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