mtorc2 activity  (Cell Signaling Technology Inc)

Journal: Brain

Article Title: Targeting spinal mechanistic target of rapamycin complex 2 alleviates inflammatory and neuropathic pain

doi: 10.1093/brain/awae275

Figure Lengend Snippet: Activation of mechanistic target of rapamycin complex 2 (mTORC2) in the spinal cord promotes pain hypersensitivity . ( A ) Schematic diagram of mechanistic target of rapamycin (mTOR) pathway. ( B ) Peripheral inflammation induced via intraplantar injection of complete Freund's adjuvant (CFA) increased spinal Akt phosphorylation at Days 1 and 7 and Rictor protein expression at Day 1 post-CFA ( n = 4 mice per group, one-way ANOVA followed by Bonferroni's post hoc comparison). ( C ) Peripheral nerve injury induced via spared nerve injury (SNI) increased Akt phosphorylation and Rictor protein levels at Day 1 and Day 28 post-injury ( n = 4 mice per group, one-way ANOVA followed by Bonferroni's post hoc comparison). ( D ) Increased p-Akt in spinal cord dorsal horn lysates of animals 6 h after intrathecal administration of A-443654 ( n = 4–5 mice per group, Student's two-tailed t -test). ( E and F ) Animals that received intrathecal administration of A-443654 show reduced mechanical and thermal thresholds in the von Frey ( E ) and radiant heat paw withdrawal ( F ) assays, respectively ( n = 9 or 10 mice per group, two-way ANOVA followed by Bonferroni's post hoc comparison) and increased spontaneous pain ( G ) ( n = 6 or 7 mice per group, Student's two-tailed t -test). All data are presented as the mean ± SEM. * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: The mTORC2 activator, compound A-443654 (MCE), was made up to a 40 mM stock in dimethyl sulfoxide, and 5 μl of 8 mM dilution made in 4% polyethylene glycol (PEG) and 4% dimethyl sulphoxide in 0.9% saline was injected intrathecally.

Techniques: Activation Assay, Injection, Adjuvant, Expressing, Comparison, Two Tailed Test

Journal: Brain

Article Title: Targeting spinal mechanistic target of rapamycin complex 2 alleviates inflammatory and neuropathic pain

doi: 10.1093/brain/awae275

Figure Lengend Snippet: Inhibition of mechanistic target of rapamycin complex 2 (mTORC2) with antisense oligonucleotide (ASO) targeting Rictor alleviates inflammatory and neuropathic pain . ( A ) Schematic diagram of Rictor-ASO intracerebroventricular (ICV) injection and behavioural time line. ( B ) Reduced Rictor and p-Akt in spinal cord dorsal horn lysates of animals 2 weeks after ICV injection of Rictor-ASO ( n = 4–5 mice per group, Student's two-tailed t -test). ( C ) There were no differences in Rictor and p-Akt protein levels between animals injected with Control- or Rictor-ASO in dorsal root ganglion (DRG) lysates ( n = 4 or 5 mice per group, Student's two-tailed t -test). ( D–G ) Animals that received Rictor-ASO show alleviation in complete Freund's adjuvant (CFA) inflammation-induced mechanical ( D ) and thermal ( E ) hypersensitivity; and carrageenan inflammation-induced mechanical ( F ) and thermal ( G ) hypersensitivity ( n = 9 or 10 mice per group, two-way ANOVA followed by Bonferroni's post hoc comparison). ( H–J ) Animals that received Rictor-ASO show alleviation in chronic constriction injury (CCI; H ) and spared nerve injury (SNI; J ) nerve injury-induced mechanical hypersensitivity and in CCI-induced thermal hypersensitivity ( I ) ( n = 9 or 10 mice per group, two-way ANOVA followed by Bonferroni's post hoc comparison). All data are presented as the mean ± SEM. n.s. = not significant; * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. Contra = contralateral; Ipsi = ipsilateral.

Article Snippet: The mTORC2 activator, compound A-443654 (MCE), was made up to a 40 mM stock in dimethyl sulfoxide, and 5 μl of 8 mM dilution made in 4% polyethylene glycol (PEG) and 4% dimethyl sulphoxide in 0.9% saline was injected intrathecally.

Techniques: Inhibition, Injection, Two Tailed Test, Control, Adjuvant, Comparison

Journal: Brain

Article Title: Targeting spinal mechanistic target of rapamycin complex 2 alleviates inflammatory and neuropathic pain

doi: 10.1093/brain/awae275

Figure Lengend Snippet: Local viral downregulation of Rictor and impairment of mechanistic target of rapamycin complex 2 (mTORC2) activity attenuates inflammatory and neuropathic pain. ( A ) Schematic diagram of AAV2/9-CAG-Cre intraspinal parenchymal injection and behavioural time line. ( B ) Reduced Rictor and p-Akt protein levels in lumbar spinal cord dorsal horn lysates of Rictor f/f animals 3 weeks after intraspinal injection of AAV2/9-CAG-Cre ( n = 4–5 mice per group, Student's two-tailed t -test). ( C–G ) Rictor f/f animals that received intraspinal injection of AAV2/9-CAG-Cre show alleviation of complete Freund's adjuvant (CFA) inflammation-induced mechanical hypersensitivity ( C ), thermal hypersensitivity ( D ) and spontaneous pain ( E ) and of spared nerve injury (SNI)-induced mechanical hypersensitivity ( F ) and spontaneous pain ( G ) ( n = 6–10 mice per group, two-way ANOVA followed by Bonferroni's post hoc comparison and Student's two-tailed t -test). Rictor f/f animals that received intraspinal injection of AAV2/9-CAG-Cre 4 weeks following SNI ( H ) show alleviation in mechanical hypersensitivity at Weeks 7 and 8 post-SNI ( I ) ( n = 12 mice per group, two-way ANOVA followed by Bonferroni's post hoc comparison). All data are presented as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. BL = baseline; WT = wild-type; MGS = Mouse Grimace Scale.

Article Snippet: The mTORC2 activator, compound A-443654 (MCE), was made up to a 40 mM stock in dimethyl sulfoxide, and 5 μl of 8 mM dilution made in 4% polyethylene glycol (PEG) and 4% dimethyl sulphoxide in 0.9% saline was injected intrathecally.

Techniques: Activity Assay, Injection, Two Tailed Test, Adjuvant, Comparison

Journal: Brain

Article Title: Targeting spinal mechanistic target of rapamycin complex 2 alleviates inflammatory and neuropathic pain

doi: 10.1093/brain/awae275

Figure Lengend Snippet: Differential regulation of mechanistic target of rapamycin complex 2 (mTORC2) in spinal cord neurons following peripheral injury. ( A ) p-Akt is increased in Vglut2 + excitatory neurons but unchanged in Pax2 + inhibitory neurons 1 day after intraplantar injection of complete Freund's adjuvant (CFA) ( n = 5 mice per group, one-way ANOVA followed by Bonferroni's post hoc comparison). ( B ) p-Akt is increased in Pax2 + inhibitory neurons but unchanged in Vglut2 + excitatory neurons 1 day after spared nerve injury (SNI) surgery ( n = 5 mice per group, one-way ANOVA followed by Bonferroni's post hoc comparison). Scale bars = 20 μm ( left ) and 200 μm ( right ). All data are presented as the mean ± SEM. n.s. = not significant; * P < 0.05 and **** P < 0.0001. Contra = contralateral; Ipsi = ipsilateral.

Article Snippet: The mTORC2 activator, compound A-443654 (MCE), was made up to a 40 mM stock in dimethyl sulfoxide, and 5 μl of 8 mM dilution made in 4% polyethylene glycol (PEG) and 4% dimethyl sulphoxide in 0.9% saline was injected intrathecally.

Techniques: Injection, Adjuvant, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Metabolic Adaptation as Potential Target in Papillary Renal Cell Carcinomas Based on Their In Situ Metabolic Characteristics

doi: 10.3390/ijms231810587

Figure Lengend Snippet: Immunohistochemical analysis of mTOR and metabolic pathway markers in papillary (PRCC) and clear cell renal cell carcinomas (CCRCC). The expression of p-mTOR (marker for mTOR kinase activity), p-S6 (mTORC1 activity marker), Rictor (marker of the amount of mTORC2), GLUT1, HXK2, PFKP (glycolysis markers), G6PD (pentose phosphate pathway marker), GLS (glutaminolysis marker), ACSS2 (marker for acetate consumption), CPT1A (fatty acid β-oxidation marker), ATPB (OXPHOS marker), COX IV, and TOM20 (mitochondrial markers) was analyzed in normal kidney, PRCCs, and CCRCCs. The detected characteristic differences in enzyme expression profiles between PRCCs and CCRCCs were highlighted by red frame. Immunohistochemistry (DAB chromogen–brown) and hematoxylin counterstaining were used. Scale bars indicate 50 μm.

Article Snippet: 60 , – , D9E , #4060 , Cell Signaling , mTORC2 activity , Anti-phospho(Ser473)-Akt.

Techniques: Immunohistochemical staining, Expressing, Marker, Activity Assay, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Metabolic Adaptation as Potential Target in Papillary Renal Cell Carcinomas Based on Their In Situ Metabolic Characteristics

doi: 10.3390/ijms231810587

Figure Lengend Snippet: Antibodies used for Protein Expression Analyses.

Article Snippet: 60 , – , D9E , #4060 , Cell Signaling , mTORC2 activity , Anti-phospho(Ser473)-Akt.

Techniques: Expressing, Activity Assay, Phospho-proteomics, Marker, Control

Journal: Scientific Reports

Article Title: Sildenafil improves hippocampal brain injuries and restores neuronal development after neonatal hypoxia–ischemia in male rat pups

doi: 10.1038/s41598-021-01097-6

Figure Lengend Snippet: Western blotting for markers of the Pl3k/AKT/MTOR pathway of the ipsilateral hippocampus in sham and HI rat pups treated with vehicle or sildenafil. Median with individual data points representation, with cropped representative western blots. Full-length blots are presented in . ( A ) p-AKT, ( B ) p-MTOR ser 2448 (mTORC1), and ( C ) p-MTOR ser 2481 (mTORC2). Significance: p value from Kruskal–Wallis test, with Dunn’s post-hoc comparison tests * p < 0.05, ** p < 0.01. Number of animals: n = 6/8/7 sham vehicle, 6/8/7 HI vehicle, and 7/8/10 HI 50 mg/kg, respectively at P12/P17/P30.

Article Snippet: To evaluate the activity of the Pl3k/AKT/mTOR pathway, we quantified the levels of phosphorylated protein kinase B (AKT) (rabbit anti-phospho-Akt Ser473 D9E, 4060; Cell Signaling Technology, Danvers, Massachusetts, USA; dilution 1:1000); the levels of phospho-mTOR serine 2448 (rabbit anti-p-mTOR Ser2448, 2971; Cell Signaling Technology, Danvers, Massachusetts, USA; dilution 1:1000), which reflect the mammalian target of rapamycin complex 1 (mTORC1) activity; the levels of phospho-mTOR Ser2481 (rabbit anti-p-mTOR Ser2481, 2974; Cell Signaling Technology, Danvers, Massachusetts, USA; dilution 1:1000), which reflect the mammalian target of rapamycin complex 2 (mTORC2) activity; and the levels of pan mTOR antibody (rabbit anti-mTOR, 2972; Cell Signaling Technology, Danvers, Massachusetts, USA; dilution 1:1000).

Techniques: Western Blot, Comparison

Journal: Experimental & Molecular Medicine

Article Title: mTORC2 activation protects retinal ganglion cells via Akt signaling after autophagy induction in traumatic optic nerve injury

doi: 10.1038/s12276-019-0298-z

Figure Lengend Snippet: Two autophagy inducers showed a dramatic difference in neuroprotective effects in the ONC model. Treatment with p62 siRNA results in a favorable neuroprotective effect by triggering specific mTORC1 deactivation and maintaining mTORC2 activation, which leads to the reduction in neuroinflammation through BOB protection and M2 macrophage polarization. Rapamycin can activate autophagy but provides no protection in the ONC model because the inactivation of mTORC2 results in BOB disruption, which may induce macrophage infiltration and pro-inflammatory cytokine-induced optic nerve damage

Article Snippet: To evaluate the therapeutic effect of mTORC2 activation in the rapamycin-treated group, mTORC2 activator SC79 (10 μg, Sigma-Aldrich, MO, USA; n = 6) was injected into rapamycin-treated rats.

Techniques: Activation Assay