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rtPA regulates the PI3K-AKT-mTOR pathway in the ICH in vitro cell model. (A, B) The DEGs between the control group and hemin group associated with the PI3K/AKT pathway (KEGG: mmu04151) and mTOR pathway (KEGG: mmu04150) were screened, and the transcriptional levels of DEGs in each group are presented as heatmaps. (C–F) Analysis of PI3K, p-AKT, AKT, p-mTOR, and mTOR of neurons ( n = 3 per group). **** P < 0.0001, vs . control group; ## P < 0.01, #### P < 0.0001, vs . hemin group. (G–J) Analysis of PI3K, p-AKT, AKT, p-mTOR, and mTOR of neurons ( n = 3 per group). (K, L) Analysis of apoptosis-associated proteins of neurons ( n = 3 per group). (M–P) Analysis of autophagy-associated proteins of neurons ( n = 3 per group). Data are shown as mean ± SEM and were analyzed by one-way analysis of variance followed by Tukey’s post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs. hemin group; & P < 0.05, && P < 0.01, &&& P < 0.001, &&&& P < 0.0001, vs . hemin + rtPA group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs. hemin + rtPA + DMSO group. AKT: RAC-alpha serine/threonine-protein kinase; DEGs: differential expression genes; DMSO: dimethyl sulfoxide; ICH: intracerebral hemorrhage; KEGG: Kyoto Encyclopedia of Genes and Genomes; LC3: microtubule-associated proteins 1A/1B light chain 3B; LY294002: PI3K inhibitor; mTOR: mammalian target of rapamycin; p62: sequestosome-1/ubiquitin-binding protein p62; 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: rtPA regulates the PI3K-AKT-mTOR pathway in the ICH in vitro cell model. (A, B) The DEGs between the control group and hemin group associated with the PI3K/AKT pathway (KEGG: mmu04151) and mTOR pathway (KEGG: mmu04150) were screened, and the transcriptional levels of DEGs in each group are presented as heatmaps. (C–F) Analysis of PI3K, p-AKT, AKT, p-mTOR, and mTOR of neurons ( n = 3 per group). **** P < 0.0001, vs . control group; ## P < 0.01, #### P < 0.0001, vs . hemin group. (G–J) Analysis of PI3K, p-AKT, AKT, p-mTOR, and mTOR of neurons ( n = 3 per group). (K, L) Analysis of apoptosis-associated proteins of neurons ( n = 3 per group). (M–P) Analysis of autophagy-associated proteins of neurons ( n = 3 per group). Data are shown as mean ± SEM and were analyzed by one-way analysis of variance followed by Tukey’s post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs. hemin group; & P < 0.05, && P < 0.01, &&& P < 0.001, &&&& P < 0.0001, vs . hemin + rtPA group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs. hemin + rtPA + DMSO group. AKT: RAC-alpha serine/threonine-protein kinase; DEGs: differential expression genes; DMSO: dimethyl sulfoxide; ICH: intracerebral hemorrhage; KEGG: Kyoto Encyclopedia of Genes and Genomes; LC3: microtubule-associated proteins 1A/1B light chain 3B; LY294002: PI3K inhibitor; mTOR: mammalian target of rapamycin; p62: sequestosome-1/ubiquitin-binding protein p62; 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, Control, Quantitative Proteomics, Ubiquitin Proteomics, Binding Assay, 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