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akt substrate 160  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc akt substrate 160
    Resistance exercise exceeds the benefits of endurance exercise in ameliorating metabolic dysfunction. Following 8 weeks of diet and exercise interventions, all mice were assessed for their metabolic function by GTT, ITT, and skeletal muscle response of Akt and <t>AS160</t> phosphorylation to injection of insulin measured by Western blot. (A–C) HOMA-IR taken after an overnight fast for baseline glucose and insulin. (D and E) GTT from 0–120 min and calculated AUC; colored * indicates significant difference from NC-SED. (F and G) ITT from 0–60 min and calculated AUC; colored * indicates significant difference from NC-SED. (H–N) pAkt stimulation, AS160 <t>S318,</t> and AS160 T642 in hindlimb muscles before and after insulin injection and the pre–post ∆ in phosphorylation. (O and P) Total and phosphorylated 4E-BP1. (Q–T) Western results for Raptor, COX4, LC3 II/I, and ubiquitin staining. Representative western blot images inset right. Data presented as mean ± standard error of the mean. Statistical analysis performed by analysis of variance between groups: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NC-SED n : 8–16 (white); HFD-SED n : 8–18 (red); HFD-R EX n : 8–16 (blue); HFD-E EX n : 8–15 (green). 4E-BP1 = Eukaryotic translation initiation factor 4E binding protein; Akt = protein kinase B; AS160 = Akt substrate 160; COX4 = cytochrome c oxidase 4; CS = citrate sythase; E EX = endurance exercise; GAPDH = glyceraldehyde 3 phosphate dehydrogenase; GTT = glucose tolerance test; HFD = high fat diet; HOMA-IR = homeostatic model assessment for insulin resistance; iAUC = integrated area under the curve; ITT = insulin tolerance test; LC3 II/I = microtubule-associated protein light chain 3; NC = normal chow; pAkt = phospho-Akt; R EX = resistance exercise; SED = sedentary; Ub = ubiquitin.
    Akt Substrate 160, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/monoclonal+rabbit+antibodies/Phospho-AS160+(Ser318)+Rabbit+mAb/pmc12816905-99-63-69
    Average 93 stars, based on 31 article reviews
    akt substrate 160 - by Bioz Stars, 2026-10
    93/100 stars

    Images

    1) Product Images from "Weightlifting outperforms voluntary wheel running for improving adiposity and insulin sensitivity in obese mice"

    Article Title: Weightlifting outperforms voluntary wheel running for improving adiposity and insulin sensitivity in obese mice

    Journal: Journal of Sport and Health Science

    doi: 10.1016/j.jshs.2025.101100

    Resistance exercise exceeds the benefits of endurance exercise in ameliorating metabolic dysfunction. Following 8 weeks of diet and exercise interventions, all mice were assessed for their metabolic function by GTT, ITT, and skeletal muscle response of Akt and AS160 phosphorylation to injection of insulin measured by Western blot. (A–C) HOMA-IR taken after an overnight fast for baseline glucose and insulin. (D and E) GTT from 0–120 min and calculated AUC; colored * indicates significant difference from NC-SED. (F and G) ITT from 0–60 min and calculated AUC; colored * indicates significant difference from NC-SED. (H–N) pAkt stimulation, AS160 S318, and AS160 T642 in hindlimb muscles before and after insulin injection and the pre–post ∆ in phosphorylation. (O and P) Total and phosphorylated 4E-BP1. (Q–T) Western results for Raptor, COX4, LC3 II/I, and ubiquitin staining. Representative western blot images inset right. Data presented as mean ± standard error of the mean. Statistical analysis performed by analysis of variance between groups: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NC-SED n : 8–16 (white); HFD-SED n : 8–18 (red); HFD-R EX n : 8–16 (blue); HFD-E EX n : 8–15 (green). 4E-BP1 = Eukaryotic translation initiation factor 4E binding protein; Akt = protein kinase B; AS160 = Akt substrate 160; COX4 = cytochrome c oxidase 4; CS = citrate sythase; E EX = endurance exercise; GAPDH = glyceraldehyde 3 phosphate dehydrogenase; GTT = glucose tolerance test; HFD = high fat diet; HOMA-IR = homeostatic model assessment for insulin resistance; iAUC = integrated area under the curve; ITT = insulin tolerance test; LC3 II/I = microtubule-associated protein light chain 3; NC = normal chow; pAkt = phospho-Akt; R EX = resistance exercise; SED = sedentary; Ub = ubiquitin.
    Figure Legend Snippet: Resistance exercise exceeds the benefits of endurance exercise in ameliorating metabolic dysfunction. Following 8 weeks of diet and exercise interventions, all mice were assessed for their metabolic function by GTT, ITT, and skeletal muscle response of Akt and AS160 phosphorylation to injection of insulin measured by Western blot. (A–C) HOMA-IR taken after an overnight fast for baseline glucose and insulin. (D and E) GTT from 0–120 min and calculated AUC; colored * indicates significant difference from NC-SED. (F and G) ITT from 0–60 min and calculated AUC; colored * indicates significant difference from NC-SED. (H–N) pAkt stimulation, AS160 S318, and AS160 T642 in hindlimb muscles before and after insulin injection and the pre–post ∆ in phosphorylation. (O and P) Total and phosphorylated 4E-BP1. (Q–T) Western results for Raptor, COX4, LC3 II/I, and ubiquitin staining. Representative western blot images inset right. Data presented as mean ± standard error of the mean. Statistical analysis performed by analysis of variance between groups: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NC-SED n : 8–16 (white); HFD-SED n : 8–18 (red); HFD-R EX n : 8–16 (blue); HFD-E EX n : 8–15 (green). 4E-BP1 = Eukaryotic translation initiation factor 4E binding protein; Akt = protein kinase B; AS160 = Akt substrate 160; COX4 = cytochrome c oxidase 4; CS = citrate sythase; E EX = endurance exercise; GAPDH = glyceraldehyde 3 phosphate dehydrogenase; GTT = glucose tolerance test; HFD = high fat diet; HOMA-IR = homeostatic model assessment for insulin resistance; iAUC = integrated area under the curve; ITT = insulin tolerance test; LC3 II/I = microtubule-associated protein light chain 3; NC = normal chow; pAkt = phospho-Akt; R EX = resistance exercise; SED = sedentary; Ub = ubiquitin.

    Techniques Used: Phospho-proteomics, Injection, Western Blot, Muscles, Ubiquitin Proteomics, Staining, Binding Assay

    Related Articles

    Membrane:

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    Article Snippet: After electrophoresis (4 h at 0.02 A), proteins were transferred onto nitrocellulose membranes (GE Healthcare Bio-Sciences, Marlborough, MA, USA) using Mini Trans-Blot Cell (Bio-Rad Laboratories, Inc., Hercules, CA) at 90 V for 1.5 h. Incubation of membranes in 5% milk solution for 1 h at 37 °C was used to block non-specific binding. .. Membrane was incubated with monoclonal rabbit antibodies raised against cleaved caspase-3, procaspase-6, cleaved caspase-6, cleaved caspase-7, Bid (Cell Signaling Technology, Inc., Danvers, MA, USA), AIF (sc-5586), Bcl-X L/S (sc-634) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), caspase-9 (BD 556585) (BD Biosciences, San Jose, CA, USA), β-actin (Sigma-Aldrich, St. Louis, MO), and monoclonal mouse antibodies against PARP-1 (BD 556434), FADD (BD 556402), Mcl-1 (BD 559027), caspase-2 (BD 611022), caspase-8 (BD 551242) (BD Biosciences, San Jose, CA, USA), caspase-10 (M059-3) (MBL International Corporation, Woburn, MA) for 12 h at 4 °C with slow shaking. ..

    Incubation:

    Article Title: Cannabimimetic N -Stearoylethanolamine as “Double-Edged Sword” in Anticancer Chemotherapy: Proapoptotic Effect on Tumor Cells and Suppression of Tumor Growth versus Its Bio-Protective Actions in Complex with Polymeric Carrier on General Toxicity of Doxorubicin In Vivo
    Article Snippet: After electrophoresis (4 h at 0.02 A), proteins were transferred onto nitrocellulose membranes (GE Healthcare Bio-Sciences, Marlborough, MA, USA) using Mini Trans-Blot Cell (Bio-Rad Laboratories, Inc., Hercules, CA) at 90 V for 1.5 h. Incubation of membranes in 5% milk solution for 1 h at 37 °C was used to block non-specific binding. .. Membrane was incubated with monoclonal rabbit antibodies raised against cleaved caspase-3, procaspase-6, cleaved caspase-6, cleaved caspase-7, Bid (Cell Signaling Technology, Inc., Danvers, MA, USA), AIF (sc-5586), Bcl-X L/S (sc-634) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), caspase-9 (BD 556585) (BD Biosciences, San Jose, CA, USA), β-actin (Sigma-Aldrich, St. Louis, MO), and monoclonal mouse antibodies against PARP-1 (BD 556434), FADD (BD 556402), Mcl-1 (BD 559027), caspase-2 (BD 611022), caspase-8 (BD 551242) (BD Biosciences, San Jose, CA, USA), caspase-10 (M059-3) (MBL International Corporation, Woburn, MA) for 12 h at 4 °C with slow shaking. ..

    Centrifugation:

    Article Title: CD45 and Basigin (CD147) Are Functional Ligands for Galectin-8 on Human Leukocytes
    Article Snippet: .. After 5 min centrifugation at 18,000× g, supernatants were boiled for 5 min, electrophoresed, and transblotted with I-Blot (Invitrogen), then probed overnight at 4 ◦C for ERK or phospho-ERK (p-ERK) with monoclonal rabbit antibodies (1:1000 in TBST-5% milk, clones 9101S and 9102, Cell Signaling, Danvers, MA, USA). ..

    Article Title: CD45 and Basigin (CD147) Are Functional Ligands for Galectin-8 on Human Leukocytes
    Article Snippet: .. After 5 min centrifugation at 18,000× g , supernatants were boiled for 5 min, electrophoresed, and transblotted with I-Blot (Invitrogen), then probed overnight at 4 °C for ERK or phospho-ERK (p-ERK) with monoclonal rabbit antibodies (1:1000 in TBST-5% milk, clones 9101S and 9102, Cell Signaling, Danvers, MA, USA). ..

    Clone Assay:

    Article Title: CD45 and Basigin (CD147) Are Functional Ligands for Galectin-8 on Human Leukocytes
    Article Snippet: .. After 5 min centrifugation at 18,000× g, supernatants were boiled for 5 min, electrophoresed, and transblotted with I-Blot (Invitrogen), then probed overnight at 4 ◦C for ERK or phospho-ERK (p-ERK) with monoclonal rabbit antibodies (1:1000 in TBST-5% milk, clones 9101S and 9102, Cell Signaling, Danvers, MA, USA). ..

    Article Title: CD45 and Basigin (CD147) Are Functional Ligands for Galectin-8 on Human Leukocytes
    Article Snippet: .. After 5 min centrifugation at 18,000× g , supernatants were boiled for 5 min, electrophoresed, and transblotted with I-Blot (Invitrogen), then probed overnight at 4 °C for ERK or phospho-ERK (p-ERK) with monoclonal rabbit antibodies (1:1000 in TBST-5% milk, clones 9101S and 9102, Cell Signaling, Danvers, MA, USA). ..

    Western Blot:

    Article Title: A novel nordihydroguaiaretic acid analog, compound 3a, alleviates acute lung injury by exerting antiapoptotic and antiinflammatory effects.
    Article Snippet: Acute lung injury (ALI) is a continuum of pulmonary changes caused by various lung insults.. Previously, we synthesized a series of nordihydroguaiaretic acid analogs; of these, compound 3a exhibited excellent antioxidant capacity in a murine model of middle cerebral artery occlusion.. However, it remains unclear whether compound 3a can modulate lipopolysaccharide (LPS)-induced ALI.

    Immunohistochemistry:

    Article Title: Complement 9 in amyloid deposits.
    Article Snippet: .. Immunohistochemistry was carried out with commercially available monoclonal antibodies directed against AA amyloid (dilution 1:2000), b amyloid (1:50; both DAKO, Hamburg, Germany), insulin (1:500; BioGenex, San Ramon, CA), apoE (1:10,000), C3 (1:400; both Abcam, Berlin, Germany), C1q (1:75,000), C3c (1:75,000; both DAKO, Hamburg, Germany), C3d (1:1,000; Abcam, Cambridge, UK), C5b-9 (1:100; DAKO, Hamburg, Germany), MASP-2 (1:200; Sigma-Aldrich, Taufkirchen, Germany), C9 (1:400; Biozol, Eching, Germany) and monoclonal rabbit antibodies directed against caspase 3 (1:100; Cell Signaling, Danvers, MA) and polyclonal rabbit antibodies directed against amyloid P-component (1:2000), kappa-light chain (1:100,000), lambda-light chain (1:14,000; all DAKO, Hamburg, Germany) and non-commercially available polyclonal rabbit antibodies directed against apolipoprotein AI (1:1000), transthyretin (TTR3, 1:2000), lambda-light chain-derived amyloid proteins (AL1 antibody, 1:250), anti-lambda-light chain peptides (AL3, 1:250; AL7, 1:200) and kappa-light chain peptides (AK3, 1:1000; all Pineda, Berlin, Germany) [6]. .. Immunostaining was done on FFPE sections with the Bond Max Leica immunostainer using the Bond Polymer Refine Detection Kit (Leica Biosystems, Wetzlar, Germany) (study cohort) or with the Ventana Benchmark immunostainer and ‘ultraView Universal DAB detection Kit’ (Roche Diagnostics Deutschland GmbH, Mannheim, Germany) (second set of controls) as described previously [21].

    Article Title: Abstracts from USCAP 2020: Genitourinary Pathology (860-1046).
    Article Snippet: .. ROS IHC was performed using monoclonal rabbit antibodies (clone D4D6, Cell Signaling Technology (CST), Danvers, MA) and Signalstain boost IHC detection method (CST) after antigen retrieval at pH8 and 125°C for 30 seconds. ..

    Bioprocessing:

    Article Title: Complement 9 in amyloid deposits.
    Article Snippet: .. Immunohistochemistry was carried out with commercially available monoclonal antibodies directed against AA amyloid (dilution 1:2000), b amyloid (1:50; both DAKO, Hamburg, Germany), insulin (1:500; BioGenex, San Ramon, CA), apoE (1:10,000), C3 (1:400; both Abcam, Berlin, Germany), C1q (1:75,000), C3c (1:75,000; both DAKO, Hamburg, Germany), C3d (1:1,000; Abcam, Cambridge, UK), C5b-9 (1:100; DAKO, Hamburg, Germany), MASP-2 (1:200; Sigma-Aldrich, Taufkirchen, Germany), C9 (1:400; Biozol, Eching, Germany) and monoclonal rabbit antibodies directed against caspase 3 (1:100; Cell Signaling, Danvers, MA) and polyclonal rabbit antibodies directed against amyloid P-component (1:2000), kappa-light chain (1:100,000), lambda-light chain (1:14,000; all DAKO, Hamburg, Germany) and non-commercially available polyclonal rabbit antibodies directed against apolipoprotein AI (1:1000), transthyretin (TTR3, 1:2000), lambda-light chain-derived amyloid proteins (AL1 antibody, 1:250), anti-lambda-light chain peptides (AL3, 1:250; AL7, 1:200) and kappa-light chain peptides (AK3, 1:1000; all Pineda, Berlin, Germany) [6]. .. Immunostaining was done on FFPE sections with the Bond Max Leica immunostainer using the Bond Polymer Refine Detection Kit (Leica Biosystems, Wetzlar, Germany) (study cohort) or with the Ventana Benchmark immunostainer and ‘ultraView Universal DAB detection Kit’ (Roche Diagnostics Deutschland GmbH, Mannheim, Germany) (second set of controls) as described previously [21].



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    DMOG-mediated HIF-1α stabilization exerts a protective effect by inhibiting <t>p53</t> signalling transduction. (A) Volcano plot of DEGs in chondrocytes treated with or without DMOG ( P < .05, |fold change| > 2) under hypoxic conditions. (B) KEGG pathway enrichment analysis of DEGs. (C) Representative immunohistochemical images of p53 in condylar chondrocytes (scale bar: 100 μm) and quantitative analysis of p53-positive area ( n = 6). (D and E) Western blot and quantitative analysis of HIF-1α, p53, p21, and BAX protein levels in chondrocytes under hypoxia and hypoxia + DMOG conditions. (F and G) Western blot images and quantitative analysis of iNOS, HIF-1α, MMP13, p53, p21, BAX, and TNF-α protein levels in chondrocytes under hypoxia, hypoxia + DMOG, and hypoxia + DMOG + NSC-207895 (HX + DMOG + NSC-207895) conditions ( n = 3). (H) Immunofluorescence staining images and quantitative analysis of IL-6 and MMP3 in chondrocytes treated with DMOG and NSC-207895 under hypoxia ( n = 3). Scale bar: 50 μm. Data are presented as mean ± SEM. Statistical significance was determined by unpaired Student’s t test (for pairwise comparisons) or one-way ANOVA (for multigroup comparisons), as appropriate. ns, no significance, * P < .05, ** P < .01, *** P < .001, **** P < .0001.
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    TMH/QG+NIR attenuates ferroptosis and matrix degradation in TBHP‐treated AFCs. (A) Schematic illustration of AF cells co‐culture system. (B) DCFH‐DA fluorescence staining images and quantitative analysis of TBHP‐pretreated AFCs after different treatments. Scale bar: 50 µm. (C) Intracellular ROS levels in TBHP‐pretreated AFCs measured by flow cytometry analysis and quantitative results. (D,E) Quantitative analysis of MDA and GSH levels in TBHP‐pretreated AFCs co‐cultured with QG, TMH and TMH/QG MN (with NIR irradiation). (F,G) Representative images and quantification of labile iron pool by FerroOrange staining and lipid peroxidation by C11‐BODIPY staining in TBHP‐pretreated AFCs co‐culture with QG, TMH and TMH/QG MN (with NIR irradiation). Scale bar: 20 µm. (H) Western blot analysis and corresponding quantitative statistics of extracellular matrix (COL1), remodeling markers (MMP3, <t>MMP13),</t> and ferroptosis‐related proteins (ACSL4, GPX4). The corresponding uncropped blot images are provided in Table . (I) Immunofluorescence staining and fluorescence intensity quantification of COL1 and MMP3 expression in TBHP‐pretreated AFCs co‐cultured with QG, TMH, and TMH/QG MN (with NIR irradiation). Scale bar: 20 µm. Data are presented as mean ± SD (n ≥ 3), and p ‐values were calculated using one‐way ANOVA with Tukey's multiple comparisons test [(B) to (I)].
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    Image Search Results


    Resistance exercise exceeds the benefits of endurance exercise in ameliorating metabolic dysfunction. Following 8 weeks of diet and exercise interventions, all mice were assessed for their metabolic function by GTT, ITT, and skeletal muscle response of Akt and AS160 phosphorylation to injection of insulin measured by Western blot. (A–C) HOMA-IR taken after an overnight fast for baseline glucose and insulin. (D and E) GTT from 0–120 min and calculated AUC; colored * indicates significant difference from NC-SED. (F and G) ITT from 0–60 min and calculated AUC; colored * indicates significant difference from NC-SED. (H–N) pAkt stimulation, AS160 S318, and AS160 T642 in hindlimb muscles before and after insulin injection and the pre–post ∆ in phosphorylation. (O and P) Total and phosphorylated 4E-BP1. (Q–T) Western results for Raptor, COX4, LC3 II/I, and ubiquitin staining. Representative western blot images inset right. Data presented as mean ± standard error of the mean. Statistical analysis performed by analysis of variance between groups: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NC-SED n : 8–16 (white); HFD-SED n : 8–18 (red); HFD-R EX n : 8–16 (blue); HFD-E EX n : 8–15 (green). 4E-BP1 = Eukaryotic translation initiation factor 4E binding protein; Akt = protein kinase B; AS160 = Akt substrate 160; COX4 = cytochrome c oxidase 4; CS = citrate sythase; E EX = endurance exercise; GAPDH = glyceraldehyde 3 phosphate dehydrogenase; GTT = glucose tolerance test; HFD = high fat diet; HOMA-IR = homeostatic model assessment for insulin resistance; iAUC = integrated area under the curve; ITT = insulin tolerance test; LC3 II/I = microtubule-associated protein light chain 3; NC = normal chow; pAkt = phospho-Akt; R EX = resistance exercise; SED = sedentary; Ub = ubiquitin.

    Journal: Journal of Sport and Health Science

    Article Title: Weightlifting outperforms voluntary wheel running for improving adiposity and insulin sensitivity in obese mice

    doi: 10.1016/j.jshs.2025.101100

    Figure Lengend Snippet: Resistance exercise exceeds the benefits of endurance exercise in ameliorating metabolic dysfunction. Following 8 weeks of diet and exercise interventions, all mice were assessed for their metabolic function by GTT, ITT, and skeletal muscle response of Akt and AS160 phosphorylation to injection of insulin measured by Western blot. (A–C) HOMA-IR taken after an overnight fast for baseline glucose and insulin. (D and E) GTT from 0–120 min and calculated AUC; colored * indicates significant difference from NC-SED. (F and G) ITT from 0–60 min and calculated AUC; colored * indicates significant difference from NC-SED. (H–N) pAkt stimulation, AS160 S318, and AS160 T642 in hindlimb muscles before and after insulin injection and the pre–post ∆ in phosphorylation. (O and P) Total and phosphorylated 4E-BP1. (Q–T) Western results for Raptor, COX4, LC3 II/I, and ubiquitin staining. Representative western blot images inset right. Data presented as mean ± standard error of the mean. Statistical analysis performed by analysis of variance between groups: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NC-SED n : 8–16 (white); HFD-SED n : 8–18 (red); HFD-R EX n : 8–16 (blue); HFD-E EX n : 8–15 (green). 4E-BP1 = Eukaryotic translation initiation factor 4E binding protein; Akt = protein kinase B; AS160 = Akt substrate 160; COX4 = cytochrome c oxidase 4; CS = citrate sythase; E EX = endurance exercise; GAPDH = glyceraldehyde 3 phosphate dehydrogenase; GTT = glucose tolerance test; HFD = high fat diet; HOMA-IR = homeostatic model assessment for insulin resistance; iAUC = integrated area under the curve; ITT = insulin tolerance test; LC3 II/I = microtubule-associated protein light chain 3; NC = normal chow; pAkt = phospho-Akt; R EX = resistance exercise; SED = sedentary; Ub = ubiquitin.

    Article Snippet: Primary antibodies used for analysis were from Cell Signaling Technologies (Danvers, MA, USA) and diluted 1:1000 unless otherwise stated as follows: protein kinase B (Akt; 1:500; #4691; Cell Signaling Technologies), phospho-Akt (pAkt) S473 (1:500; #9271; Cell Signaling Technologies), Ubiquitin (#3933; Cell Signaling Technologies), microtubule-associated protein light chain 3 (LC3 II/I; #4018; Cell Signaling Technologies), cytochrome c oxidase subunit 4(COX4; #11967; Cell Signaling Technologies), Akt substrate 160 (AS160 S318; #8619; Cell Signaling Technologies), AS160 T642 (#8881; Cell Signaling Technologies), eukaryotic translation initiation factor 4E binding protein (4E-BP1; #9452; Cell Signaling Technologies), and glyceraldehyde 3 phosphate dehydrogenase (GAPDH; #2118; Cell Signaling Technologies).

    Techniques: Phospho-proteomics, Injection, Western Blot, Muscles, Ubiquitin Proteomics, Staining, Binding Assay

    Resistance exercise exceeds the benefits of endurance exercise in ameliorating metabolic dysfunction. Following 8 weeks of diet and exercise interventions, all mice were assessed for their metabolic function by GTT, ITT, and skeletal muscle response of Akt and AS160 phosphorylation to injection of insulin measured by Western blot. (A–C) HOMA-IR taken after an overnight fast for baseline glucose and insulin. (D and E) GTT from 0–120 min and calculated AUC; colored * indicates significant difference from NC-SED. (F and G) ITT from 0–60 min and calculated AUC; colored * indicates significant difference from NC-SED. (H–N) pAkt stimulation, AS160 S318, and AS160 T642 in hindlimb muscles before and after insulin injection and the pre–post ∆ in phosphorylation. (O and P) Total and phosphorylated 4E-BP1. (Q–T) Western results for Raptor, COX4, LC3 II/I, and ubiquitin staining. Representative western blot images inset right. Data presented as mean ± standard error of the mean. Statistical analysis performed by analysis of variance between groups: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NC-SED n : 8–16 (white); HFD-SED n : 8–18 (red); HFD-R EX n : 8–16 (blue); HFD-E EX n : 8–15 (green). 4E-BP1 = Eukaryotic translation initiation factor 4E binding protein; Akt = protein kinase B; AS160 = Akt substrate 160; COX4 = cytochrome c oxidase 4; CS = citrate sythase; E EX = endurance exercise; GAPDH = glyceraldehyde 3 phosphate dehydrogenase; GTT = glucose tolerance test; HFD = high fat diet; HOMA-IR = homeostatic model assessment for insulin resistance; iAUC = integrated area under the curve; ITT = insulin tolerance test; LC3 II/I = microtubule-associated protein light chain 3; NC = normal chow; pAkt = phospho-Akt; R EX = resistance exercise; SED = sedentary; Ub = ubiquitin.

    Journal: Journal of Sport and Health Science

    Article Title: Weightlifting outperforms voluntary wheel running for improving adiposity and insulin sensitivity in obese mice

    doi: 10.1016/j.jshs.2025.101100

    Figure Lengend Snippet: Resistance exercise exceeds the benefits of endurance exercise in ameliorating metabolic dysfunction. Following 8 weeks of diet and exercise interventions, all mice were assessed for their metabolic function by GTT, ITT, and skeletal muscle response of Akt and AS160 phosphorylation to injection of insulin measured by Western blot. (A–C) HOMA-IR taken after an overnight fast for baseline glucose and insulin. (D and E) GTT from 0–120 min and calculated AUC; colored * indicates significant difference from NC-SED. (F and G) ITT from 0–60 min and calculated AUC; colored * indicates significant difference from NC-SED. (H–N) pAkt stimulation, AS160 S318, and AS160 T642 in hindlimb muscles before and after insulin injection and the pre–post ∆ in phosphorylation. (O and P) Total and phosphorylated 4E-BP1. (Q–T) Western results for Raptor, COX4, LC3 II/I, and ubiquitin staining. Representative western blot images inset right. Data presented as mean ± standard error of the mean. Statistical analysis performed by analysis of variance between groups: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NC-SED n : 8–16 (white); HFD-SED n : 8–18 (red); HFD-R EX n : 8–16 (blue); HFD-E EX n : 8–15 (green). 4E-BP1 = Eukaryotic translation initiation factor 4E binding protein; Akt = protein kinase B; AS160 = Akt substrate 160; COX4 = cytochrome c oxidase 4; CS = citrate sythase; E EX = endurance exercise; GAPDH = glyceraldehyde 3 phosphate dehydrogenase; GTT = glucose tolerance test; HFD = high fat diet; HOMA-IR = homeostatic model assessment for insulin resistance; iAUC = integrated area under the curve; ITT = insulin tolerance test; LC3 II/I = microtubule-associated protein light chain 3; NC = normal chow; pAkt = phospho-Akt; R EX = resistance exercise; SED = sedentary; Ub = ubiquitin.

    Article Snippet: Primary antibodies used for analysis were from Cell Signaling Technologies (Danvers, MA, USA) and diluted 1:1000 unless otherwise stated as follows: protein kinase B (Akt; 1:500; #4691; Cell Signaling Technologies), phospho-Akt (pAkt) S473 (1:500; #9271; Cell Signaling Technologies), Ubiquitin (#3933; Cell Signaling Technologies), microtubule-associated protein light chain 3 (LC3 II/I; #4018; Cell Signaling Technologies), cytochrome c oxidase subunit 4(COX4; #11967; Cell Signaling Technologies), Akt substrate 160 (AS160 S318; #8619; Cell Signaling Technologies), AS160 T642 (#8881; Cell Signaling Technologies), eukaryotic translation initiation factor 4E binding protein (4E-BP1; #9452; Cell Signaling Technologies), and glyceraldehyde 3 phosphate dehydrogenase (GAPDH; #2118; Cell Signaling Technologies).

    Techniques: Phospho-proteomics, Injection, Western Blot, Muscles, Ubiquitin Proteomics, Staining, Binding Assay

    DMOG-mediated HIF-1α stabilization exerts a protective effect by inhibiting p53 signalling transduction. (A) Volcano plot of DEGs in chondrocytes treated with or without DMOG ( P < .05, |fold change| > 2) under hypoxic conditions. (B) KEGG pathway enrichment analysis of DEGs. (C) Representative immunohistochemical images of p53 in condylar chondrocytes (scale bar: 100 μm) and quantitative analysis of p53-positive area ( n = 6). (D and E) Western blot and quantitative analysis of HIF-1α, p53, p21, and BAX protein levels in chondrocytes under hypoxia and hypoxia + DMOG conditions. (F and G) Western blot images and quantitative analysis of iNOS, HIF-1α, MMP13, p53, p21, BAX, and TNF-α protein levels in chondrocytes under hypoxia, hypoxia + DMOG, and hypoxia + DMOG + NSC-207895 (HX + DMOG + NSC-207895) conditions ( n = 3). (H) Immunofluorescence staining images and quantitative analysis of IL-6 and MMP3 in chondrocytes treated with DMOG and NSC-207895 under hypoxia ( n = 3). Scale bar: 50 μm. Data are presented as mean ± SEM. Statistical significance was determined by unpaired Student’s t test (for pairwise comparisons) or one-way ANOVA (for multigroup comparisons), as appropriate. ns, no significance, * P < .05, ** P < .01, *** P < .001, **** P < .0001.

    Journal: International Dental Journal

    Article Title: Hypoxia-Inducible Factor-1α Stabilization Alleviates Hypoxia-Induced Temporomandibular Joint Osteoarthritis by Activating MDM2 to Suppression of p53 Signalling

    doi: 10.1016/j.identj.2026.109805

    Figure Lengend Snippet: DMOG-mediated HIF-1α stabilization exerts a protective effect by inhibiting p53 signalling transduction. (A) Volcano plot of DEGs in chondrocytes treated with or without DMOG ( P < .05, |fold change| > 2) under hypoxic conditions. (B) KEGG pathway enrichment analysis of DEGs. (C) Representative immunohistochemical images of p53 in condylar chondrocytes (scale bar: 100 μm) and quantitative analysis of p53-positive area ( n = 6). (D and E) Western blot and quantitative analysis of HIF-1α, p53, p21, and BAX protein levels in chondrocytes under hypoxia and hypoxia + DMOG conditions. (F and G) Western blot images and quantitative analysis of iNOS, HIF-1α, MMP13, p53, p21, BAX, and TNF-α protein levels in chondrocytes under hypoxia, hypoxia + DMOG, and hypoxia + DMOG + NSC-207895 (HX + DMOG + NSC-207895) conditions ( n = 3). (H) Immunofluorescence staining images and quantitative analysis of IL-6 and MMP3 in chondrocytes treated with DMOG and NSC-207895 under hypoxia ( n = 3). Scale bar: 50 μm. Data are presented as mean ± SEM. Statistical significance was determined by unpaired Student’s t test (for pairwise comparisons) or one-way ANOVA (for multigroup comparisons), as appropriate. ns, no significance, * P < .05, ** P < .01, *** P < .001, **** P < .0001.

    Article Snippet: The membranes were blocked with 5% nonfat milk and incubated with the following primary antibodies: HIF-1α (1:1000; Abcam), p53 (1:1000; ABclonal), TNF-α (1:1000; Huabio), MMP13 (1:1000; Abcam), iNOS (1:1000; Servicebio), BAX (1:1000; Proteintech), p21 (1:1000; Abcam), MDM2 (1:1000; Immunoway), and α-Tubulin (1:5000; Servicebio).

    Techniques: Transduction, Immunohistochemical staining, Western Blot, Immunofluorescence, Staining

    Stabilized HIF-1α transcriptionally activates Mdm2 to promote p53 ubiquitin-proteasomal degradation in hypoxic condylar. (A) Co-IP assay detecting endogenous p53 ubiquitination in hypoxic chondrocytes treated with or without 1 mM DMOG. (B and C) qRT-PCR analysis of Tp53 and Mdm2 mRNA expression in chondrocytes cultured under hypoxia with or without DMOG intervention. (D) Reciprocal Co-IP assay for detecting physical interaction between MDM2 and p53 in hypoxic chondrocytes with or without DMOG treatment. (E) Western blot analysis of p53 protein abundance in hypoxic chondrocytes incubated with DMOG in the presence or absence of 10 μM MG132. (F) JASPAR database-based in silico prediction of potential HIF-1α binding sites on rat Mdm2 promoter. (G) ChIP-qPCR assay measuring HIF-1α occupancy on the Mdm2 promoter in hypoxic chondrocytes with or without DMOG. (H) Schematic diagram. Data are presented as mean ± SEM ( n = 3 per group). Statistical significance was determined by unpaired Student’s t test. ** P < .01, *** P < .001.

    Journal: International Dental Journal

    Article Title: Hypoxia-Inducible Factor-1α Stabilization Alleviates Hypoxia-Induced Temporomandibular Joint Osteoarthritis by Activating MDM2 to Suppression of p53 Signalling

    doi: 10.1016/j.identj.2026.109805

    Figure Lengend Snippet: Stabilized HIF-1α transcriptionally activates Mdm2 to promote p53 ubiquitin-proteasomal degradation in hypoxic condylar. (A) Co-IP assay detecting endogenous p53 ubiquitination in hypoxic chondrocytes treated with or without 1 mM DMOG. (B and C) qRT-PCR analysis of Tp53 and Mdm2 mRNA expression in chondrocytes cultured under hypoxia with or without DMOG intervention. (D) Reciprocal Co-IP assay for detecting physical interaction between MDM2 and p53 in hypoxic chondrocytes with or without DMOG treatment. (E) Western blot analysis of p53 protein abundance in hypoxic chondrocytes incubated with DMOG in the presence or absence of 10 μM MG132. (F) JASPAR database-based in silico prediction of potential HIF-1α binding sites on rat Mdm2 promoter. (G) ChIP-qPCR assay measuring HIF-1α occupancy on the Mdm2 promoter in hypoxic chondrocytes with or without DMOG. (H) Schematic diagram. Data are presented as mean ± SEM ( n = 3 per group). Statistical significance was determined by unpaired Student’s t test. ** P < .01, *** P < .001.

    Article Snippet: The membranes were blocked with 5% nonfat milk and incubated with the following primary antibodies: HIF-1α (1:1000; Abcam), p53 (1:1000; ABclonal), TNF-α (1:1000; Huabio), MMP13 (1:1000; Abcam), iNOS (1:1000; Servicebio), BAX (1:1000; Proteintech), p21 (1:1000; Abcam), MDM2 (1:1000; Immunoway), and α-Tubulin (1:5000; Servicebio).

    Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Expressing, Cell Culture, Western Blot, Quantitative Proteomics, Incubation, In Silico, Binding Assay, ChIP-qPCR

    TMH/QG+NIR attenuates ferroptosis and matrix degradation in TBHP‐treated AFCs. (A) Schematic illustration of AF cells co‐culture system. (B) DCFH‐DA fluorescence staining images and quantitative analysis of TBHP‐pretreated AFCs after different treatments. Scale bar: 50 µm. (C) Intracellular ROS levels in TBHP‐pretreated AFCs measured by flow cytometry analysis and quantitative results. (D,E) Quantitative analysis of MDA and GSH levels in TBHP‐pretreated AFCs co‐cultured with QG, TMH and TMH/QG MN (with NIR irradiation). (F,G) Representative images and quantification of labile iron pool by FerroOrange staining and lipid peroxidation by C11‐BODIPY staining in TBHP‐pretreated AFCs co‐culture with QG, TMH and TMH/QG MN (with NIR irradiation). Scale bar: 20 µm. (H) Western blot analysis and corresponding quantitative statistics of extracellular matrix (COL1), remodeling markers (MMP3, MMP13), and ferroptosis‐related proteins (ACSL4, GPX4). The corresponding uncropped blot images are provided in Table . (I) Immunofluorescence staining and fluorescence intensity quantification of COL1 and MMP3 expression in TBHP‐pretreated AFCs co‐cultured with QG, TMH, and TMH/QG MN (with NIR irradiation). Scale bar: 20 µm. Data are presented as mean ± SD (n ≥ 3), and p ‐values were calculated using one‐way ANOVA with Tukey's multiple comparisons test [(B) to (I)].

    Journal: Advanced Science

    Article Title: Acid‐Responsive Nanobot‐Integrated Core‐Shell Microneedles Reprogram the Degenerative Annulus Fibrosus Microenvironment Through Epigenetic Suppression of Ferroptosis

    doi: 10.1002/advs.77829

    Figure Lengend Snippet: TMH/QG+NIR attenuates ferroptosis and matrix degradation in TBHP‐treated AFCs. (A) Schematic illustration of AF cells co‐culture system. (B) DCFH‐DA fluorescence staining images and quantitative analysis of TBHP‐pretreated AFCs after different treatments. Scale bar: 50 µm. (C) Intracellular ROS levels in TBHP‐pretreated AFCs measured by flow cytometry analysis and quantitative results. (D,E) Quantitative analysis of MDA and GSH levels in TBHP‐pretreated AFCs co‐cultured with QG, TMH and TMH/QG MN (with NIR irradiation). (F,G) Representative images and quantification of labile iron pool by FerroOrange staining and lipid peroxidation by C11‐BODIPY staining in TBHP‐pretreated AFCs co‐culture with QG, TMH and TMH/QG MN (with NIR irradiation). Scale bar: 20 µm. (H) Western blot analysis and corresponding quantitative statistics of extracellular matrix (COL1), remodeling markers (MMP3, MMP13), and ferroptosis‐related proteins (ACSL4, GPX4). The corresponding uncropped blot images are provided in Table . (I) Immunofluorescence staining and fluorescence intensity quantification of COL1 and MMP3 expression in TBHP‐pretreated AFCs co‐cultured with QG, TMH, and TMH/QG MN (with NIR irradiation). Scale bar: 20 µm. Data are presented as mean ± SD (n ≥ 3), and p ‐values were calculated using one‐way ANOVA with Tukey's multiple comparisons test [(B) to (I)].

    Article Snippet: Following blocking with protein‐free rapid blocking solution (Servicebio, G2052‐500ML), the membranes were incubated overnight at 4°C with primary antibodies (1:1000 dilution) against ACSL4 (proteintech, 22401‐1‐AP), GPX4 (Abclonal, A1933), MMP3 (proteintech, 17873‐1‐AP), MMP13 (Abclonal, A11148), COL1 (proteintech, 14695‐1‐AP), DNMT1 (proteintech, 24206‐1‐AP), DNMT3A (proteintech, 20954‐1‐AP), DNMT3B (proteintech, 26971‐1‐AP), LaminB1 (proteintech, 12987‐1‐AP), ATF3 (Abclonal, A13469) and β‐actin (BOSTER, BA2305).

    Techniques: Co-culture Assay, Fluorescence, Staining, Analysis, Flow Cytometry, Cell Culture, Irradiation, Western Blot, Immunofluorescence, Expressing

    Transcriptomic profiling identifies ATF3 as a key regulator of TMH/QG+NIR‐mediated ferroptosis inhibition in degenerative AFCs. (A) RNA sequencing of AFCs treated with either TBHP+NIR or TBHP+NIR+TMH/QG. (B) Volcano plot identifying 3571 DEGs between TBHP+NIR and TBHP+NIR+TMH/QG groups. (C,D) Functional enrichment analysis of DEGs using GO biological processes and KEGG pathways. (E–G) GSEA plots demonstrating significant enrichment of processes related to extracellular matrix degradation, oxidative stress and ferroptosis. (H) 25 key genes identified from the intersection of ferroptosis driver genes and DEG_Down, 14 key genes identified from the intersection of ferroptosis suppressor genes and DEG_Up. (I) Cluster analysis of DEGs highlighting specific ferroptosis driver and suppressor genes. (J) PPI network of the top 28 hub genes with Atf3 ranked as the top hub gene. (K) 2 key genes ( Atf3 and Trib2 ) identified from the intersection of key genes and DEG_Top50. (L) Western blot analysis of ATF3 and TRIB2 protein expression levels in TBHP+NIR and TBHP+NIR+TMH/QG groups. The corresponding uncropped blot images are provided in Table . (M) Cluster analysis of DEGs showing the Integrated Stress Response between TBHP+NIR and TBHP+NIR+TMH/QG groups. (N) Relative mRNA expression levels of Ddit3 and Chac1 . (O) Representative images and quantitative analysis of C11‐BODIPY staining to assess lipid peroxidation in shAtf3 and LV‐Atf3 groups. Scale bar: 20 µm. (P) Immunofluorescence staining of ATF3 in the TBHP+TMH/QG+NIR+LV‐Atf3 group compared to the TBHP+TMH/QG+NIR+LV‐NC control group. Scale bar: 20 µm. (Q) Western blot analysis of ECM (COL1, MMP3, and MMP13) and ferroptosis (ACSL4 and GPX4) markers in TBHP+TMH/QG+NIR+LV‐Atf3 and TBHP+TMH/QG+NIR+LV‐NC groups. The corresponding uncropped blot images are provided in Table . (R) Flow cytometry analysis of intracellular ROS levels in TBHP+TMH/QG+NIR+LV‐Atf3 and TBHP+TMH/QG+NIR+LV‐NC groups. (S,T) C11‐BODIPY and FerroOrange staining images in TBHP+TMH/QG+NIR+LV‐Atf3 and TBHP+TMH/QG+NIR+LV‐NC groups. Scale bar: 20 µm. Data are presented as mean ± SD (n ≥ 3), and p ‐values were calculated using two‐tailed Student's t‐ test (N).

    Journal: Advanced Science

    Article Title: Acid‐Responsive Nanobot‐Integrated Core‐Shell Microneedles Reprogram the Degenerative Annulus Fibrosus Microenvironment Through Epigenetic Suppression of Ferroptosis

    doi: 10.1002/advs.77829

    Figure Lengend Snippet: Transcriptomic profiling identifies ATF3 as a key regulator of TMH/QG+NIR‐mediated ferroptosis inhibition in degenerative AFCs. (A) RNA sequencing of AFCs treated with either TBHP+NIR or TBHP+NIR+TMH/QG. (B) Volcano plot identifying 3571 DEGs between TBHP+NIR and TBHP+NIR+TMH/QG groups. (C,D) Functional enrichment analysis of DEGs using GO biological processes and KEGG pathways. (E–G) GSEA plots demonstrating significant enrichment of processes related to extracellular matrix degradation, oxidative stress and ferroptosis. (H) 25 key genes identified from the intersection of ferroptosis driver genes and DEG_Down, 14 key genes identified from the intersection of ferroptosis suppressor genes and DEG_Up. (I) Cluster analysis of DEGs highlighting specific ferroptosis driver and suppressor genes. (J) PPI network of the top 28 hub genes with Atf3 ranked as the top hub gene. (K) 2 key genes ( Atf3 and Trib2 ) identified from the intersection of key genes and DEG_Top50. (L) Western blot analysis of ATF3 and TRIB2 protein expression levels in TBHP+NIR and TBHP+NIR+TMH/QG groups. The corresponding uncropped blot images are provided in Table . (M) Cluster analysis of DEGs showing the Integrated Stress Response between TBHP+NIR and TBHP+NIR+TMH/QG groups. (N) Relative mRNA expression levels of Ddit3 and Chac1 . (O) Representative images and quantitative analysis of C11‐BODIPY staining to assess lipid peroxidation in shAtf3 and LV‐Atf3 groups. Scale bar: 20 µm. (P) Immunofluorescence staining of ATF3 in the TBHP+TMH/QG+NIR+LV‐Atf3 group compared to the TBHP+TMH/QG+NIR+LV‐NC control group. Scale bar: 20 µm. (Q) Western blot analysis of ECM (COL1, MMP3, and MMP13) and ferroptosis (ACSL4 and GPX4) markers in TBHP+TMH/QG+NIR+LV‐Atf3 and TBHP+TMH/QG+NIR+LV‐NC groups. The corresponding uncropped blot images are provided in Table . (R) Flow cytometry analysis of intracellular ROS levels in TBHP+TMH/QG+NIR+LV‐Atf3 and TBHP+TMH/QG+NIR+LV‐NC groups. (S,T) C11‐BODIPY and FerroOrange staining images in TBHP+TMH/QG+NIR+LV‐Atf3 and TBHP+TMH/QG+NIR+LV‐NC groups. Scale bar: 20 µm. Data are presented as mean ± SD (n ≥ 3), and p ‐values were calculated using two‐tailed Student's t‐ test (N).

    Article Snippet: Following blocking with protein‐free rapid blocking solution (Servicebio, G2052‐500ML), the membranes were incubated overnight at 4°C with primary antibodies (1:1000 dilution) against ACSL4 (proteintech, 22401‐1‐AP), GPX4 (Abclonal, A1933), MMP3 (proteintech, 17873‐1‐AP), MMP13 (Abclonal, A11148), COL1 (proteintech, 14695‐1‐AP), DNMT1 (proteintech, 24206‐1‐AP), DNMT3A (proteintech, 20954‐1‐AP), DNMT3B (proteintech, 26971‐1‐AP), LaminB1 (proteintech, 12987‐1‐AP), ATF3 (Abclonal, A13469) and β‐actin (BOSTER, BA2305).

    Techniques: Profiling, Inhibition, RNA Sequencing, Functional Assay, Analysis, Western Blot, Expressing, Staining, Immunofluorescence, Control, Flow Cytometry, Two Tailed Test

    DNMT3A‐mediated ATF3 promoter methylation underlies the anti‐ferroptotic effect of TMH/QG+NIR in degenerative AFCs. (A) GSEA plots identify significant enrichment of DEGs in the DNA methylation pathway. (B,C) Methylation‐specific PCR and corresponding quantification of the Atf3 promoter methylation level in degenerative AFCs treated with the TMH/QG+NIR microneedle system. The corresponding uncropped gel images are provided in Table . (D) Western blot analysis of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) in the TBHP+NIR and TBHP+NIR+TMH/QG groups. The corresponding uncropped blot images are provided in Table . (E) ChIP‐qPCR analysis of DNMT1, DNMT3A, and DNMT3B enrichment at the Atf3 promoter region. (F) ChIP‐qPCR analysis specifically quantifying DNMT3A enrichment at the Atf3 promoter. (G) Measurement of MDA content. (H) Western blot analysis of SLC7A11 and ATF3 protein levels in the TBHP+NIR+TMH/QG group with or without DY‐46‐2. The corresponding uncropped blot images are provided in Table . (I) Relative mRNA expression level of Slc7a11 and Atf3 in the TBHP+NIR+TMH/QG group with or without the DNMT3A inhibitor DY‐46‐2. (J) Flow cytometry analysis of intracellular ROS levels. (K) Representative FerroOrange staining images in the TBHP+NIR+TMH/QG group with or without DY‐46‐2. Scale bar: 20 µm. (L) Western blot analysis of key ferroptosis (ACSL4 and GPX4) and extracellular matrix (COL1, MMP3 and MMP13) markers in the TBHP+NIR+TMH/QG group with or without DY‐46‐2. The corresponding uncropped blot images are provided in Table . (M) IHC staining of ATF3, SLC7A11 and DNMT3A expression in human disc tissues from mild and severe degeneration groups. Scale bar: 20 µm. (N) MSP of the ATF3 promoter methylation level in human mild and severe degeneration groups. The corresponding uncropped gel images are provided in Table . (O) Western blot analysis of ATF3, SLC7A11 and DNMT3A protein expression in human disc tissues from mild and severe degeneration groups. The corresponding uncropped blot images are provided in Table . (P) Relative mRNA expression of ATF3 in human mild and severe degeneration groups. (Q–S) Correlation analysis of relative ATF3 expression in AF tissue and relative GPX4 , SLC7A11 and DNMT3A expression. (T) Schematic model illustrating the proposed mechanism. Data are presented as mean ± SD (n ≥ 3), and p ‐values were calculated using two‐tailed Student's t ‐test [(C), (F), (G), (I) and (P)] and one‐way ANOVA with Tukey's multiple comparisons test (E).

    Journal: Advanced Science

    Article Title: Acid‐Responsive Nanobot‐Integrated Core‐Shell Microneedles Reprogram the Degenerative Annulus Fibrosus Microenvironment Through Epigenetic Suppression of Ferroptosis

    doi: 10.1002/advs.77829

    Figure Lengend Snippet: DNMT3A‐mediated ATF3 promoter methylation underlies the anti‐ferroptotic effect of TMH/QG+NIR in degenerative AFCs. (A) GSEA plots identify significant enrichment of DEGs in the DNA methylation pathway. (B,C) Methylation‐specific PCR and corresponding quantification of the Atf3 promoter methylation level in degenerative AFCs treated with the TMH/QG+NIR microneedle system. The corresponding uncropped gel images are provided in Table . (D) Western blot analysis of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) in the TBHP+NIR and TBHP+NIR+TMH/QG groups. The corresponding uncropped blot images are provided in Table . (E) ChIP‐qPCR analysis of DNMT1, DNMT3A, and DNMT3B enrichment at the Atf3 promoter region. (F) ChIP‐qPCR analysis specifically quantifying DNMT3A enrichment at the Atf3 promoter. (G) Measurement of MDA content. (H) Western blot analysis of SLC7A11 and ATF3 protein levels in the TBHP+NIR+TMH/QG group with or without DY‐46‐2. The corresponding uncropped blot images are provided in Table . (I) Relative mRNA expression level of Slc7a11 and Atf3 in the TBHP+NIR+TMH/QG group with or without the DNMT3A inhibitor DY‐46‐2. (J) Flow cytometry analysis of intracellular ROS levels. (K) Representative FerroOrange staining images in the TBHP+NIR+TMH/QG group with or without DY‐46‐2. Scale bar: 20 µm. (L) Western blot analysis of key ferroptosis (ACSL4 and GPX4) and extracellular matrix (COL1, MMP3 and MMP13) markers in the TBHP+NIR+TMH/QG group with or without DY‐46‐2. The corresponding uncropped blot images are provided in Table . (M) IHC staining of ATF3, SLC7A11 and DNMT3A expression in human disc tissues from mild and severe degeneration groups. Scale bar: 20 µm. (N) MSP of the ATF3 promoter methylation level in human mild and severe degeneration groups. The corresponding uncropped gel images are provided in Table . (O) Western blot analysis of ATF3, SLC7A11 and DNMT3A protein expression in human disc tissues from mild and severe degeneration groups. The corresponding uncropped blot images are provided in Table . (P) Relative mRNA expression of ATF3 in human mild and severe degeneration groups. (Q–S) Correlation analysis of relative ATF3 expression in AF tissue and relative GPX4 , SLC7A11 and DNMT3A expression. (T) Schematic model illustrating the proposed mechanism. Data are presented as mean ± SD (n ≥ 3), and p ‐values were calculated using two‐tailed Student's t ‐test [(C), (F), (G), (I) and (P)] and one‐way ANOVA with Tukey's multiple comparisons test (E).

    Article Snippet: Following blocking with protein‐free rapid blocking solution (Servicebio, G2052‐500ML), the membranes were incubated overnight at 4°C with primary antibodies (1:1000 dilution) against ACSL4 (proteintech, 22401‐1‐AP), GPX4 (Abclonal, A1933), MMP3 (proteintech, 17873‐1‐AP), MMP13 (Abclonal, A11148), COL1 (proteintech, 14695‐1‐AP), DNMT1 (proteintech, 24206‐1‐AP), DNMT3A (proteintech, 20954‐1‐AP), DNMT3B (proteintech, 26971‐1‐AP), LaminB1 (proteintech, 12987‐1‐AP), ATF3 (Abclonal, A13469) and β‐actin (BOSTER, BA2305).

    Techniques: Methylation, DNA Methylation Assay, Western Blot, Analysis, ChIP-qPCR, Expressing, Flow Cytometry, Staining, Immunohistochemistry, Tissue, Two Tailed Test

    Key resources table.

    Journal: Advanced Science

    Article Title: AI‐Designed TREM1‐Targeted LYTAC Nanoparticles Reprogram the Neuroimmune Microenvironment in Traumatic Brain Injury

    doi: 10.1002/advs.77972

    Figure Lengend Snippet: Key resources table.

    Article Snippet: βIII‐Tubulin Rabbit mAb , ABclonal , A17913.

    Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Software

    Antibodies used in this study.

    Journal: Pediatric Discovery

    Article Title: Early Growth Response 1‐Associated Epithelial Autophagy‐Related Changes in Allergic Airway Inflammation

    doi: 10.1002/pdi3.70075

    Figure Lengend Snippet: Antibodies used in this study.

    Article Snippet: LC3B rabbit mAb , ABclonal , A19665 , ARC0144.

    Techniques: Cell-Signaling

    House dust mite (HDM) stimulation induces early growth response 1 (EGR1) expression and autophagy‐associated changes in airway epithelial cells. (A–C) EGR1 mRNA and protein levels in BEAS‐2B cells stimulated with HDM extract (30 μg/mL, 3 hours) or vehicle control, assessed by quantitative polymerase chain reaction (qPCR) and Western blot. (D–F) Western blot analysis of LC3B and p62 protein levels. The LC3B‐II/I ratio was quantified by densitometry. (G) Representative immunofluorescence images of LC3B puncta (green) in BEAS‐2B cells. Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. (H) IL‐33 release into culture supernatants measured by ELISA. (I) Airway resistance in response to increasing concentrations of methacholine in HDM‐challenged mice and controls. Data were analyzed using repeated‐measures ANOVA. (J) Representative hematoxylin and eosin‐stained lung tissue sections. Scale bar, 100 μm. (K–M) Immunofluorescence detection of EGR1 (red) and LC3B (green) in lung tissue sections. Nuclei were stained with DAPI (blue). Fluorescence intensity was quantified and normalized to the control. Data are presented as mean ± standard deviation. For in vitro experiments: (A, G) n = 3 independent experiments; (B–F) n = 4 independent experiments. For in vivo experiments: n = 4 mice per group. Statistical significance was determined by an unpaired Student's t ‐test unless otherwise indicated. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant.

    Journal: Pediatric Discovery

    Article Title: Early Growth Response 1‐Associated Epithelial Autophagy‐Related Changes in Allergic Airway Inflammation

    doi: 10.1002/pdi3.70075

    Figure Lengend Snippet: House dust mite (HDM) stimulation induces early growth response 1 (EGR1) expression and autophagy‐associated changes in airway epithelial cells. (A–C) EGR1 mRNA and protein levels in BEAS‐2B cells stimulated with HDM extract (30 μg/mL, 3 hours) or vehicle control, assessed by quantitative polymerase chain reaction (qPCR) and Western blot. (D–F) Western blot analysis of LC3B and p62 protein levels. The LC3B‐II/I ratio was quantified by densitometry. (G) Representative immunofluorescence images of LC3B puncta (green) in BEAS‐2B cells. Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. (H) IL‐33 release into culture supernatants measured by ELISA. (I) Airway resistance in response to increasing concentrations of methacholine in HDM‐challenged mice and controls. Data were analyzed using repeated‐measures ANOVA. (J) Representative hematoxylin and eosin‐stained lung tissue sections. Scale bar, 100 μm. (K–M) Immunofluorescence detection of EGR1 (red) and LC3B (green) in lung tissue sections. Nuclei were stained with DAPI (blue). Fluorescence intensity was quantified and normalized to the control. Data are presented as mean ± standard deviation. For in vitro experiments: (A, G) n = 3 independent experiments; (B–F) n = 4 independent experiments. For in vivo experiments: n = 4 mice per group. Statistical significance was determined by an unpaired Student's t ‐test unless otherwise indicated. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant.

    Article Snippet: LC3B rabbit mAb , ABclonal , A19665 , ARC0144.

    Techniques: Expressing, Control, Real-time Polymerase Chain Reaction, Western Blot, Analysis, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Staining, Tissue, Fluorescence, Standard Deviation, In Vitro, In Vivo

    Validation of early growth indicaton 1 ( EGR1 ) overexpression and knockdown efficiency, and the effects on autophagy‐related proteins and IL‐33 release in house dust mite (HDM)‐stimulated BEAS‐2B cells. (A–C) EGR1 mRNA and EGR1 protein levels in cells transfected with the EGR1 overexpression plasmid (oe‐EGR1) or empty vector, assessed by quantitative polymerase chain reaction (qPCR) and Western blot. (D–F) EGR1 mRNA and ERG1 protein levels in cells transfected with EGR1‐targeting siRNA (si‐EGR1) or scrambled control, assessed by qPCR and Western blot. (G–I) Western blot analysis of LC3B and p62 in oe‐EGR1‐transfected cells, followed by HDM stimulation (30 μg/mL, 3 h). The LC3B‐II/I ratio and p62 levels were quantified by densitometry. (J) IL‐33 release into culture supernatants under the same conditions, measured by ELISA. (K–M) Western blot analysis of LC3B and p62 in si‐EGR1‐transfected cells with HDM stimulation. (N) Corresponding IL‐33 release measured by ELISA. (O–P) Immunofluorescence detection of LC3B puncta (green) in oe‐EGR1 and si‐EGR1 cells stimulated with HDM. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. All data are presented as mean ± standard deviation ( n = 3 independent experiments). Statistical significance for panels (A–P) was determined by one‐way ANOVA followed by appropriate post‐hoc tests. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant.

    Journal: Pediatric Discovery

    Article Title: Early Growth Response 1‐Associated Epithelial Autophagy‐Related Changes in Allergic Airway Inflammation

    doi: 10.1002/pdi3.70075

    Figure Lengend Snippet: Validation of early growth indicaton 1 ( EGR1 ) overexpression and knockdown efficiency, and the effects on autophagy‐related proteins and IL‐33 release in house dust mite (HDM)‐stimulated BEAS‐2B cells. (A–C) EGR1 mRNA and EGR1 protein levels in cells transfected with the EGR1 overexpression plasmid (oe‐EGR1) or empty vector, assessed by quantitative polymerase chain reaction (qPCR) and Western blot. (D–F) EGR1 mRNA and ERG1 protein levels in cells transfected with EGR1‐targeting siRNA (si‐EGR1) or scrambled control, assessed by qPCR and Western blot. (G–I) Western blot analysis of LC3B and p62 in oe‐EGR1‐transfected cells, followed by HDM stimulation (30 μg/mL, 3 h). The LC3B‐II/I ratio and p62 levels were quantified by densitometry. (J) IL‐33 release into culture supernatants under the same conditions, measured by ELISA. (K–M) Western blot analysis of LC3B and p62 in si‐EGR1‐transfected cells with HDM stimulation. (N) Corresponding IL‐33 release measured by ELISA. (O–P) Immunofluorescence detection of LC3B puncta (green) in oe‐EGR1 and si‐EGR1 cells stimulated with HDM. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. All data are presented as mean ± standard deviation ( n = 3 independent experiments). Statistical significance for panels (A–P) was determined by one‐way ANOVA followed by appropriate post‐hoc tests. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant.

    Article Snippet: LC3B rabbit mAb , ABclonal , A19665 , ARC0144.

    Techniques: Biomarker Discovery, Over Expression, Knockdown, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Control, Analysis, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Standard Deviation

    PI3K‐III (Vps34) protein levels in response to house dust mite (HDM) and early growth response 1 (EGR1) manipulation, and the impact of 3‐methyladenine (3‐MA) on autophagy and IL‐33 release in EGR1‐overexpressing cells. (A–B) Western blot analysis and quantification of Vps34 protein levels in BEAS‐2B cells stimulated with HDM extract (30 μg/mL, 3 h) or vehicle control. (C–D) Western blot analysis of Vps34 protein levels in cells transfected with oe‐EGR1 or empty vector, with or without HDM stimulation. (E–F) Western blot analysis of Vps34 protein levels in cells transfected with si‐EGR1 or scrambled control, with or without HDM stimulation. (G–I) Western blot analysis of LC3B and p62 in oe‐EGR1‐transfected cells treated with or without Class III PI3K inhibitor 3‐methyladenine (3‐MA, 10 mmol/L, 6 h). (J) IL‐33 release in the same experimental setting, measured by ELISA. All data are presented as mean ± standard deviation ( n = 3 independent experiments). For panels (A–B), statistical significance was determined by an unpaired Student's t ‐test. For panels (C–J), one‐way ANOVA followed by appropriate post‐hoc tests was used. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant.

    Journal: Pediatric Discovery

    Article Title: Early Growth Response 1‐Associated Epithelial Autophagy‐Related Changes in Allergic Airway Inflammation

    doi: 10.1002/pdi3.70075

    Figure Lengend Snippet: PI3K‐III (Vps34) protein levels in response to house dust mite (HDM) and early growth response 1 (EGR1) manipulation, and the impact of 3‐methyladenine (3‐MA) on autophagy and IL‐33 release in EGR1‐overexpressing cells. (A–B) Western blot analysis and quantification of Vps34 protein levels in BEAS‐2B cells stimulated with HDM extract (30 μg/mL, 3 h) or vehicle control. (C–D) Western blot analysis of Vps34 protein levels in cells transfected with oe‐EGR1 or empty vector, with or without HDM stimulation. (E–F) Western blot analysis of Vps34 protein levels in cells transfected with si‐EGR1 or scrambled control, with or without HDM stimulation. (G–I) Western blot analysis of LC3B and p62 in oe‐EGR1‐transfected cells treated with or without Class III PI3K inhibitor 3‐methyladenine (3‐MA, 10 mmol/L, 6 h). (J) IL‐33 release in the same experimental setting, measured by ELISA. All data are presented as mean ± standard deviation ( n = 3 independent experiments). For panels (A–B), statistical significance was determined by an unpaired Student's t ‐test. For panels (C–J), one‐way ANOVA followed by appropriate post‐hoc tests was used. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant.

    Article Snippet: LC3B rabbit mAb , ABclonal , A19665 , ARC0144.

    Techniques: Western Blot, Analysis, Control, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Standard Deviation