anti p tfeb 87932s  (Cell Signaling Technology Inc)

Journal: Biomaterials Research

Article Title: Bimetallic Copper–Manganese Zeolitic Imidazolate Framework Nanozyme Scavenges Reactive Oxygen Species to Alleviate Osteoarthritis via Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Axis and Autophagic Flux Restoration

doi: 10.34133/bmr.0306

Figure Lengend Snippet: CuMn-ZIF nanozymes mitigate oxidative stress and protect chondrocytes in vitro. (A) Schematic diagram illustrating the proposed mechanism of CuMn-ZIF nanozymes in treating OA. CuMn-ZIF scavenges ROS, inhibits the phosphorylation of the PI3K–AKT–mTOR pathway, promotes TFEB nuclear translocation, enhances autophagic flux, and ultimately restores the balance between anabolism and catabolism in chondrocytes. (B) Intracellular ROS levels were measured using the DCFH-DA probe and (C) analyzed by flow cytometry following treatment with PBS, H 2 O 2 , H 2 O 2 + ZIF, or H 2 O 2 + CuMn-ZIF. (D) Representative fluorescence images and (E) quantitative analysis of mitochondrial membrane potential assessed by JC-1 staining (scale bar, 75 μm). Red fluorescence indicates JC-1 aggregates (healthy mitochondria), and green fluorescence indicates JC-1 monomers (depolarized mitochondria). (F) Representative WB images and (G) corresponding quantitative analysis of the protein expression levels of catabolic markers (ADAMTS-5, MMP-13) and anabolic markers (COL2A1, AGGRECAN). (H) Cell viability was determined by CCK-8 assay after treatment with PBS, H 2 O 2 , H 2 O 2 + ZIF, or H 2 O 2 + CuMn-ZIF. (I) Live/dead cell staining images (scale bar, 250 μm) using calcein-AM (green, live cells) and PI (red, dead cells), and the corresponding quantitative analysis of (J) calcein-AM and (K) PI fluorescence intensity. Statistical analysis was conducted using one-way ANOVA analysis. Data were represented as mean ± SD ( n = 3). **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns: not significant.

Article Snippet: For protein separation, electrophoresis was conducted at 90 V and the voltage was increased to 150 V. The proteins were transferred onto a nitrocellulose (NC) membrane and treated with blocking solution for 2 h, followed by overnight incubation with primary antibodies at 4 °C : AKT (CST, 9272), p-AKT (CST, 4060S), mTOR (CST, 2983), p-mTOR (CST, 5536), PI3K (Abclonal, A0982), p-PI3K (Abbkine, ABP50495 ), LC3B (Abcam, ab192890), P62 (Abcam, ab56416), TFEB (CST, 37681), and p-TFEB. glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Absin, abs830030; Affinit, AF7021) was used as the housekeeping protein.

Techniques: In Vitro, Phospho-proteomics, Translocation Assay, Flow Cytometry, Fluorescence, Membrane, Staining, Expressing, CCK-8 Assay

Journal: Biomaterials Research

Article Title: Bimetallic Copper–Manganese Zeolitic Imidazolate Framework Nanozyme Scavenges Reactive Oxygen Species to Alleviate Osteoarthritis via Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Axis and Autophagic Flux Restoration

doi: 10.34133/bmr.0306

Figure Lengend Snippet: CuMn-ZIF nanozymes enhance autophagic flux and promote TFEB nuclear translocation in chondrocytes in vitro. (A) Representative WB images and (B) quantitative analysis of autophagy-related proteins (LC3-II/I and P62) in chondrocytes treated with PBS, H 2 O 2 , H 2 O 2 + ZIF, or H 2 O 2 + CuMn-ZIF. (C) Representative confocal images and (D) quantitative analysis of autophagic flux using the mCherry-GFP-LC3 fluorescent probe. Yellow puncta (mCherry + GFP + ) represent autophagosomes, and red puncta (mCherry + GFP − ) represent autolysosomes (scale bar, 250 μm). (E) Immunofluorescence images showing the subcellular localization of TFEB (FITC, green) with nuclei counterstained by DAPI (blue), and (F) the corresponding statistical analysis of nuclear-to-cytoplasmic TFEB fluorescence intensity ratio (scale bar, 75 μm). (G) Representative WB images of p-TFEB and total TFEB and (H) quantitative analysis of p-TFEB/TFEB protein levels. (I) Flow cytometry analysis and (J) quantification of lysosomal pH using LysoTracker Red staining. (K) Representative confocal images and (L) quantitative analysis of autophagic flux using the mCherry-GFP-LC3 fluorescent probe according to siNC and siTFEB treatment (scale bar, 100 μm). Statistical analysis was conducted using one-way ANOVA analysis. Data were represented as mean ± SD ( n = 3). **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns: not significant.

Article Snippet: For protein separation, electrophoresis was conducted at 90 V and the voltage was increased to 150 V. The proteins were transferred onto a nitrocellulose (NC) membrane and treated with blocking solution for 2 h, followed by overnight incubation with primary antibodies at 4 °C : AKT (CST, 9272), p-AKT (CST, 4060S), mTOR (CST, 2983), p-mTOR (CST, 5536), PI3K (Abclonal, A0982), p-PI3K (Abbkine, ABP50495 ), LC3B (Abcam, ab192890), P62 (Abcam, ab56416), TFEB (CST, 37681), and p-TFEB. glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Absin, abs830030; Affinit, AF7021) was used as the housekeeping protein.

Techniques: Translocation Assay, In Vitro, Immunofluorescence, Fluorescence, Flow Cytometry, Staining