Journal: Signal Transduction and Targeted Therapy

Article Title: Abrogation of aberrant glycolytic interactions eliminates senescent cells and alleviates aging-related dysfunctions

doi: 10.1038/s41392-025-02502-6

Figure Lengend Snippet: Senescent cells (SnCs) display increased interaction between PGAM1 and Chk1 and increased glycolysis. Evaluation of the PGAM1-Chk1 interaction and glycolytic activity during cellular senescence ( a – f , n = 3, biological replicates). a Diagram of the NanoBiT assay used to detect PGAM1-Chk1 interactions. LgBiT-tagged PGAM1 and SmBiT-tagged Chk1 were introduced into MCR5-RasER cells. b MCR5-RasER cells were treated with 20 nM 4-hydroxytamoxifen (4-OHT). The cells were cultured for 16 days. SA-β GAL and γH2AX staining are shown in the upper right and lower panels, respectively. The bars indicate 100 μm and 10 μm, respectively. c . Senescent profiles were assessed during 4-OHT treatment. Immunoblotting for several senescence markers is shown: p16 Ink4a , Lamin B1, p53 and phospho-p53 (Ser15) protein. d Evaluation of SA-β GAL staining (black) and glucose flux (blue) in MCR5-RasER cells after tamoxifen treatment. The cells were collected at the indicated time points. e Assessment of glycolytic activity during cellular senescence. On the indicated days, the following glycolytic parameters were assessed: lactate production (black) and glucose consumption (green bar). f Evaluation of the PGAM1-Chk1 interaction via the NanoBiT assay (orange) in MCR5-RasER cells after tamoxifen treatment. ( g – j Effects of Nutlin 3a and 3b on the PGAM1-Chk1 interaction and glycolytic features in SnCs. Senescence was provoked in IMR90 cells by the ectopic expression of Ras G12V. g Diagram of the chemical structure of Nutlin isomers 3a and 3b (left and right panels, respectively). h Immunoprecipitation was used to detect PGAM1-Chk1 interactions in SnCs. SnCs were treated with Nutlin 3a or 3b at 20 or 40 μM for 48 h. Controls received neither compound. The data are representative of two independent experiments. i Glucose consumption and lactate production were measured in SnCs treated with Nutlin 3a (green) or 3b (blue) ( n = 3 biological replicates). j Evaluation of 2NBDG (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose) uptake in SnCs after Nutlin 3a (green) or 3b (blue) treatment. 2NBDG was detected as green fluorescence (left panel). The bar indicates 100 μm. Fluorescence intensities were assessed in each cell (right panel). The data represent two independent experiments. The data represent the means ± SEMs of three biological replicates. Single (*) and double (**) asterisks indicate statistical significance at p < 0.05 and p < 0.01, respectively. Statistical analyses were performed with one-way analysis of variance (ANOVA) and Dunnett’s multiple comparison test. The part of the image was created with BIORENDER

Article Snippet: Anti-mouse Chk1 (25887-1-AP, 1:1000) and anti-mouse FOXM1 (13147-1-AP, 1:1000) were purchased from Proteintech (USA).

Techniques: Activity Assay, Cell Culture, Staining, Western Blot, Expressing, Immunoprecipitation, Fluorescence, Comparison

Journal: Signal Transduction and Targeted Therapy

Article Title: Abrogation of aberrant glycolytic interactions eliminates senescent cells and alleviates aging-related dysfunctions

doi: 10.1038/s41392-025-02502-6

Figure Lengend Snippet: Abrogation of the PGAM1-Chk1 interaction eliminated senescent cells. a – c Senolytic effect of Nutlin 3b. Ras G12V-expressing senescent IMR90 cells were prepared. a γH2AX staining of control and SnCs. The indicated cells were treated with Nutlin 3a or 3b. The ratio of γH2AX foci-positive cells is shown in the right panel. b Cleavage of caspase 3, p53, and p21 CIP1 in stress-induced senescent IMR90 cells treated with Nutlin 3a or 3b. The data represent two independent experiments. c Relative counts of senescent and control cells under Nutlin 3a or 3b treatment. The control and SnCs were treated with Nutlin 3a or 3b at different concentrations (0–40 μM) for 96 h. The blue and orange lines indicate the control and SnCs, respectively. Triangle; Nutlin 3a, Circle; Nutlin 3b. ( n = 3, biological replicates). d Metabolomic analysis of control and SnCs with or without Nutlin 3b treatment. Metabolites in the glycolytic pathway are shown in black boxes, including G6P glucose-6-phosphate, F6P fructose 6-phosphate, 3-PG 3-phosphoglyceric acid, 2-PG 2-phosphoglyceric acid, and PEP phosphoenolpyruvate. Metabolites in the pentose phosphate pathway (PPP) are shown in the blue box, including 6GP 6-phospho-gluconate, Ru5P ribulose 5-phosphate, R5P ribose 5-phosphate, and PRPP phosphoribosyl diphosphate. e Effects of CM on the PGAM1-Chk1 interaction. Conditioned medium was collected from either control or SnCs. IMR90 cells expressing LgBiT-PGAM1 or Chk1-SmBiT were exposed to each conditioned medium. A NanoBit assay was performed to detect the interaction between PGAM and Chk1 ( n = 3, biological replicates). This image was created with BIORENDER. f The impact of lactate on the PGAM1-Chk1 interaction. IMR90 cells expressing LgBiT-PGAM1 and Chk1-SmBiT were exposed to increasing amounts of IL6, IL1β, or lactate. The NanoBit signal was normalized against the protein content ( n = 3, biological replicates). The data represent the means ± SEMs of three biological replicates. Single (*) and double (**) asterisks indicate statistical significance at p < 0.05 and p < 0.01, respectively. Statistical analyses were performed with one-way analysis of variance (ANOVA) and Dunnett’s multiple comparison test

Article Snippet: Anti-mouse Chk1 (25887-1-AP, 1:1000) and anti-mouse FOXM1 (13147-1-AP, 1:1000) were purchased from Proteintech (USA).

Techniques: Expressing, Staining, Control, Comparison

Journal: Signal Transduction and Targeted Therapy

Article Title: Abrogation of aberrant glycolytic interactions eliminates senescent cells and alleviates aging-related dysfunctions

doi: 10.1038/s41392-025-02502-6

Figure Lengend Snippet: Aging-related dysfunctions in vivo were alleviated by Nutlin 3b treatment. Administration of Nutlin 3b to aged mice. Twenty-month-old female mice were treated with weekly i.p . injections of Nutlin 3b for three months. One week later, the physiological impact of Nutlin 3b in the mice was assessed ( n = 6, mice). a Schematic diagram of the in vivo protocol for Nutlin-3b treatment. b Wire-hang test for the evaluation of muscle strength. c Blood parameters of young or aged mice with or without Nutlin-3b treatment. d Immunohistochemical examination of the liver for p21 CIP1 and F4/80 staining. Representative images of stained samples from the indicated mice are shown (upper panels). The ratio of p21 CIP1 - or F4/80-positive cells was assessed (lower panel). The bar indicates 50 μm. e Comparison of the mRNA levels of p16 Ink4a and SASP factors (Il6, Cxcl1, Tnfα, and Ccl5). Liver extracts from the indicated mice were analyzed via RT‒PCR. f Immunoprecipitation was used to detect PGAM1-Chk1 interactions in aged livers with or without Nutlin 3b treatment and in young livers. g Evaluation of SA-β GAL staining in aged livers. Representative pictures (left panels) and positivity of SA-β GAL staining in the indicated samples. Each group comprised female ( n = 3) and male ( n = 3) mice. The data represent the means ± SEMs. Single (*) and double (**) asterisks indicate statistical significance at p < 0.05 and p < 0.01, respectively. Statistical analyses were performed via unpaired Student’s two-tailed t tests ( b ) or one-way analysis of variance (ANOVA) and Dunnett’s multiple comparison test ( c – e and g )

Article Snippet: Anti-mouse Chk1 (25887-1-AP, 1:1000) and anti-mouse FOXM1 (13147-1-AP, 1:1000) were purchased from Proteintech (USA).

Techniques: In Vivo, Immunohistochemical staining, Staining, Comparison, Immunoprecipitation, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Abrogation of aberrant glycolytic interactions eliminates senescent cells and alleviates aging-related dysfunctions

doi: 10.1038/s41392-025-02502-6

Figure Lengend Snippet: The HIF-2α protein accumulates in senescent cells and is downregulated by inhibiting the PGAM1-Chk1 interaction. a Protein levels of glycolytic enzymes after Nutlin 3b treatment. SnCs induced by oncogenic Ras were exposed to different concentrations of Nutlin 3b (0–40 μM). Aldo aldolase, GAPDH glyceraldehyde 3-phosphate dehydrogenase, PGK phosphoglycerate kinase, ENO enolase. b Transcription factors for glycolysis were evaluated in control and Ras-induced SnCs. Western blot with antibodies against HIF-2α, HIF-1α, E2F1, c-Myc and Lamin B1 in the indicated cells. c Effect of Nutlin 3b on the HIF-2α protein. The protein levels of HIF-2α are shown via western blotting. Senescent and control cells were treated with Nutlin 3b for 48 h. d Diagram of the amino-terminal sequence of HIF-2α in several organisms. Conserved amino acids are highlighted. Conserved motifs comprise the consensus sequence for phosphorylation by Chk1 (K/R-K/R-x-x-S/T), which is surrounded by a red box. Motifs 1 and 2 include Ser-12 and Ser-19, respectively. Hs Homo sapiens , Mm Mus musculus , Rn Rattus norvegicus , Gg Gallus gallus , Xt Xenopus tropicalis . e Ubiquitination assay for the HIF-2α protein. Extracts were collected from SnCs expressing His-ubiquitin and various versions of HA-HIF-2α. Purification on Ni-NTA agarose beads was followed by immunoblotting with an HA antibody. The data are representative of two independent experiments. f Phosphorylation status of HIF-2α at Ser12 in SnCs. Control or SnCs were transfected with Chk1 siRNA. The resulting extracts were probed with an anti-phospho-S12-HIF-2α antibody. g The impact of lactate on HIF-2α status. IMR90 cells were exposed to increasing amounts of lactate. Western blotting was performed. h NanoBit assay for assessing the PGAM1-Chk1 interaction after ablation of GPR81 ( n = 3, biological replicates). IMR90 cells expressing LgBiT-PGAM1 and Chk1-SmBiT were transfected with the indicated siRNAs. i Evaluation of Chk1 and Rsk1 activation after lactate treatment by western blotting. Antibodies against phospho-Chk1-S280 and phospho-RSK1-Thr573 were applied. j Impact of the Chk1-S280 status on the PGAM1-Chk1 interaction. Several retroviruses bearing LgBiT-PGAM1, Chk1WT-SmBiT, or Chk1-SmBiT (S280A or S280D) mutants have been produced. The indicated retroviruses were introduced into IMR90 cells. Senescence was induced by Ras G12V expression. NanoBit assays detected interactions between PGAM1 and Chk1-WT or Chk1- S280A in SnCs (left panel) and between PGAM1 and Chk1-WT or Chk1- S280D in non-SnCs (right panel) ( n = 3, biological replicates). k Effect of RSK1 knockdown on the PGAM1-Chk1 interaction in oncogene-induced SnCs. A NanoBit assay was performed to detect the interaction between PGAM1 and Chk1 ( n = 3, biological replicates). l A schematic diagram of the mechanism that regulates the interaction between PGAM1 and Chk1. This image was created with BIORENDER. The data represent the means ± SEMs. Single (*) and double (**) asterisks indicate statistical significance at p < 0.05 and p < 0.01, respectively. Statistical analyses were performed via unpaired Student’s two-tailed t tests ( j ) or one-way analysis of variance (ANOVA) and Dunnett’s multiple comparison test ( h and k )

Article Snippet: Anti-mouse Chk1 (25887-1-AP, 1:1000) and anti-mouse FOXM1 (13147-1-AP, 1:1000) were purchased from Proteintech (USA).

Techniques: Control, Western Blot, Sequencing, Phospho-proteomics, Ubiquitin Proteomics, Expressing, Purification, Transfection, Activation Assay, Produced, Knockdown, Two Tailed Test, Comparison

Journal: Signal Transduction and Targeted Therapy

Article Title: Abrogation of aberrant glycolytic interactions eliminates senescent cells and alleviates aging-related dysfunctions

doi: 10.1038/s41392-025-02502-6

Figure Lengend Snippet: A PGAM1-Chk1 antagonist suppresses FOXM1 during senescence. a , b Assessment of the proapoptotic BH family in SnCs after Nutlin 3b treatment. a RT‒PCR analysis of SnCs with or without Nutlin 3b. ( n = 3, biological replicates). b p53 siRNA-transfected SnCs were exposed to Nutlin 3b. RT‒PCR analysis of the indicated cells. c Knockdown of BIM in SnCs. SnCs were transfected with the indicated siRNA, siBIM, or scramble control RNA. The transfectants were then subjected to Nutlin 3b treatment. d – f Comparison of the results of the microarray analysis between the control and Nutlin 3b-treated SnCs ( n = 3, biological replicates). d Fold enrichment analysis by comparison between 1168 genes downregulated by Nutlin 3b in SnCs and 9442 datasets of chromatin immunoprecipitation (ChIP) information reported in the National Center for Biotechnology Information (NCBI), European Bioinformatics Institute (EBI) and DNA Data Bank of Japan (DDBJ). Eighty-six of the 9442 datasets displayed high enrichment (>3-fold). Twenty-one datasets for FOXM1 are shown in red (left panel). e Heatmap of 37 FOXM1 target genes downregulated by Nutlin 3b. SnCs with or without Nutlin 3b were compared. f Gene expression of the FOX family in SnCs after Nutlin 3b treatment, as determined via microarray data. Left panel, heatmap analysis of the FOX family. Right panel: Red and blue bars indicate reduced and increased expression, respectively. Dagger (†) indicates |FC|≥2 and Lpe. P < 0.05 is presented in Supplementary Fig. . Evaluation of FOXM1 protein levels in SnCs after Nutlin 3b treatment ( g ) and after transfection with HIF-2α siRNA ( h ). The data are representative of two independent experiments. i Evaluation of BIM mRNA in SnCs after FOXM1 knockdown. FOXM1 siRNA was transfected into SnCs ( n = 3, biological replicates). j Schematic diagram of the promoter region in the human BIM gene. Several cis-elements for FKH are shown. The indicated regions were subsequently cloned and inserted into luciferase reporter plasmids (left lower panel). Luciferase reporter assays were performed in SnCs after Nutlin 3b treatment (middle panel). A reporter assay for the proximal region was conducted in SnCs after FOXM1 knockdown (right panel) ( n = 3, biological replicates). The data represent the means ± SEMs. Single (*) and double (**) asterisks indicate statistical significance at p < 0.05 and p < 0.01, respectively. Statistical analyses were performed via unpaired Student’s two-tailed t test ( a ) or one-way analysis of variance (ANOVA) and Dunnett’s multiple comparison test ( b , c , i and j )

Article Snippet: Anti-mouse Chk1 (25887-1-AP, 1:1000) and anti-mouse FOXM1 (13147-1-AP, 1:1000) were purchased from Proteintech (USA).

Techniques: Transfection, Knockdown, Control, Comparison, Microarray, Chromatin Immunoprecipitation, Gene Expression, Expressing, Clone Assay, Luciferase, Reporter Assay, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Abrogation of aberrant glycolytic interactions eliminates senescent cells and alleviates aging-related dysfunctions

doi: 10.1038/s41392-025-02502-6

Figure Lengend Snippet: Chemical or genetic interference with PGAM1-Chk1 binding alleviated lung fibrosis in vivo. Generation of Pgam1 W68A knock-in (KI) mice via the CRISPR/Cas9 system ( a – c ). Bleomycin (BLM)-induced lung fibrosis was evaluated in Pgam1 KI/KI mice ( d – f ). a Schematic diagram of Pgam1 (exons 1–4) alleles; the Nutlin-consensus motif ([L/I/V/M]-[W/Y/F]-X-X- [L/I/V/M]) is located in exon 2. The W68A mutation was introduced via CRISPR/Cas9. b Genomic PCR was used to detect the W68A mutation in Pgam1 KI/KI mice. c Representative images of Pgam1 +/+ and KI/KI mice. The bar indicates 1 cm. d Histological images of BLM-induced lung fibrosis in Pgam1 KI/KI mice obtained via Masson’s trichrome (MT) staining. Lung tissues were collected on day 18 after 2.5 mg/kg BLM injection. The bar indicates 100 μm. e Evaluation of fibrotic areas in Pgam1 KI/KI mice via MT staining. f Assessment of fibrotic markers in lungs from Pgam1 KI/KI mice via RT‒PCR analysis. Administration of Nutlin 3b to a mouse model of lung fibrosis ( g – k ). BLM was intratracheally instilled into the lungs of 12-week-old CD1 mice, resulting in DNA damage-induced pulmonary fibrosis. g Schematic diagram of the in vivo protocol of BLM-induced pulmonary fibrosis induced by Nutlin 3b treatment or vehicle. Lung tissues were collected on day 18 after BLM injection. h Representative images of MT staining of the lungs of Nutlin 3b-treated mice. The bar indicates 100 μm. i Evaluation of fibrotic areas via MT staining. j Heatmap analysis of fibrosis markers, senescence and SASP among the control, BLM + vehicle, and BLM + Nutlin 3b groups. k Assessment of the expression of the fibrotic markers col3a1, col5a2, fibronectin, and tropoelastin in BLM-induced lung fibrosis after Nutlin 3b treatment via RT‒PCR analysis. l A schematic diagram of the mechanism by which a novel senolytic pathway targets aberrant PGAM1-Chk1 interactions. This image was created with BIORENDER

Article Snippet: Anti-mouse Chk1 (25887-1-AP, 1:1000) and anti-mouse FOXM1 (13147-1-AP, 1:1000) were purchased from Proteintech (USA).

Techniques: Binding Assay, In Vivo, Knock-In, CRISPR, Mutagenesis, Staining, Injection, Control, Expressing

Journal: NAR Cancer

Article Title: PARP inhibitor response is enhanced in prostate cancer when XRCC1 expression is reduced

doi: 10.1093/narcan/zcaf015

Figure Lengend Snippet: Antibodies used for immunoblotting experiments

Article Snippet: CHK1 (#2360) , 1:1000 , Cell Signaling Technology.

Techniques: Western Blot