Journal: Bone Research

Article Title: Chaperone-mediated autophagy directs a dual mechanism to balance premature senescence and senolysis to prevent intervertebral disc degeneration

doi: 10.1038/s41413-025-00441-0

Figure Lengend Snippet: CMA is essential for maintaining the youthful state of the nucleus pulposus. a , b The protein and mRNA levels of L2A (LAMP2A) in human intervertebral discs with different degeneration grades. c The protein levels of L2A, P53, P21, and P16 in different senescent NPC models, N-Sen (Nutlin-3a-induced senescence), R-Sen (Replicative senescence, passage 9), and I-Sen (IL1B-induced senescence). d Immunofluorescence shows the levels of SA-β-gal and CMA activity in different senescent NPC models. CMA activity is represented by the average number of red spots per cell. e The protein levels of L2A, P53, P21, and P16 in NPC treated with ATRA or sg-L2A transfection. f Flow cytometry results of the cell cycle of each group. g The effect of CMA activation on IL1B-induced IDD. MRI imaging and histological staining were performed after sampling. h The grade of IDD in each group was evaluated by Pfirrmann scores, DHI, and histological scores, as described before. i Immunofluorescence showing levels of P53, P21, and P16 in the rat discs. j – l The effect of L2A-KD on rat IDD, evaluated by Pfirrmann grading and DHI. m The effect of L2A-KD on in vivo levels of P53, P21, and P16. All graphs show the mean ± SEM of at least three independent experiments. * P <0.05, ** P <0.01, *** P <0.001

Article Snippet: AR7 (#HY-101106), Chloroquine/CQ (#HY-17589A), 3MA (#HY-19312), BPTES (#HY-12683), apoptosis inducer-10/AI-10 (#HY-146255), IL1B (#HY- P73149 ), All-trans Retinoic acid/ATRA (#HY-14649), Rapamycin/Rap (#HY-10219) and MG132 (#HY-13259) were purchased from MedChemExpress company.

Techniques: Immunofluorescence, Activity Assay, Transfection, Flow Cytometry, Activation Assay, Imaging, Staining, Sampling, In Vivo

Journal: Bone Research

Article Title: Chaperone-mediated autophagy directs a dual mechanism to balance premature senescence and senolysis to prevent intervertebral disc degeneration

doi: 10.1038/s41413-025-00441-0

Figure Lengend Snippet: Elevated DYRK1A drives premature senescence in CMA-downregulated NPC. a TMT-MS analysis showing that DYRK1A levels were increased in NPC after L2A knockout. b Schematic diagram of the sequence of DYRK1A. c The protein level of DYRK1A in NPC treated with ATRA, si-L2A, or sg-L2A. d Immunofluorescence shows the level of DYRK1A in NPC after L2A knockout. e Immunoblotting shows the levels of L2A, DYRK1A, P53, P21, and P16 in NPC treated as indicated. NPC were treated with IL1B (10 ng/mL, 48 h) and Har (Harmine, 20 μg/mL, 48 h). f Flow cytometry showing the cell cycle of NPC treated as indicated. The proportion of NPC in the S phase was used for comparison. g Relative BrdU levels of NPC treated as indicated, measured at 450 nm. h SA-β-gal fluorescence levels detected by Fluorescein diβ-D-galactopyranoside (FDG) in NPC treated as indicated. i Western blot showing the protein levels of DYRK1A and senescence markers in NPC of each group. j Flow cytometry showing the cell cycle changes of NPC treated as indicated. k Relative BrdU levels of NPC treated as indicated. l Immunofluorescence showing SA-β-gal levels in NPC after DYRK1A and L2A overexpression. All graphs show the mean ± SEM of at least three independent experiments. * P <0.05, ** P <0.01, *** P <0.001

Article Snippet: AR7 (#HY-101106), Chloroquine/CQ (#HY-17589A), 3MA (#HY-19312), BPTES (#HY-12683), apoptosis inducer-10/AI-10 (#HY-146255), IL1B (#HY- P73149 ), All-trans Retinoic acid/ATRA (#HY-14649), Rapamycin/Rap (#HY-10219) and MG132 (#HY-13259) were purchased from MedChemExpress company.

Techniques: Knock-Out, Sequencing, Immunofluorescence, Western Blot, Flow Cytometry, Comparison, Fluorescence, Over Expression

Journal: Bone Research

Article Title: Chaperone-mediated autophagy directs a dual mechanism to balance premature senescence and senolysis to prevent intervertebral disc degeneration

doi: 10.1038/s41413-025-00441-0

Figure Lengend Snippet: CMA induces a fate switch of senescent NPC from cellular senescence to apoptosis. a , b Immunofluorescence showing the effect of L2A overexpression on P53 and senescence levels in I-Sen NPC. c The effect of L2A overexpression on the cell cycle of NPC in the control or I-Sen group. d Western blot showing the effect of L2A overexpression on the levels of senescence-related proteins (P53, P21, P16) in I-Sen NPC. e Western blot showing the effect of L2A overexpression on the levels of SASP inflammatory factors (IL6, IL-8, IL1B, TNF) and MMP3 in I-Sen NPC. f After treatment with CMA activator AR7 in I-Sen NPC, RNA-seq revealed that apoptosis-related pathways were significantly enriched. g , h TUNEL staining shows the apoptosis level in I-Sen NPC after L2A overexpression. i , j Immunoblotting showing the effect of L2A overexpression on the expression of apoptosis-related proteins in I-Sen NPC. k , l The effect of L2A overexpression on mitochondrial membrane potential in NPC of each group. m The apoptosis levels in normal and senescent NPC (R-Sen and I-Sen) treated with AI-10 or combined with L2A overexpression. n The effect of L2A overexpression or combined with BNIP3 knockdown on the apoptosis of senescent NPC. All graphs show the mean ± SEM of at least three independent experiments. * P <0.05, ** P <0.01, *** P <0.001, ns indicates not significant

Article Snippet: AR7 (#HY-101106), Chloroquine/CQ (#HY-17589A), 3MA (#HY-19312), BPTES (#HY-12683), apoptosis inducer-10/AI-10 (#HY-146255), IL1B (#HY- P73149 ), All-trans Retinoic acid/ATRA (#HY-14649), Rapamycin/Rap (#HY-10219) and MG132 (#HY-13259) were purchased from MedChemExpress company.

Techniques: Immunofluorescence, Over Expression, Control, Western Blot, RNA Sequencing, TUNEL Assay, Staining, Expressing, Membrane, Knockdown

Journal: Nature Communications

Article Title: Macrophage SUCLA2 coupled glutaminolysis manipulates obesity through AMPK

doi: 10.1038/s41467-025-57044-w

Figure Lengend Snippet: a Schematic diagram of mice feeding and this image was created in BioRender. Zang, Y. (2025) https://BioRender.com/e16k510 . b–n Male AMPKα fl/fl mice and LysM-Cre, AMPKα fl/fl mice with C57BL/6 background at the age of 6 weeks were fed HFD with or without IL-1β neutralizing antibody (1 mg/kg, twice one week) to explore the obesity development. Immunofluorescent staining of IL-1β in BAT and ScWAT ( b ), body weight gain ( c , n = 5 mice), relative fat and lean mass ( d , n = 5 mice), the weight of Liver ( e , n = 5 mice), BAT ( f , n = 5 mice), and ScWAT ( g , n = 5 mice), representative H&E staining of the liver, BAT and ScWAT ( h ), insulin tolerance test ( i , n = 5 mice), the rectal temperature in cold exposure at 4 °C for different times ( j , k , n = 5 mice), immunohistochemical staining of UCP-1 in BAT ( l ), the proinflammatory genes of ScWAT ( m , Il1b, Tnfa, Nos2, Ccl2 and F4/80: n = 5 mice in each group, Il6: n = 4 mice in LysM-Cre, AMPKα fl/fl + IL-1β mAb group and n = 5 mice in other group), and the immunohistochemical staining of F4/80 in BAT and ScWAT ( n ). Data are presented as the mean ± SEM, groups were compared by two-way ANOVA followed by Fisher’s LSD test ( c – g , i – k , m ). P < 0.05 was considered to be statistically significant.

Article Snippet: To trigger the activation of the inflammasome and detect secreted IL-1β, macrophages were exposed to 2 mM ATP (#HY-B2176, MCE) for 30 min after the incubation with 100 ng/mL LPS, and the secreted IL-1β was detected by mouse IL-1β Elisa kit (#abs520001, Absin).

Techniques: Staining, Immunohistochemical staining

Journal: Nature Communications

Article Title: Macrophage SUCLA2 coupled glutaminolysis manipulates obesity through AMPK

doi: 10.1038/s41467-025-57044-w

Figure Lengend Snippet: a–t Male LysM-Cre, IL-1β fl/fl mice, LysM-Cre, IL-1β fl/fl , AMPKα fl/fl mice, AMPKα fl/fl mice, IL-1β fl/fl mice, and LysM-Cre, AMPKα fl/fl mice with C57BL/6 background at the age of 8 weeks were fed HFD to explore the obesity and related phenotype. Body weight change ( a , n = 8 mice), body weight gain ( b , n = 8 mice), representative mice image ( c ), relative fat and lean mass ( d , n = 8 mice), the representative image of liver, BAT and ScWAT ( e ), the metabolic organ weight of liver ( f , n = 8 mice), BAT ( g , n = 8 mice) and ScWAT ( h , n = 8 mice), representative H&E staining of liver, BAT and ScWAT ( i ), insulin tolerance test ( j , n = 8 mice), the rectal temperature in cold exposure at 4 °C for different times ( k , l , n = 8 mice), immunohistochemical staining of UCP-1 in BAT ( m ), the proinflammatory genes of ScWAT ( n – s , n = 8 mice) and immunohistochemical staining of F4/80 in BAT and ScWAT ( t ). Data are presented as the mean ± SEM, groups were compared by one-way ANOVA followed by Fisher’s LSD test ( b , d , f – h , right of j , l , n – s ) or two-way ANOVA followed by Fisher’s LSD test ( a ). P < 0.05 was considered to be statistically significant.

Article Snippet: To trigger the activation of the inflammasome and detect secreted IL-1β, macrophages were exposed to 2 mM ATP (#HY-B2176, MCE) for 30 min after the incubation with 100 ng/mL LPS, and the secreted IL-1β was detected by mouse IL-1β Elisa kit (#abs520001, Absin).

Techniques: Staining, Immunohistochemical staining

Journal: Nature Communications

Article Title: Macrophage SUCLA2 coupled glutaminolysis manipulates obesity through AMPK

doi: 10.1038/s41467-025-57044-w

Figure Lengend Snippet: a Schematic representation of the potential substrate of AMPK in succinate formation, and this image was created in BioRender. Zang, Y. (2025) https://BioRender.com/e16k510 . Co-immunoprecipitation analysis of the interaction of AMPKα with SUCLA2 ( b ) or SUCLG2 ( c ) in RAW 264.7 cells. d Immunoblot analysis of indicated proteins in BMDMs that are transfected with siRNA for 30 h followed by stimulation with 100 ng/mL LPS for an additional 12 h. e Immunoblot analysis of indicated proteins in AMPKα fl/fl BMDMs (Flox) and LysM-Cre, AMPKα fl/fl BMDMs (MKO) that are transfected with siRNA for 18 h followed by stimulation with 100 ng/mL LPS for an additional 12 h. f Immunoblot analysis of the expression of SUCLG1 and SUCLA2 after AMPKα was knocked down by siRNA for 48 h in RAW 264.7 cells. g The relative enzymatic activity of SUCLA2 (the direction from succinyl-CoA to succinate) was detected after AMPKα was knocked down by siRNA for 36 h followed by stimulation with 100 ng/mL LPS for an additional 12 h in RAW264.7 cells ( n = 3 biological replicates). h In vitro phosphorylation analysis was performed by mixing purified His-CAMKKβ, His-AMPKα1β1γ1 and His-SUCLA2 in the presence of ATP-g-S, and immunoblot analysis of indicated proteins with indicated antibodies. i Venn diagram was used to integrate the phosphorylation site that was detected by mass spectrometry and predicted by GPS 5.0 ( http://gps.biocuckoo.cn/ ) or HPRD . j Protein sequence alignment indicated the conservation of SUCLA2 at Ser60 across multiple species. k Immunoblot analysis of indicated proteins in the in vitro kinase assay that mixed the purified His-CAMKKβ, His-AMPKα1β1γ1 with His-SUCLA2 (WT) or His-SUCLA2 (S60A). l Immunoblot analysis of indicated proteins in BMDMs after incubation with 200 µM A-769662 for 3 h. m In vitro phosphorylation analysis was performed by mixing purified His-CAMKKβ, His-AMPKα1β1γ1 and His-SUCLA2 (WT) or His-SUCLA2 (S60A) in the presence of ATP-g-S, and immunoblot analysis of indicated proteins with indicated antibodies. The enzymatic activity (the direction from succinyl-CoA to succinate) of purified WT-SUCLA2 ( n ) or S60A-SUCLA2 ( o ) after incubation with AMPK in vitro (n = 3 biological replicates). p The enzymatic activity (the direction from succinyl-CoA to succinate) of purified WT-SUCLA2, S60A-SUCLA2, and S60D-SUCLA2 in the same protein content (n = 4 biological replicates). q–r The relative gene expression of IL-1β in RAW264.7 cells that overexpress human WT-SUCLA2, S60A-SUCLA2 and S60D-SUCLA2 through lentivirus infection for 48 h followed the treatment by 100 ng/mL LPS for 6 h in the condition of glutamine replete ( q ) or deprived ( r ) condition (n = 4 biological replicates). The relative gene expression of IL-1β in RAW264.7 cells that overexpress human WT-SUCLA2 with S60A-SUCLA2 ( s ) or S60D-SUCLA2 ( t ) through lentivirus infection for 48 h, the cells were treated with 200 µM A-769662 for 3 h in advance followed by 100 ng/mL LPS for 6 h (n = 4 biological replicates). Data are presented as the mean ± SEM, groups were compared by the unpaired two-tailed Student’s t test (g) or one-way ANOVA followed by Bonferroni’s multiple-comparisons test ( q , r ) or two-way ANOVA followed by Bonferroni’s multiple-comparisons test ( n , o , p , s , t ), representative data are shown from one of the three independent experiments ( b – f , h , k – m ). P < 0.05 was considered to be statistically significant.

Article Snippet: To trigger the activation of the inflammasome and detect secreted IL-1β, macrophages were exposed to 2 mM ATP (#HY-B2176, MCE) for 30 min after the incubation with 100 ng/mL LPS, and the secreted IL-1β was detected by mouse IL-1β Elisa kit (#abs520001, Absin).

Techniques: Immunoprecipitation, Western Blot, Transfection, Expressing, Activity Assay, In Vitro, Phospho-proteomics, Purification, Mass Spectrometry, Sequencing, Kinase Assay, Incubation, Gene Expression, Infection, Two Tailed Test

Journal: Nature Communications

Article Title: Macrophage SUCLA2 coupled glutaminolysis manipulates obesity through AMPK

doi: 10.1038/s41467-025-57044-w

Figure Lengend Snippet: a Immunofluorescent staining of CD68, pT 172 -AMPK, pS 60 -SUCLA2 and IL-1β in the subcutaneous adipose tissue from lean (BMI = 21.5), overweight (BMI = 25.7) or obese (BMI = 32.0) subjects, representative data are shown from one of the three independent experiments. b Model of how macrophage AMPK responds to glutaminolysis and regulates glutaminolysis-coupled IL-1β expression, and this image was created in BioRender. Zang, Y. (2025) https://BioRender.com/e16k510 .

Article Snippet: To trigger the activation of the inflammasome and detect secreted IL-1β, macrophages were exposed to 2 mM ATP (#HY-B2176, MCE) for 30 min after the incubation with 100 ng/mL LPS, and the secreted IL-1β was detected by mouse IL-1β Elisa kit (#abs520001, Absin).

Techniques: Staining, Expressing