ps211 tfeb  (Cell Signaling Technology Inc)

Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: FGL2 disruption promoted the expression of nuclear TFEB. The cytosolic and nuclear distribution of TFEB ( A ) and MITF ( B ) in MHCC97H cells was detected by WB following FGL2 knockdown. Quantification of nuclear TFEB or MITF to Histone-H3 was shown. ( C ) Images and quantification of nuclear TFEB in MHCC97H cells were displayed. Scale bar, 50 μm. The expression levels of TFEB ( D ) and MITF ( E ) were determined by WB in mouse liver cancer tissues ( n = 6). The transcriptional levels of Lamp1, Atp6v0d2 , Ctns and Ctsb were detected by qPCR in mouse liver cancer tissues ( F ) and MHCC97H cells ( G ). TFEB siRNA or control siRNA was transfected to MHCC97H cells pretreated with FGL2 shRNA or control shRNA. The protein levels of LAMP1, CTSD ( H ), and PD-L1 ( I ) were determined by WB. The relative quantification of LAMP1, CTSD or PD-L1 to GAPDH was shown. ( J ) The expression of TFEB in an HCC tissue microarray was detected by mIHC. Representative images were shown. Scale bar, 20 μm. Kaplan-Meier analysis of the progression-free survival ( K ) and overall survival ( L ) of HCC patients based on nuclear TFEB expression ( n = 85). Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Next, membranes were incubated with the following antibodies: FGL2 (1:500) (sc-100276, Santa Cruz Biotechnology), PD-L1 (1:4000) (66248-1-Ig, Proteintech), DDDDK-Tag (1:5000) (AE063, Abclonal), LAMP1 (1:10000) (67300-1-Ig, Proteintech), CTSD (1:10000) (21327-1-AP, Proteintech), TFEB (1:5000) (68632-1-Ig, Proteintech), phospho-TFEB (1:1000) (37681, CST), TFE3 (1:500) (A7936, Abclonal), MITF (1:1000) (13092-1-AP, Proteintech), GAPDH (1:50000) (60004-1-Ig, Proteintech), Histone H3 (1:5000) (17168-1-AP, Proteintech), p70S6K (1:1000) (2708, CST), phospho-p70S6K (1:1000) (9234, CST), 4EBP1 (1:1000) (9644, CST), phospho-4EBP1 (1:1000) (2855, CST), AKT (1:1000) (9272, CST), phospho-AKT (1:1000) (4060, CST), GSK3β(1:1000) (12456, CST), phospho-GSK3β (1:1000) (5558, CST), PKC (1:4000) (12919-1-AP, Proteintech), phospho-PKC (1:1000) (38938, CST), ERK1/2 (1:1000) (16443-1-AP, Proteintech), phospho-p44/42 MAPK (Erk1/2) (1:2000) (4370, CST), ATG5 (1:2000) (10181-2-AP, Proteintech), ATG7 (1:1000) (10088-2-AP, Proteintech), HRP goat anti-rabbit IgG (1:10000) (A21020, Abbkine) and HRP goat anti-mouse IgG (1:10000) (A21010, Abbkine).

Techniques: Disruption, Expressing, Knockdown, Control, Transfection, shRNA, Quantitative Proteomics, Microarray

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