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

Journal: Nature Communications

Article Title: The PIK3CA/AKT pathway drives therapy resistance in rhabdomyosarcoma

doi: 10.1038/s41467-025-66632-9

Figure Lengend Snippet: A Histopathology of xenografted tumors. Representative RD parental sensitive (S1) and resistant models (R1, R4) showing co-immunofluorescence staining of PI3Kα (Alexa Fluor 546) and MDR1 (Alexa Fluor 488); IHC for p-AKT (Ser473) and p-NRF2 (Ser40; A , left). Quantification of percent positive cells within each model mean +/− ST noted on images or in graphical analysis (right). N = 3 in sensitive models and n = 6 in resistant models (biological replicates, RD and Rh41). Data are mean ± SD. B Western blot analysis of RD and Rh41 cell line grown after selection in xenografted mice. Data shown are from one representative experiment and was repeated three times using biological replicates with similar results. The lysates derive from the same experiment but were run on different gels. One gel was used for analysis of PI3K p110, Nrf2, p-Nrf2, AKT, p-AKT, p-4EBP1, S6, another for EGFR, PI3K p85, p-PI3K p85, MDR1, MRP1, BCRP, p-PTEN, PTEN, 4EBP1, and p-S6. Both gels were processed in parallel. C Quantification of cell viability following drug treatment in RD and Rh41 models. Cells treated for 4 days. Grey denotes comparisons that were not done. Scale (1.0 growth/viable and 0.0 dead). Data shown as mean from three independent biological replicates. D Representative confocal image of RD therapy sensitive and resistant model following treatment with PARPi-FL (red) and temozolomide (TMZ) or in combination with alpelisib PIK3CAα inhibitor (OT + AL) or Tariquidar ABC transport inhibitor (OT + TA). Samples were counterstained with NucBlue. Z-stack image (top, D ) and cell cross-section (bottom, D ). E Quantification of relative PARPi-FL uptake in therapy sensitive and OT resistant RD and Rh41 cells subjected to OT, OT + alpelisib (AL), OT + tariquidar (TA). n > 248 cells per condition. Shown is a representative example from one of three independent, biological replicate experiments; similar results observed across all experiments ( D , E ). F Representative images and G flow cytometry analysis of RD parental sensitive (S1) and resistant cells (R1, R4) showing fluorescence eFluxx-ID® signals after treating with DMSO or alpelisib PIK3CAα inhibitor (AL). The fluorescence signal of the dye negatively correlates with the activity of the ABC transporters. Images are representative of three independent biological replicates ( F , G ). P < 0.05 was considered statistically significant. ANOVA followed by Dunnett post hoc test was used to make comparisons between sensitive and resistant clones ( A ). ANOVA followed by two-sided Student’s T -test comparisons within each resistant model ( E ). Scale bar equals 25 µm ( A ), 10 µm ( D , upper; F ), and 2 µm ( D , lower).

Article Snippet: Primary antibodies used included PTEN (CST, 9559), p-PTEN (Ser380/Thr382/383) (CST, 9551), EGFR (abcam, ab52894), PI3K p85 (CST, 4292), p-PI3K p85 (Tyr458) (CST, 17466), AKT (CST, 4691), p-AKT (Ser473) (CST, 4060), NRF2 (abcam, ab62352), p-NRF2 (Ser40) (abcam, ab76026), S6 (CST, 2317), p-S6 (Ser235/236) (CST, 81736), 4EBP1 (CST, 9644), p-4EBP1 (Thr37/46) (CST, 2855), MDR1/ABCB1 (CST, 13342), MRP1/ABCC1 (CST, 72202), BCRP/ABCG2 (CST, 42078), GAPDH (CST, 5174).

Techniques: Histopathology, Immunofluorescence, Staining, Western Blot, Selection, Flow Cytometry, Fluorescence, Activity Assay, Clone Assay