam, Cat# ab48394, RRID: AB_881433), endoplasmic reticulum chaperone BiP (Grp78/BIP; mouse, 1:1000, Proteintech, Cat# 66574-1-Ig, RRID: AB_2881934), cyclic AMP-dependent transcription factor ATF-6 alpha (ATF6; rabbit, 1:1000, Proteintech, Cat# 24169-1-AP, RRID: AB_2876891), PRKR-like endoplasmic reticulum kinase (PERK; rabbit, 1:1000, Cell Signaling Technology, Cat# 3192S, RRID: AB_2095847), phospho-PERK (rabbit, 1:1000, Cell Signaling Technology, Cat# 3179S, RRID: AB_2095853), eukaryotic translation initiation factor 2 subunit alpha (eIF2α; rabbit, 1:1000, Cell Signaling Technology, Cat# 9722S, RRID: AB_2230924), phospho-eIF2α (rabbit, 1:1000, Cell Signaling Technology, 9721S, RRID: AB_330951), phosphatidylinositol 3-kinase regulatory subunit alpha (PI3 kinase p85; rabbit, 1:1000, Cell Signaling Technology, Cat# 4257S, RRID: AB_659889), RAC-alpha serine/threonine-protein kinase (AKT; rabbit, 1:1000, Cell Signaling Technology, Cat# 4691S, RRID: AB_915783), phospho-AKT (rabbit, 1:1000, Cell Signaling Technology, Cat# 4060S, RRID: AB_2315049), mammalian target of rapamycin (mTOR; rabbit, 1:1000, Cell Signaling Technology, Cat# 2983S, RRID: AB_2105622), phospho-mTOR (rabbit, 1:1000, Cell Signaling Technology, Cat# 2971S, RRID: AB_330970), and β-actin (mouse, 1:1000, Proteintech, Cat# 66009-1-Ig, RRID: AB_2687938).

Techniques: In Vitro, Control, Transmission Assay, Electron Microscopy, Quantitative Proteomics, Ubiquitin Proteomics, Binding Assay, Recombinant

rtPA ameliorates endoplasmic reticulum stress in the in vitro ICH cell model. (A) Confocal images and three-dimensional reconstruction of endoplasmic reticulum continuity of neurons by ER tracker after hemin and rtPA treatment. Scale bars: 3 µm. (B–F) Quantitative analysis of ER stress–associated proteins of neurons. Data are shown as mean ± SEM ( n = 3 per group). * P < 0.05, ** P < 0.01, vs. control group; # P < 0.05, ## P < 0.01, vs . hemin group (one-way analysis of variance followed by Tukey’s post hoc test). (G) Immunofluorescence staining images of p-PERK (red, labeled by Cy3) in neurons after hemin and rtPA treatment. Scale bars: 50 µm. 3D: Three-dimensional; ATF6: cyclic AMP-dependent transcription factor ATF-6 alpha; DAPI: 4′,6-diamidino-2-phenylindole; eIF2α: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; Grp78: endoplasmic reticulum chaperone BiP; PERK: PRKR-like endoplasmic reticulum kinase; rtPA: recombinant tissue plasminogen activator.

Journal: Neural Regeneration Research

Article Title: Recombinant tissue plasminogen activator protects neurons after intracerebral hemorrhage through activating the PI3K/AKT/mTOR pathway

doi: 10.4103/NRR.NRR-D-23-01953

Figure Lengend Snippet: rtPA ameliorates endoplasmic reticulum stress in the in vitro ICH cell model. (A) Confocal images and three-dimensional reconstruction of endoplasmic reticulum continuity of neurons by ER tracker after hemin and rtPA treatment. Scale bars: 3 µm. (B–F) Quantitative analysis of ER stress–associated proteins of neurons. Data are shown as mean ± SEM ( n = 3 per group). * P < 0.05, ** P < 0.01, vs. control group; # P < 0.05, ## P < 0.01, vs . hemin group (one-way analysis of variance followed by Tukey’s post hoc test). (G) Immunofluorescence staining images of p-PERK (red, labeled by Cy3) in neurons after hemin and rtPA treatment. Scale bars: 50 µm. 3D: Three-dimensional; ATF6: cyclic AMP-dependent transcription factor ATF-6 alpha; DAPI: 4′,6-diamidino-2-phenylindole; eIF2α: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; Grp78: endoplasmic reticulum chaperone BiP; PERK: PRKR-like endoplasmic reticulum kinase; rtPA: recombinant tissue plasminogen activator.

Article Snippet: The following primary antibodies were used for analysis: bcl2 (rabbit, 1:1000, Proteintech, Cat# 12789-1-AP, RRID: AB_2227948), bax (rabbit, 1:1000, Proteintech, Cat# 50599-2-Ig, RRID: AB_2061561), coiled-coil myosin-like bcl2-interacting protein (beclin1; rabbit, 1:1000, Proteintech, Cat# 11306-1-AP, RRID: AB_2259061), sequestosome-1/ubiquitin-binding protein p62 (SQSTM1/p62; rabbit, 1:1000, Abclonal, Cat# A11250, RRID: AB_2758477), microtubule-associated proteins 1A/1B light chain 3B (LC3; rabbit, 1:1000, Abcam, Cat# ab48394, RRID: AB_881433), endoplasmic reticulum chaperone BiP (Grp78/BIP; mouse, 1:1000, Proteintech, Cat# 66574-1-Ig, RRID: AB_2881934), cyclic AMP-dependent transcription factor ATF-6 alpha (ATF6; rabbit, 1:1000, Proteintech, Cat# 24169-1-AP, RRID: AB_2876891), PRKR-like endoplasmic reticulum kinase (PERK; rabbit, 1:1000, Cell Signaling Technology, Cat# 3192S, RRID: AB_2095847), phospho-PERK (rabbit, 1:1000, Cell Signaling Technology, Cat# 3179S, RRID: AB_2095853), eukaryotic translation initiation factor 2 subunit alpha (eIF2α; rabbit, 1:1000, Cell Signaling Technology, Cat# 9722S, RRID: AB_2230924), phospho-eIF2α (rabbit, 1:1000, Cell Signaling Technology, 9721S, RRID: AB_330951), phosphatidylinositol 3-kinase regulatory subunit alpha (PI3 kinase p85; rabbit, 1:1000, Cell Signaling Technology, Cat# 4257S, RRID: AB_659889), RAC-alpha serine/threonine-protein kinase (AKT; rabbit, 1:1000, Cell Signaling Technology, Cat# 4691S, RRID: AB_915783), phospho-AKT (rabbit, 1:1000, Cell Signaling Technology, Cat# 4060S, RRID: AB_2315049), mammalian target of rapamycin (mTOR; rabbit, 1:1000, Cell Signaling Technology, Cat# 2983S, RRID: AB_2105622), phospho-mTOR (rabbit, 1:1000, Cell Signaling Technology, Cat# 2971S, RRID: AB_330970), and β-actin (mouse, 1:1000, Proteintech, Cat# 66009-1-Ig, RRID: AB_2687938).

Techniques: In Vitro, Control, Immunofluorescence, Staining, Labeling, Recombinant

The PI3K inhibitor LY294002 reverses the anti-ER stress effect of rtPA and the EGF domain of rtPA may mediate the PI3K/AKT pathway in the ICH in vitro cell model. (A–C) Analysis of ER stress–associated proteins ( n = 3 per group). (D) Confocal images and three-dimensional reconstruction of endoplasmic reticulum continuity of neurons by ER tracker after rtPA and PI3K inhibitor LY294002 treatment. Scale bars: 3 µm. (E) Immunofluorescence images of p-PERK (red, labeled by Cy3) in neurons after rtPA and PI3K inhibitor LY294002 treatment. Scale bars: 50 µm. (F–H) Analysis of PI3K p85 and p-AKT. Data are represented as mean ± SEM ( n = 3 per group). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . hemin group; & P < 0.05, && P < 0.01, vs . hemin + rtPA group; # P < 0.05, vs . hemin + rtPA + DMSO group (one-way analysis of variance followed by Tukey’s post hoc test). (I) Transmission electron microscopy images of cells after rtPA and rtPA domain inhibitor treatment. Scale bar: 100 µm. 3D: Three-dimensional; AKT: RAC-alpha serine/threonine-protein kinase; ATF6: cyclic AMP-dependent transcription factor ATF-6 alpha; DAPI: 4′,6-diamidino-2-phenylindole; EGF: epidermal growth factor; eIF2α: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; LY294002: PI3K inhibitor; mTOR: mammalian target of rapamycin; PERK: PRKR-like endoplasmic reticulum kinase; PI3K: phosphatidylinositol 3-kinase regulatory subunit alpha; rtPA: recombinant tissue plasminogen activator.

Journal: Neural Regeneration Research

Article Title: Recombinant tissue plasminogen activator protects neurons after intracerebral hemorrhage through activating the PI3K/AKT/mTOR pathway

doi: 10.4103/NRR.NRR-D-23-01953

Figure Lengend Snippet: The PI3K inhibitor LY294002 reverses the anti-ER stress effect of rtPA and the EGF domain of rtPA may mediate the PI3K/AKT pathway in the ICH in vitro cell model. (A–C) Analysis of ER stress–associated proteins ( n = 3 per group). (D) Confocal images and three-dimensional reconstruction of endoplasmic reticulum continuity of neurons by ER tracker after rtPA and PI3K inhibitor LY294002 treatment. Scale bars: 3 µm. (E) Immunofluorescence images of p-PERK (red, labeled by Cy3) in neurons after rtPA and PI3K inhibitor LY294002 treatment. Scale bars: 50 µm. (F–H) Analysis of PI3K p85 and p-AKT. Data are represented as mean ± SEM ( n = 3 per group). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . hemin group; & P < 0.05, && P < 0.01, vs . hemin + rtPA group; # P < 0.05, vs . hemin + rtPA + DMSO group (one-way analysis of variance followed by Tukey’s post hoc test). (I) Transmission electron microscopy images of cells after rtPA and rtPA domain inhibitor treatment. Scale bar: 100 µm. 3D: Three-dimensional; AKT: RAC-alpha serine/threonine-protein kinase; ATF6: cyclic AMP-dependent transcription factor ATF-6 alpha; DAPI: 4′,6-diamidino-2-phenylindole; EGF: epidermal growth factor; eIF2α: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; LY294002: PI3K inhibitor; mTOR: mammalian target of rapamycin; PERK: PRKR-like endoplasmic reticulum kinase; PI3K: phosphatidylinositol 3-kinase regulatory subunit alpha; rtPA: recombinant tissue plasminogen activator.

Article Snippet: The following primary antibodies were used for analysis: bcl2 (rabbit, 1:1000, Proteintech, Cat# 12789-1-AP, RRID: AB_2227948), bax (rabbit, 1:1000, Proteintech, Cat# 50599-2-Ig, RRID: AB_2061561), coiled-coil myosin-like bcl2-interacting protein (beclin1; rabbit, 1:1000, Proteintech, Cat# 11306-1-AP, RRID: AB_2259061), sequestosome-1/ubiquitin-binding protein p62 (SQSTM1/p62; rabbit, 1:1000, Abclonal, Cat# A11250, RRID: AB_2758477), microtubule-associated proteins 1A/1B light chain 3B (LC3; rabbit, 1:1000, Abcam, Cat# ab48394, RRID: AB_881433), endoplasmic reticulum chaperone BiP (Grp78/BIP; mouse, 1:1000, Proteintech, Cat# 66574-1-Ig, RRID: AB_2881934), cyclic AMP-dependent transcription factor ATF-6 alpha (ATF6; rabbit, 1:1000, Proteintech, Cat# 24169-1-AP, RRID: AB_2876891), PRKR-like endoplasmic reticulum kinase (PERK; rabbit, 1:1000, Cell Signaling Technology, Cat# 3192S, RRID: AB_2095847), phospho-PERK (rabbit, 1:1000, Cell Signaling Technology, Cat# 3179S, RRID: AB_2095853), eukaryotic translation initiation factor 2 subunit alpha (eIF2α; rabbit, 1:1000, Cell Signaling Technology, Cat# 9722S, RRID: AB_2230924), phospho-eIF2α (rabbit, 1:1000, Cell Signaling Technology, 9721S, RRID: AB_330951), phosphatidylinositol 3-kinase regulatory subunit alpha (PI3 kinase p85; rabbit, 1:1000, Cell Signaling Technology, Cat# 4257S, RRID: AB_659889), RAC-alpha serine/threonine-protein kinase (AKT; rabbit, 1:1000, Cell Signaling Technology, Cat# 4691S, RRID: AB_915783), phospho-AKT (rabbit, 1:1000, Cell Signaling Technology, Cat# 4060S, RRID: AB_2315049), mammalian target of rapamycin (mTOR; rabbit, 1:1000, Cell Signaling Technology, Cat# 2983S, RRID: AB_2105622), phospho-mTOR (rabbit, 1:1000, Cell Signaling Technology, Cat# 2971S, RRID: AB_330970), and β-actin (mouse, 1:1000, Proteintech, Cat# 66009-1-Ig, RRID: AB_2687938).

Techniques: In Vitro, Immunofluorescence, Labeling, Transmission Assay, Electron Microscopy, Recombinant

SPH regulates NLRP3 inflammasome, ER stress, oxidative stress, and apoptosis/survival pathways in the hearts of hypertensive mice. (A, B) Representative Western blots and densitometric analysis for ER stress (GRP78), NLRP3 inflammasome components (NLRP3, ASC, C-Caspase-1, C-IL-1β, C-GSDMD), and oxidative stress (SOD1). (C–H) Relative mRNA expression of Nlrp3, Asc, Casp1, Gsdmd, IL1b, and IL18 by qRT-PCR. (I, J) Representative Western blots and densitometric analysis for apoptosis markers (BAX, BCL2, C-Caspase-3) and PI3K/AKT pathway proteins (p-PI3K, PI3K, p-AKT, AKT). GAPDH or total proteins were used for normalization. Data are mean ± SEM (n = 3 per group). Statistical analysis was by one-way ANOVA with Tukey's post-hoc test.

Journal: International Journal of Cardiology. Cardiovascular Risk and Prevention

Article Title: The sphingolipid metabolite sphingosine protects against hypertension by targeting metabolic-inflammatory crosstalk via the NLRP3 inflammasome

doi: 10.1016/j.ijcrp.2025.200562

Figure Lengend Snippet: SPH regulates NLRP3 inflammasome, ER stress, oxidative stress, and apoptosis/survival pathways in the hearts of hypertensive mice. (A, B) Representative Western blots and densitometric analysis for ER stress (GRP78), NLRP3 inflammasome components (NLRP3, ASC, C-Caspase-1, C-IL-1β, C-GSDMD), and oxidative stress (SOD1). (C–H) Relative mRNA expression of Nlrp3, Asc, Casp1, Gsdmd, IL1b, and IL18 by qRT-PCR. (I, J) Representative Western blots and densitometric analysis for apoptosis markers (BAX, BCL2, C-Caspase-3) and PI3K/AKT pathway proteins (p-PI3K, PI3K, p-AKT, AKT). GAPDH or total proteins were used for normalization. Data are mean ± SEM (n = 3 per group). Statistical analysis was by one-way ANOVA with Tukey's post-hoc test.

Article Snippet: The following primary antibodies were used: NLRP3 (Abcam, Cat# ab270449), ASC (Abcam, Cat#ab307560), Caspase-1 (Proteintech, Cat# 31020-1-AP), Cleaved Caspase-1 (CST, Cat# 89332), IL-1β(Abcam, Cat# ab254360), Cleaved IL-1β (CST, Cat#63124), IL- 18 (Proteintech, Cat# 33710-1-AP), SOD1 (Proteintech, Cat# 10269-1-AP), BCL2 (Proteintech, Cat# 26593-1-AP), BAX(Proteintech, Cat# 50599-2-Ig), and GRP78 (Proteintech, Cat# 11587-1-AP),GAPDH (Proteintech, Cat#60004-1-Ig), Phospho-PI3 Kinase p85 (CST, Cat#4228T), Phospho-AKT(Proteintech, Cat#66444-1-Ig), AKT(Proteintech, Cat#60203-2-Ig), GSDMD (N-Terminal) (Affinity,Cat# DF13758),GSDMD (Proteintech,Cat# 20770-1-AP).

Techniques: Western Blot, Expressing, Quantitative RT-PCR

SPH protects human umbilical vein endothelial cells (HUVECs) from Angiotensin IIinduced injury, pyroptosis, and oxidative stress by inhibiting the NLRP3 inflammasome pathway. (A) Cell viability of HUVECs treated with AngII (1 μM) for 12 h followed by various concentrations of SPH (0.5–6 μM) for 24 h. (B) Cell viability of HUVECs treated with SPH alone for 24 h. (C, D) Representative Western blots and densitometric analysis for NLRP3 inflammasome components (NLRP3, ASC, c-Caspase-1, c-IL-1β), the pyroptosis effector N-GSDMD, ER stress marker GRP78, and oxidative stress marker SOD1. (E–J) Relative mRNA expression of NLRP3, ASC, CASP1, IL1B, IL18, and GSDMD by RT-qPCR. Data are the mean ± SEM of three independent experiments. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 vs. the AngII-only group, unless otherwise indicated.

Journal: International Journal of Cardiology. Cardiovascular Risk and Prevention

Article Title: The sphingolipid metabolite sphingosine protects against hypertension by targeting metabolic-inflammatory crosstalk via the NLRP3 inflammasome

doi: 10.1016/j.ijcrp.2025.200562

Figure Lengend Snippet: SPH protects human umbilical vein endothelial cells (HUVECs) from Angiotensin IIinduced injury, pyroptosis, and oxidative stress by inhibiting the NLRP3 inflammasome pathway. (A) Cell viability of HUVECs treated with AngII (1 μM) for 12 h followed by various concentrations of SPH (0.5–6 μM) for 24 h. (B) Cell viability of HUVECs treated with SPH alone for 24 h. (C, D) Representative Western blots and densitometric analysis for NLRP3 inflammasome components (NLRP3, ASC, c-Caspase-1, c-IL-1β), the pyroptosis effector N-GSDMD, ER stress marker GRP78, and oxidative stress marker SOD1. (E–J) Relative mRNA expression of NLRP3, ASC, CASP1, IL1B, IL18, and GSDMD by RT-qPCR. Data are the mean ± SEM of three independent experiments. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 vs. the AngII-only group, unless otherwise indicated.

Article Snippet: The following primary antibodies were used: NLRP3 (Abcam, Cat# ab270449), ASC (Abcam, Cat#ab307560), Caspase-1 (Proteintech, Cat# 31020-1-AP), Cleaved Caspase-1 (CST, Cat# 89332), IL-1β(Abcam, Cat# ab254360), Cleaved IL-1β (CST, Cat#63124), IL- 18 (Proteintech, Cat# 33710-1-AP), SOD1 (Proteintech, Cat# 10269-1-AP), BCL2 (Proteintech, Cat# 26593-1-AP), BAX(Proteintech, Cat# 50599-2-Ig), and GRP78 (Proteintech, Cat# 11587-1-AP),GAPDH (Proteintech, Cat#60004-1-Ig), Phospho-PI3 Kinase p85 (CST, Cat#4228T), Phospho-AKT(Proteintech, Cat#66444-1-Ig), AKT(Proteintech, Cat#60203-2-Ig), GSDMD (N-Terminal) (Affinity,Cat# DF13758),GSDMD (Proteintech,Cat# 20770-1-AP).

Techniques: Western Blot, Marker, Expressing, Quantitative RT-PCR