Journal: Signal Transduction and Targeted Therapy

Article Title: Hyperglutaminolysis drives senescence and aging through arginine-mTORC1 axis activation

doi: 10.1038/s41392-026-02576-w

Figure Lengend Snippet: Glutaminolysis activates mTORC1 and suppresses autophagy via mTORC1 activation. Representative immunoblotting images ( a ) and quantitation ( b ) for phosphorylated p70/S6K (P-p70/S6K) and 4EBP1(P-4EBP) in NIH3T3 cells with 24 hours of serum starvation. Pro: proliferating cells, Sen: senescent cells induced by H 2 O 2 . Senescence induction was combined with 10 μM DON or siGls1 treatment for three days in NIH3T3 cells. mTORC1 activity in senescent cells was examined by testing p70/S6K and 4EBP1 phosphorylation ( c , d ) and mTOR-LAMP2 colocalization ( e , f ). Autophagy flux was examined by detecting RFP+/GFP- (red) puncta in senescent populations stably expressing LC3-RFP-GFP protein ( g , h ), and p62 protein abundance in senescent cells, without ( i , j ) or with ( k , l ) 25 nM BafA1 treatment combined during the last 24 hours. m – q Autophagy flux in senescent cells under conditions with 20 mM glutamine loading (Gln, 24 hours), or mTORC1 inactivation caused by siRNA for Raptor (siRap) and rapamycin (Rapa, 0.1 μM) treatments. m RT-qPCR based Raptor knockdown verification. RFP + /GFP- cell ratio ( n , o ), and p62 protein abundance ( p , q ). p70/S6K phosphorylation levels in serum-starved proliferating (Pro) and H 2 O 2 -induced senescent cells (Sen), with 2 μM DON treatment for 2 hours ( r , s ), and 20 mM Gln treatment for 6 hours ( t , u ). All assays were conducted three times. Scale bar = 50 μm. All histogram data are shown as mean ± SD. Student’s t-test: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, n.s . non-significant

Article Snippet: Antibodies against p16 ( P21212 ) were purchased from ProMab Biotechnologies, Inc (Hunan, China); antibodies against p62 (ab109012) were purchased from Abcam (Cambridge, UK); antibodies against GLS1 (A5125) and 4EBP1 (A5090) were purchased from Selleck (Houston, USA) were purchased from Absin (Shanghai, China); antibodies against phosphorylated (P)-p70/S6K (9205S) were purchased from CST (Massachusetts, USA); antibodies against p70/S6K (A4898) and P-4EBP1 (AP0030) were purchased from ABclonal Technology (Hubei, China); antibody against mTOR (R1510-21) was purchased from Huabio (Zhejiang, China); antibody against LAMP2 (MA1-165) was purchased from Thermo Fisher Scientific (Massachusetts, USA); antibodies against β-actin (bs-10966R) were purchased from Bioss (Beijing, China); goat anti-rabbit IgG (H&L) (HRP conjugate) (701051) was purchased from Zenbio (Chengdu, China); and goat anti-rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor TM Plus 488 (A32731) and goat anti-rat IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor TM Plus 594 ( A48264 ) were purchased from Thermo Fisher Scientific (Massachusetts, USA).

Techniques: Activation Assay, Western Blot, Quantitation Assay, Activity Assay, Phospho-proteomics, Stable Transfection, Expressing, Quantitative Proteomics, Quantitative RT-PCR, Knockdown

Journal: Signal Transduction and Targeted Therapy

Article Title: Hyperglutaminolysis drives senescence and aging through arginine-mTORC1 axis activation

doi: 10.1038/s41392-026-02576-w

Figure Lengend Snippet: Glutaminolysis-driven arginine biosynthesis promotes mTORC1 activation in cells and mice. a – i Proliferating NIH3T3 cells were used for assays. Representative immunoblotting images ( a ) and quantitation ( b ) of p70/S6K and 4EBP1 phosphorylation in cells treated with 4 mM of the indicated metabolites alone or in combination for five hours after a one-hour DON (2 μM) pretreatment. Glu: glutamate, NH 4 Cl: ammonium, Asp: aspartate, Cit: citrulline, Arg: arginine. Three biological replicates were tested. c A sketch map showing the enzymes involved in the glutaminolysis-arginine synthesis pathway. GLS1: glutaminase 1, OTC: ornithine transcarbamylase, GOT2: glutamic-oxaloacetic transaminase 2, ASS1: argininosuccinate synthase 1, ASL: argininosuccinate lyase. d RT-qPCR based detections for mRNAs of Got2 , Otc, Ass1 , and Asl genes in cells transfected with indicated siRNAs. p70/S6K phosphorylation ( e , f ) and mTOR-LAMP2 colocalization ( g , h ) in siRNAs-transfected cells cultured in fresh medium with 4 mM glutamine for 6 hours after a 24-hour period of glutamine starvation. i Arginine levels in cells transfected with indicated siRNAs. All cellular experiments were repeated three times. j – l Pooled homogenates of indicated tissues from 4-5 mice per group were assayed with at least three technical repetitions. Arginine levels in kidney, spleen and muscle of young (3 months) and old mice (27 months), measured by using pooled homogenates of tissues from 4-5 mice ( j ). p70/S6K phosphorylation levels in gastrocnemius muscle of mice ( k , l ). m – q Mice with AAV9-shAsl or AAV9-GFP injection (1 × 10¹¹ viral genomes) were applied for assays after one to three months of viral injection, with three mice per group. Fluorescence intensity in gastrocnemius muscle with AAV9-GFP injection ( m ). mRNA levels of the Asl gene in gastrocnemius muscle of mice infected with AAV9-shAsl (shAsl) and control mice (shNC) ( n ). Arginine levels ( o ) and p70/S6K phosphorylation levels ( p , q ) in gastrocnemius muscle. Scale bar = 50 μm. Histogram data are shown as mean ± SD. Student’s t -test: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

Article Snippet: Antibodies against p16 ( P21212 ) were purchased from ProMab Biotechnologies, Inc (Hunan, China); antibodies against p62 (ab109012) were purchased from Abcam (Cambridge, UK); antibodies against GLS1 (A5125) and 4EBP1 (A5090) were purchased from Selleck (Houston, USA) were purchased from Absin (Shanghai, China); antibodies against phosphorylated (P)-p70/S6K (9205S) were purchased from CST (Massachusetts, USA); antibodies against p70/S6K (A4898) and P-4EBP1 (AP0030) were purchased from ABclonal Technology (Hubei, China); antibody against mTOR (R1510-21) was purchased from Huabio (Zhejiang, China); antibody against LAMP2 (MA1-165) was purchased from Thermo Fisher Scientific (Massachusetts, USA); antibodies against β-actin (bs-10966R) were purchased from Bioss (Beijing, China); goat anti-rabbit IgG (H&L) (HRP conjugate) (701051) was purchased from Zenbio (Chengdu, China); and goat anti-rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor TM Plus 488 (A32731) and goat anti-rat IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor TM Plus 594 ( A48264 ) were purchased from Thermo Fisher Scientific (Massachusetts, USA).

Techniques: Activation Assay, Western Blot, Quantitation Assay, Phospho-proteomics, Quantitative RT-PCR, Transfection, Cell Culture, Injection, Fluorescence, Infection, Control