Journal: bioRxiv

Article Title: Inhibition of S6K lowers age-related inflammation and immunosenescence and increases lifespan through the endolysosomal system

doi: 10.1101/2022.08.25.505264

Figure Lengend Snippet: a , Adult-onset repression of S6K ubiquitously using actGS>S6K RNAi extended lifespan (n=200). b , Adult-onset repression of S6K in the fat body using Lsp2GS>S6K RNAi extended lifespan (n=180). c , Rapamycin extended lifespan of control flies, but not of flies with ubiquitous overexpression of constitutively active S6K ( daGS>S6K CA ). Ubiquitous overexpression of constitutively active S6K significantly attenuated the response to rapamycin treatment (rapamycin: p<0.0001, daGS>S6K CA induction: p<0.0001, interaction p=0.0236, n=200). d , Adult-onset S6K activation in the fat body ( Lsp2GS>S6K CA ) significantly attenuated rapamycin-related longevity (rapamycin: p<0.0001, Lsp2GS>S6K CA induction: p<0.0001, interaction p=0.0331, n=200). Log-rank test and Cox Proportional Hazards (CPH) test.

Article Snippet: Membranes were blocked by Intercept TBS Blocking Buffer (LI-COR, #927-60001) for 1 h and probed with the following primary antibodies diluted in Intercept T20 TBS Antibody Diluent (LI-COR, #927-65001): anti-S6K (1:1,000), anti-pS6K T398 (Cell Signaling Technology, #9209, 1:1,000), anti-Relish (Developmental Studies Hybridoma Bank, #21F3, 1:100), anti-tubulin (Sigma, #T9026,1:5,000), anti-puromycin (Sigma, #MABE343, 1:1,000), anti-Stx12 (Synaptic Systems, #110 132, 1:1000).

Techniques: Over Expression, Activation Assay

Journal: bioRxiv

Article Title: Inhibition of S6K lowers age-related inflammation and immunosenescence and increases lifespan through the endolysosomal system

doi: 10.1101/2022.08.25.505264

Figure Lengend Snippet: a , Lysotracker staining of fat bodies from young (day 10) flies treated with rapamycin and overexpressing constitutively active S6K ( Lsp2GS>S6K CA ). Fat body-specific adult-onset overexpression of constitutively active S6K significantly increased acidic organelle size in response to rapamycin treatment (rapamycin: p<0.0001, Lsp2GS>S6K CA induction: p<0.0001, interaction p=0.0246, n=12). b , Lysotracker-positive enlarged acidic organelles (red) colocalized with the lysosomal marker Lamp1 ( Lsp2GS>S6K CA ; GFP-Lamp1 , green, upper panel) and partially colocalized with Rab7 ( Lsp2GS>S6K CA ; YFP-Rab7 , green, lower panel), a marker for late endosomes, in young flies treated with rapamycin and overexpressing S6K CA . c , Electron microscopy imaging of fat bodies of young flies treated with rapamycin and overexpressing S6K CA . Overexpression of S6K CA in the fat body attenuated the effect of rapamycin on multilamellar lysosomes (rapamycin: p=0.0395, Lsp2GS>S6K CA induction: p=0.2798, interaction p=0.0388, n=5). d , Lysotracker staining of young fat bodies overexpressing dominant negative Rab7 ( Lsp2GS>Rab7 DN ). Fat body-specific overexpression of Rab7 DN significantly increased acidic organelle size (p=0.0064, n=12). e , Fat body-specific overexpression of dominant negative Rab5 ( Lsp2GS>Rab5 DN ) also increased acidic organelle size (p=0.0798, n=12). Data are displayed as Tukey box plot (a, d-e) or mean□±□s.e.m. (c ). Each data point represents an average value per fat body. Scale bar, 10 μm (a-b, d-e) or 1 μm (c) . Linear mixed model (a, d-e) or negative binomial generalized linear model (c) followed by Tukey’s multiple comparison test.

Article Snippet: Membranes were blocked by Intercept TBS Blocking Buffer (LI-COR, #927-60001) for 1 h and probed with the following primary antibodies diluted in Intercept T20 TBS Antibody Diluent (LI-COR, #927-65001): anti-S6K (1:1,000), anti-pS6K T398 (Cell Signaling Technology, #9209, 1:1,000), anti-Relish (Developmental Studies Hybridoma Bank, #21F3, 1:100), anti-tubulin (Sigma, #T9026,1:5,000), anti-puromycin (Sigma, #MABE343, 1:1,000), anti-Stx12 (Synaptic Systems, #110 132, 1:1000).

Techniques: Staining, Over Expression, Marker, Electron Microscopy, Imaging, Dominant Negative Mutation

Journal: bioRxiv

Article Title: Inhibition of S6K lowers age-related inflammation and immunosenescence and increases lifespan through the endolysosomal system

doi: 10.1101/2022.08.25.505264

Figure Lengend Snippet: a , Syx13 protein level was increased upon rapamycin treatment in young fat body cells and this increase was partly reverted by S6K overexpression ( Lsp2GS>S6K CA ; rapamycin: p=0.0003, Lsp2GS>S6K CA induction: p=0.0138, interaction p=0.0086, n=5). b , Syx13 protein level was increased upon S6K repression ( Lsp2GS>S6K RNAi ; p=0.0226, n=5) in young fat body cells. Syx13 protein levels were measured by mass spectrometry-based proteomics. c , Knock-down of Syx13 expression ( Lsp2GS>Syx13 RNAi ) resulted in enlarged lysosomes in the fat body of young flies, depicted by lysotracker staining (p=0.0057, n=14). d , Overexpression of Syx13 ( Lsp2GS>Syx13 ) did not affect lysosomal enlargement (p=0.2894, n=12) in young fat bodies. e , Overexpression of Syx13 ( Lsp2GS>S6K CA ;Syx13 ) rescued the enlarged lysosomes of flies overexpressing S6K ( Lsp2GS>S6K CA ) treated with rapamycin ( Lsp2GS>S6K CA induction: p<0.0001, Lsp2GS>Syx13 induction: p<0.0001, interaction p=0.0429, n=12). f , Overexpression of Syx13 ( Lsp2GS>S6K CA ;Syx13 ) partially rescued the multilamellar lysosomes of S6K overexpressing ( Lsp2GS>S6K CA ) flies treated with rapamycin, depicted by electron microscopy ( Lsp2GS>S6K CA induction: p=0.0005, Lsp2GS>Syx13 induction: p=0.7989, interaction p=0.3068, n=5). Data are displayed as mean□±□s.e.m. (a, b, f) or displayed as Tukey box plot (c-e) . Each data point represents an average value per five fat bodies (a-b ) or per fat body (c-f) . Scale bar, 10 μm (c-e) or 1 μm (f) . Linear mixed model (a-e) or negative binomial generalized linear model (f) followed by Tukey’s multiple comparison test.

Article Snippet: Membranes were blocked by Intercept TBS Blocking Buffer (LI-COR, #927-60001) for 1 h and probed with the following primary antibodies diluted in Intercept T20 TBS Antibody Diluent (LI-COR, #927-65001): anti-S6K (1:1,000), anti-pS6K T398 (Cell Signaling Technology, #9209, 1:1,000), anti-Relish (Developmental Studies Hybridoma Bank, #21F3, 1:100), anti-tubulin (Sigma, #T9026,1:5,000), anti-puromycin (Sigma, #MABE343, 1:1,000), anti-Stx12 (Synaptic Systems, #110 132, 1:1000).

Techniques: Over Expression, Mass Spectrometry, Expressing, Staining, Electron Microscopy

Journal: bioRxiv

Article Title: Inhibition of S6K lowers age-related inflammation and immunosenescence and increases lifespan through the endolysosomal system

doi: 10.1101/2022.08.25.505264

Figure Lengend Snippet: a , Cleaved Relish (Rel49, 49 kDa) in fat bodies of young (day 10), middle (day 30), and old (day 50) flies (age effect p=0.0034, n=4). b-c , Relish protein localisation ( b , n=9 in young and n=13 in old) and DptA transcript expression ( c , n=4) in fat bodies of young and old flies. d-f , S6K inhibition ( Lsp2GS>S6K RNAi ) suppressed the age-related increase in activated Rel49 ( d , n=5), accumulation of Relish in the nucleus ( e , n=14), and the increase in DptA expression ( f , n=3). g-h , Rapamycin treatment suppressed age-related Relish localisation (g) and the increase in DptA (h) . Overexpression of S6K ( Lsp2GS>S6K CA ) blocked the effect of rapamycin on Relish localisation ( g , rapamycin: p=0.0025, Lsp2GS>S6K CA induction: p<0.0001, interaction p=0.2345, n=14) and DptA expression ( h , n=4). i , Rapamycin treatment improved bacterial clearance in old flies infected with Ecc15 . This effect was blocked by S6K overexpression ( Lsp2GS>S6K CA ) (rapamycin: p=0.0185, Lsp2GS>S6K CA induction: p=0.0024, interaction p=0.0620, n=12). Data are displayed as mean□±□s.e.m. (a, c-d, f, h) or Tukey box plot (b, e, g, i) . Each data point represents an average value per fat body (b, e, g ), per five fat bodies (a, c, d, f, h) , or per three whole flies (i) . Scale bar, 10 μm. One-way ANOVA followed by Dunnett’s multiple comparison test (a); linear mixed model (b, e, g) or two-way ANOVA with log transformation (i) followed by Tukey’s multiple comparison test; two-sided Student’s t-test with (c, f, h) or without log-transformation (d) .

Article Snippet: Membranes were blocked by Intercept TBS Blocking Buffer (LI-COR, #927-60001) for 1 h and probed with the following primary antibodies diluted in Intercept T20 TBS Antibody Diluent (LI-COR, #927-65001): anti-S6K (1:1,000), anti-pS6K T398 (Cell Signaling Technology, #9209, 1:1,000), anti-Relish (Developmental Studies Hybridoma Bank, #21F3, 1:100), anti-tubulin (Sigma, #T9026,1:5,000), anti-puromycin (Sigma, #MABE343, 1:1,000), anti-Stx12 (Synaptic Systems, #110 132, 1:1000).

Techniques: Expressing, Inhibition, Over Expression, Infection, Transformation Assay

Journal: bioRxiv

Article Title: Inhibition of S6K lowers age-related inflammation and immunosenescence and increases lifespan through the endolysosomal system

doi: 10.1101/2022.08.25.505264

Figure Lengend Snippet: a-c , Rapamycin treatment suppressed the age-related nuclear localisation of Relish in old fat body cells. Dominant negative Rab7 ( a , rapamycin: p=0.0638, Lsp2GS>Rab7 DN induction: p<0.0001, interaction p=0.0160, n=14) and Rab5 ( b , rapamycin: p<0.0001, Lsp2GS>Rab5 DN induction: p<0.0001, interaction: p=0.0003, n=14) blocked the effect of rapamycin on the age-related nuclear localisation of Relish. c-d , Knockdown of Syx13 ( Lsp2GS>Syx13 RNAi ) blocked the effect of rapamycin on ( c ) Relish nuclear localisation (rapamycin: p=0.0012, Lsp2GS>Syx13 RNAi induction: p<0.0001, interaction p=0.2930, n=14) and ( d) bacterial clearance (rapamycin: p=0.0093, Lsp2GS>Syx13 RNAi induction: p=0.0057, interaction p=0.2226, n=8). e , Knockdown of Syx13 ( Lsp2GS>S6K RNAi ;Syx13 RNAi ) blocked the effect of S6K knockdown on Relish nuclear localisation ( Lsp2GS>S6K RNAi induction: p=0.0004, Lsp2GS>Syx13 RNAi induction: p<0.0001, interaction: p=0.0088, n=14). f-g , Overexpression of Syx13 ( Lsp2GS>S6K CA ;Syx13 ) rescued the effect of S6K activation on Relish localisation ( f , Lsp2GS>S6K CA induction: p<0.0001, Lsp2GS>Syx13 induction: p<0.0001, interaction p<0.0001, n=14) and bacterial clearance ( g , Lsp2GS>S6K CA induction: p=0.0644, Lsp2GS>Syx13 induction: p=0.7042, interaction: p=0.0429, n=10) in old flies treated with rapamycin. h-i , Middle-age-onset repression of Relish using Lsp2GS>Relish RNAi improved bacterial clearance ( h , n=8 in 0 h.p.i. group and n=12 in 12 h.p.i. groups) and extended lifespan ( i , p=0.0091, n=200). Data are displayed as Tukey box plot. Each data point represents an average value per fat body (a-c, e-f) or per three whole flies (d, g-h) . Scale bar, 10 μm. Linear mixed model (a-c, e-f) , two-way ANOVA with log transformation (d, g) followed by Tukey’s multiple comparison test; two-sided Student’s t-test (h) ; log-rank test (i) .

Article Snippet: Membranes were blocked by Intercept TBS Blocking Buffer (LI-COR, #927-60001) for 1 h and probed with the following primary antibodies diluted in Intercept T20 TBS Antibody Diluent (LI-COR, #927-65001): anti-S6K (1:1,000), anti-pS6K T398 (Cell Signaling Technology, #9209, 1:1,000), anti-Relish (Developmental Studies Hybridoma Bank, #21F3, 1:100), anti-tubulin (Sigma, #T9026,1:5,000), anti-puromycin (Sigma, #MABE343, 1:1,000), anti-Stx12 (Synaptic Systems, #110 132, 1:1000).

Techniques: Dominant Negative Mutation, Over Expression, Activation Assay, Transformation Assay

Journal: Endocrinology

Article Title: AKT activation promotes metastasis in a mouse model of follicular thyroid carcinoma.

doi: 10.1210/en.2005-0172

Figure Lengend Snippet: FIG. 6. A, Expression of a dominant-negative AKT (DNAKT) in cells reduces downstream targets of AKT. Infection of a primary thyroid cell line using either a control adenovirus containing -gal or an HA-tagged DNAKT was performed at an MOI of 100. Western blot of the cell lysates confirms the presence of the HA epitope only in the DNAKT infected cells. The level of phosphorylated mammalian target of rapamycin (phos-mTOR), and phosphorylated p70 S6K (phos-p70 S6K) downstream targets of AKT, is reduced in the DNAKT-infected cells. Protein disulfide isomerase (PDI) was used as a control for loading. Representative data from two independent experiments are shown. B, Inhibition of AKT signaling impairs primary thyroid cell motility. Cell motility assay of the adenovirus-infected cell lines, com- pared with control, show a 35% reduction of cell motility in cells expressing the DNAKT. Data are presented as the mean SEM of two independent experiments performed in triplicate (n 6).

Article Snippet: Antibodies used according to the manufacturers’ manuals include phospho-AKT (Ser473) no. 9271 (Cell Signaling Technologies, Beverly, MA), AKT no. 9272 (Cell Signaling Technologies) at 1:1000 dilution, hemagglutinin (HA) sc-7392 (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:200, phospho-mammalian target of rapamycin (mTOR) (Ser2448) no. 2971 (Cell Signaling Technologies) at 1:1000, and phosphop70 S6K (Thr421/Ser424) no. 9204 (Cell Signaling Technologies) at 1:1000 dilution.

Techniques: Expressing, Dominant Negative Mutation, Infection, Control, Western Blot, Inhibition, Motility Assay

Journal: PLoS ONE

Article Title: 2-Deoxyglucose Suppresses ERK Phosphorylation in LKB1 and Ras Wild-Type Non-Small Cell Lung Cancer Cells

doi: 10.1371/journal.pone.0168793

Figure Lengend Snippet: A. H522, Calu-1 and H520 cells were pretreated with or without 10 μM Compound C (C.C) for 30 minutes before 2-DG treatment. P-S6K and p-ERK levels were examined by Western blot. B. H522, H520, Calu-1 and H1650 cells were transiently transfected with control siRNA or AMPKα siRNA. AMPK protein expression and p-ERK levels were examined by Western blot. C. H596 cells were transiently transfected with control siRNA or AMPKα siRNA, and treated with 25 mM 2-DG for 2 hours. AMPK protein expression and p-ERK levels were examined by Western blot. D. Calu-1 cells were transiently transfected with plasmid encoding control vector or dominant negative AMPKα 1(DN-AMPK), and AMPKα protein expression and p-ERK levels were examined by Western blot. E. H596 cells were transiently transfected with plasmid encoding control vector or dominant negative AMPK α 1 (DN-AMPK), and treated with 25 mM 2-DG for 2 hours. AMPKα protein expression and p-ERK levels were examined by Western blot.

Article Snippet: Antibodies against total AMPK (#2532), p-AMPKα Thr172 (#2535), p-ACC (phospho-acetyl-CoA carboxylase) Ser79 (#3661), total ERK1/2 (#9102), p-ERK1/2 Thr202/Tyr204 (#9101), total AKT (#9272), p-AKT Thr473 (#9271), p-S6K Thr389 (#9105) and Kras (#3965) were purchased from Cell Signaling Technology, USA.

Techniques: Western Blot, Transfection, Control, Expressing, Plasmid Preparation, Dominant Negative Mutation

Journal: Molecular carcinogenesis

Article Title: Involvement of the Akt/mTOR pathway on EGF-induced cell transformation.

doi: 10.1002/mc.10140

Figure Lengend Snippet: Figure 4. Effect of rapamycin on EGF-induced activation of Akt and p70 S6K. JB6 Cl 41 cells (80% confluence) were starved by replacing the medium with 0.1% FBS/MEM and culturing for 48 h. The cells were then pretreated with rapamycin, LY294002, or wortmannin for 1 h at the indicated concentration. The cells were treated with EGF (10 ng/mL) and subsequently cultured for 30 min. Then, the cells were lysed and Akt or p70 S6K was immunopreci- pitated using anti–Akt1/2 antibody or anti–p70 S6K antibody, respectively. Activities are assessed with their respective substrate peptide and g-32P ATP. Each bar indicates the mean SE (n ¼ 3–4). *Significantly different from the EGF-stimulated control (P < 0.05).

Article Snippet: Materials Eagle’s minimal essential medium (MEM), L-glutamine and basal medium Eagle (BME) were from Life Technologies, Inc. (Rockville, MD); fetal bovine serum (FBS) and gentamicin were from BioWhittaker Biosciences (Walkersville, MD); PI3K inhibitors wortmannin and LY29402 and the mTOR inhibitor rapamycin were from Calbiochem (La Jolla, CA); the Akt immunoprecipitation kinase assay kit, S6 kinase assay kit, and dominant negative mutants of Akt1 (DNM-Akt1) were from Upstate Biotechnology, Inc. (Lake Placid, NY); the Akt antibody and phospho-specific Akt (serine 473), p70 S6K antibody, and phospho-specific p70 S6K (threonine 389), and PhosphoPlus p44/42 mitogen activator protein (MAP) kinase antibody kits were from Cell Signaling Technology, Inc. (Beverly, MA); the anti–Akt1/2 antibody was from Santa Cruz (Santa Cruz, CA).

Techniques: Activation Assay, Concentration Assay, Cell Culture, Control

Journal:

Article Title: Macrophage infiltration into adipose tissue may promote angiogenesis for adipose tissue remodeling in obesity

doi: 10.1152/ajpendo.90296.2008

Figure Lengend Snippet: Mammalian target of rapamycin (mTOR) in PDGF-induced tube formation. The role of mTOR/S6K in PDGF-induced tube formation was tested with mTOR inhibitor (rapamycin) and protein synthesis inhibitor cycloheximide (CHX). A: representative images of tube formation in the presence of rapamycin and CHX. Insulin was used as a positive control in induction of mTOR activation. Rapamycin (200 nM) and CHX (20 ng/ml) were used to pretreat cells for 30 min, followed by treatment of PDGF (50 ng/ml) or insulin (100 nM) for 10 h. B: quantification of tube formation. C: representative Western blots for mTOR/S6K signaling pathway in endothelial cells. Phosphorylation of S6K was determined in SVEC4-10 cells after treatment with PDGF or insulin. D: quantification of Western blot signals. In this figure, all experiments were repeated 3 times with consistent results. Each data point represents mean ± SE (n = 3 experiments).

Article Snippet: The SVEC4-10 endothelial cells were cotransfected with 3 μg of pRK7-HA-S6K1-KR plasmid for dominant-negative S6K (8985, Addgene) and 0.5 μg of pcDNA3.1 plasmid for neomycin resistance.

Techniques: Positive Control, Activation Assay, Western Blot

Journal:

Article Title: Macrophage infiltration into adipose tissue may promote angiogenesis for adipose tissue remodeling in obesity

doi: 10.1152/ajpendo.90296.2008

Figure Lengend Snippet: S6K in PDGF-induced tube formation. A: expression of dominant-negative S6K (S6K-DN) in stable cells. B: inhibition of S6K activity by S6K-DN mutant. C: tube formation in stable cells in response to PDGF. D: quantification of tube formation. In this figure, all experiments were repeated 3 times with consistent results. Each data point represents mean ± SE (n = 3 experiments).

Article Snippet: The SVEC4-10 endothelial cells were cotransfected with 3 μg of pRK7-HA-S6K1-KR plasmid for dominant-negative S6K (8985, Addgene) and 0.5 μg of pcDNA3.1 plasmid for neomycin resistance.

Techniques: Expressing, Dominant Negative Mutation, Inhibition, Activity Assay, Mutagenesis

Journal:

Article Title: Macrophage infiltration into adipose tissue may promote angiogenesis for adipose tissue remodeling in obesity

doi: 10.1152/ajpendo.90296.2008

Figure Lengend Snippet: S6K in tube formation induced by albumin. A: Induction of phosphorylation of S6K by albumin. Phosphorylation of S6K was examined in whole cell lysate in Western blot. B: BSA-stimulated tube formation and inhibition by rapamycin (200 nM). C: quantification of tube formation. D: cell viability after rapamycin treatment. Cell viability was determined with MTT assay at 24 h after cell exposure to rapamycin. In this figure, all experiments were repeated 3 times with consistent results. Each data points represents mean ± SE (n = 3 experiments).

Article Snippet: The SVEC4-10 endothelial cells were cotransfected with 3 μg of pRK7-HA-S6K1-KR plasmid for dominant-negative S6K (8985, Addgene) and 0.5 μg of pcDNA3.1 plasmid for neomycin resistance.

Techniques: Western Blot, Inhibition, MTT Assay

Journal: Scientific Reports

Article Title: mTORC1 phosphorylates LARP6 to stimulate type I collagen expression

doi: 10.1038/srep41173

Figure Lengend Snippet: ( a ) LARP6 phosphorylation changes in activation of HSCs. Primary rat HSCs were transduced by adenovirus expressing wt LARP6 on day 2 or on day 4 and analyzed by 2DGE on day 3 or on day 5 (CON, panels 1 and 2). The same cells were also treated with rapamycin on day 2 and day 4 and analyzed on day 3 and day 5 (panels 3 and 4). Panels 5 and 6; cells transduced with S348A/S409A mutant. ( b ) Increased AKT and mTORC1 signaling during HSCs activation. HSCs at day 3 and day 5 in culture were analyzed for expression of phospho-AKT and phospho-S6K. p-AKT: phopho-AKT. AKT: total AKT. p-S6K: phospho-S6K. S6K: total S6K. ACT: β-actin, loading control. ( c ) Dominant negative effect of S348A/S409A in HSCs. HSCs were transduced on day 3 with wt LARP6 and S348A/S409A mutant and on day 5 cellular (CELL) and medium (MED) level of COL1A1 was analyzed by Western blot. Loading controls: β-actin (ACT) and fibronectin (FIB). HA-LARP6, expression of the transduced proteins. ( d ) Rapamycin reduces expression of collagen by HSCs. HSCs cultured for 5 days were treated with or without RAPA for 2 h and COL1A1 polypeptide was analyzed intracellularly (CELL) or in the medium (MED).

Article Snippet: Antibodies used were: anti-LARP6 antibody from Abnova (H00055323-B01P), anti-HA antibody from Sigma-Aldrich (H9658), anti-phospho AKT (S473), anti-pan AKT, anti-phospho S6K (T389), and anti-S6K antibodies from Cell Signaling Technology (12694, 4691, 9205, and 2708, respectively), anti-collagen α1(I) antibody from Rockland (600-401-103), anti-collagen α2(I) antibody from Santa Cruz Biotechnology (sc-8786), anti-calnexin antibody, human anti-fibronectin antibody and anti-STRAP antibody from BD Transduction Laboratories (610523, 610077, and 611346, respectively), rat anti-fibronectin from Millipore (AB1954), anti-β-actin antibody from Abnova (ab8227), anti-Sec61β antibody from Thermo Scientific (PA3-015) and anti-raptor antibody from Bethyl Laboratories (A300-553A-M).

Techniques: Phospho-proteomics, Activation Assay, Expressing, Transduction, Mutagenesis, Control, Dominant Negative Mutation, Western Blot, Cell Culture

Journal: Cell Death and Differentiation

Article Title: Autophagy and phagocytosis-like cell cannibalism exert opposing effects on cellular survival during metabolic stress

doi: 10.1038/cdd.2012.37

Figure Lengend Snippet: Increased TOR activity and the lack of negative regulatory feedback between autophagy and AMPK in alc mutants. (a) TOR activity is stimulated in alc mutants, as evidenced by increased phosphorylation of its downstream target S6 kinase (dS6K).15 Depicted is a representative immunoblot of head extracts (∼100 of each genotype) from heterozygous control (left) and alc mutant flies (right), using an antibody specific for phosphorylated dS6K. The bar graph depicts quantification of the increased phosphorylation corrected for levels of non-phosphorylated dS6K and normalized to control values. Represented are mean values (n=6)±S.D., **P<0.01 in Student's t-test. Genotypes as in Figure 1. (b) Downregulation of TOR using a dominant-negative allele (TORDN, top row) or TSC2 overexpression (middle row) results in aggravation of the neurodegenerative phenotype at day 1 (not shown) and day 7 (shown here). Expression of a constitutively active dS6K (S6KCA) also exacerbates alc-mediated neurodegeneration. Flies were reared in the dark so as to sensitize the background for investigation of additive effects (see comment to Figure 3). Quantification: **P<0.01, ***P<0.001 when compared with alc−/− (alcΔ125). Data are given as mean values±S.D. Genotypes: FRT42D,alcΔ125/+ EGUF/UAS-TORFRB and FRT42D,alcΔ125/+ EGUF/UAS-TSC2 and FRT42D,alcΔ125/+ EGUF/UAS-S6KSTDEplus corresponding homozygous alc mutants. alcΔ125 as in Figure 1. (c) Increased autophagy reduces activating phosphorylation of α-AMPK, suggesting a negative regulatory feedback loop. Shown is a representative immunoblot from control lines (act5C-Gal4 driver line (lane 1) and atg8aEP line (lane 3)) and flies in which autophagy was induced by ubiquitous overexpression of atg8a (lane 2). All flies originated from the same cross. Genotypes: yw/Y; act5C-Gal4/+ and Atg8aEP/yw; act5C-Gal4/+ and Atg8aEP/yw

Article Snippet: Lysates were cleared by centrifugation, boiled in 1X SDS sample buffer, resolved by SDS-PAGE, transferred onto nitrocellulose membranes and probed with anti-phospho- Drosophila- S6K (Thr398; 1 : 1000, #9209, Cell Signaling, Leiden, The Netherlands), anti-S6K- α (1 : 200, sc-230, Santa Cruz Biotechnology, Heidelberg, Germany), anti-phospho-AMPK-α (Thr172; 1 : 2000, #4188, Cell signaling), anti-AMPK- α 1/2 (1 : 500, ab80039, Abcam, Cambridge, UK) and with anti- α -tubulin (1 : 2500, ab4074, Abcam), and subsequently with peroxidase-conjugated secondary antibody (1 : 10 000; Jackson ImmunoResearch).

Techniques: Activity Assay, Phospho-proteomics, Western Blot, Control, Mutagenesis, Dominant Negative Mutation, Over Expression, Expressing