Journal: Aging Cell

Article Title: Activated mTOR Signaling in the RPE Drives EMT , Autophagy, and Metabolic Disruption, Resulting in AMD ‐Like Pathology in Mice

doi: 10.1111/acel.70018

Figure Lengend Snippet: Generation and characterization of mLST8 KI mice. (a) Cartoon showing that in addition to mTOR, mLST8 is a major component of both mTOR complexes 1 and 2. (b) Schematic showing the strategy for generating mLST8 KI mice. Briefly, TGA stop codon in the mouse Best1 gene was replaced with the “T2A‐mouse mLST8 CDS (coding sequence)” cassette. The targeting vector was generated by PCR using BAC clone RP23‐340G9 from the C57BL/6 library as a template. The targeting vector had Neo cassette, which was flanked by SDA (self‐deletion anchor) sites. DTA (diptheria toxin A) was used for negative selection. C57BL/6 embryonic stem cells were used for gene targeting. (c) Western blot analysis indicating that RPE lysates shows overexpression of mLST8 relative to WT. Such changes were not seen in retina lysates from the same mice. Retina RPE lysate preparation was confirmed by evaluating the levels of GFAP and RPE65 in the respective lysates n = 3. ** p < 0.01. (d) Western blot from RPE lysates of mLST8 KI RPE cells further revealed an increased ratio of p‐S6K/S6K, p‐4EBP/4EBP, p‐ULK1/ULK1, and p‐Akt1/Akt1 in these cells, compared to controls (WT) n = 3. * p < 0.05. (e) Western blot showing that mLST8 overexpression (AAV2‐ mLST8 ) in Raptor or Rictor KO MEF cells activates mTORC2 target (p‐Akt1) and mTORC1 target (p‐P70S6K), respectively, compared to controls n = 3. **** p < 0.001, ns = not significant.

Article Snippet: The cells from all the experimental groups were lysed in the CHAPS lysis buffer, and Raptor and Rictor antibodies were used to immunoprecipitate mTOR bound complexes using protein Ig beads (Thermo Fisher, 88802). mTORC1 and mTORC2 target proteins S6K1 (Sino Biological, 10099‐H09B‐50) and Akt1 (EMD Milipore, 14–279) were given to the immune‐precipitated complexes and incubated for 15 min in 37°C.

Techniques: Sequencing, Plasmid Preparation, Generated, Selection, Western Blot, Over Expression

Journal: Aging Cell

Article Title: Activated mTOR Signaling in the RPE Drives EMT , Autophagy, and Metabolic Disruption, Resulting in AMD ‐Like Pathology in Mice

doi: 10.1111/acel.70018

Figure Lengend Snippet: βA3/A1‐crystallin overexpression in the RPE rescues autophagy and melanosome alterations and retinal structure/function in mLST8 KI mice. (a) Cartoon showing the strategy for subretinal injection of the AAV2‐m Cryba1 construct into one eye of 8‐month‐old mLST8 KI mice, with the contralateral eyes receiving PBS vehicle. Animals were euthanized 2 and 4 months after injection. (b, c) Western blot analysis and densitometry showing that Cryba1 overexpression in the RPE (confirmed by western blot in b) of mLST8 KI mice could rescue the abnormal levels of both mTORC1 (p‐S6K) and mTORC2 (p‐Akt1) targets (b), and the melanosome marker (PMEL; b), as well as rescued the levels of major regulators of autophagosome formation (Atg9b, Atg7; c) in these animals (b, c), relative to PBS injected eyes (b, c). n = 3. * p < 0.05, ** p < 0.01. AAV2‐m Cryba1 treatment (subretinal injection) to mLST8 KI mice for 4 months rescued retinal function as evident from increase in scotopic (d) a‐ and (e) b‐wave amplitudes after the treatment, compared to PBS‐injected contralateral eyes of the KI mouse. n = 4. **** p < 0.0001, * p < 0.05. (f) AAV2‐m Cryba1 treatment to mLST8 KI mice for 4 months also rescued early RPE changes (arrows) like the patchy appearance of the monolayer and decline in thickness (spider plot), compared to PBS–treated contralateral eyes of the mLST8 KI mouse. n = 4. Scale bar = 20 μm. * p < 0.05. (g) Cartoon depicting overexpression of mLST8 in RPE cells ( mLST8 KI mice) activated both mTORC1 and mTORC2, disrupting glucose metabolism, mitochondrial function, autophagy, and melanosome function, leading to debris accumulation, EMT activation, and age‐related retinal degeneration resembling AMD. Targeting mTOR with inhibitors or modulators rescued these changes, suggesting a potential therapeutic strategy for retinal diseases by modulating mTOR signaling. Created with BioRender.com .

Article Snippet: The cells from all the experimental groups were lysed in the CHAPS lysis buffer, and Raptor and Rictor antibodies were used to immunoprecipitate mTOR bound complexes using protein Ig beads (Thermo Fisher, 88802). mTORC1 and mTORC2 target proteins S6K1 (Sino Biological, 10099‐H09B‐50) and Akt1 (EMD Milipore, 14–279) were given to the immune‐precipitated complexes and incubated for 15 min in 37°C.

Techniques: Over Expression, Injection, Construct, Western Blot, Marker, Activation Assay

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: FASEB BioAdvances

Article Title: NCAPD3 ‐mediated AKT activation regulates prostate cancer progression

doi: 10.1096/fba.2024-00073

Figure Lengend Snippet: NCAPD3 increased the level of STAT3 and recruited more STAT3 to the promoters of EZH2 and JAK2 and then activated EZH2/NSD2/mTORC2 and JAK2/PI3K pathways, which phosphorylated AKT at Thr 308 and Ser 473. Moreover, there is a positive mutual activation between STAT3 and JAK2, further enhanced by NCAPD3 to promote PCa progression. The graph was drawn by Figdraw.

Article Snippet: The inhibitors for PI3K (LY294002; Cat#HY‐10108), JAK2 (Z3; Cat#HY‐15480), mTORC2 (Torin1; Cat#HY‐13003), STAT3 (Stattic; Cat#HY‐13818), and AKT (MK‐2206; Cat#HY‐10385) were from MedChemExpress (Newark, NJ, USA) and inhibitor for EZH2 (GSK126; Cat#500561) were from Merck (Shanghai, China).

Techniques: Activation Assay

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Curbing Breast Cancer by Altering V-ATPase Action on F-Actin, Heterochromatin, ETV7 and mTORC2 Signaling.

doi: 10.33594/000000706

Figure Lengend Snippet: Fig. 12. Proton pump inhibition reduces mTOR signaling but has variable effects on AKT. (A, B) Left: Representative cropped western blots for p-mTORC1, p- mTORC2, mTOR and β-actin loading control in CTRL and BAF treated lowly metastatic MCF7 (A) and highly metastatic MB231 cells (B). Right: average band densities ± SEM of p- mTORC1, p-mTORC2, and mTOR proteins for CTRL and BAF treated MCF7 (A) and MB231 cells (B), expressed as percent of control. p-mTORC1 and p-mTORC2 were normalized by mTOR; mTOR was normalized by β-actin. (C, D) Left: Representative cropped western blots for p-AKT, AKT, and β-actin loading control in CTRL and BAF treated MCF7 (C) and MB231 cells (D). Right: average band densities of p-AKT and AKT ± SEM for CTRL and BAF treated MCF7 (C) and MB231 cells (D), expressed as percent of control. p-AKT was normalized by AKT; AKT was normalized by β-actin. * P<0.05, N = 4, statistical comparisons used the two-sample Kolmogorov-Smirnov test.

Article Snippet: Primary antibodies for actin (DSHB JLA20s), lamin A/C (DSHB MANLAC1(4A7)), HP1α for heterochromatin (CST 2616), ETV7 (DSHB PCRP-ETV71A1-s), V-ATPase (DSHB 224-256-2-s), p-mTORC1 (Ser2448) (CST 5536), p-mTORC2 (Ser2481) (CST 2974), mTOR (DSHB CPTC-MTOR-1-s), p-AKT (Ser473) (CST 4060), and AKT (DSHB CPTC-AKT1-2-s) were used.

Techniques: Inhibition, Western Blot, Control