Journal: Gene expression

Article Title: Bile Duct Ligation Induces ATZ Globule Clearance In a Mouse Model of Alpha-1 Antitrypsin Deficiency

doi: 10.3727/105221616X692991

Figure Lengend Snippet: List of antibodies used in this study

Article Snippet: TFEB , Goat pAb V-17 (Santa Cruz) , 1:1000 , WB.

Techniques:

Journal: Ecotoxicology and environmental safety

Article Title: Graphene oxide induced dynamic changes of autophagy-lysosome pathway and cell apoptosis via TFEB dysregulation in F98 cells.

doi: 10.1016/j.ecoenv.2022.114172

Figure Lengend Snippet: Fig. 3. Effects of GO on TFEB nuclear translocation and transcriptional activity. (A) Cells were treated with PBS (control), EBSS (starved) or 30 μg/mL GO from 6 to 24 h. TFEB nuclear translocation was observed by immunofluorescence using a TFEB antibody. Scale bar: 10 µm. Nuclear TFEB intensity after cells were exposed to different durations of GO was quantified in the right histogram. (B-C) After GO exposure, the expression of TFEB in nuclear and cytosolic fractions was detected using western blotting. TFEB expression levels in the nuclear and cytosolic fractions were normalized to Lamin B and GAPDH expression levels, respectively. Quantification of western blotting data was presented in the right histograms. (D) Cells were exposed to 30 μg/mL GO for 24 h. RT-PCR analysis showed the relative mRNA expression of TFEB target genes (Maplc3b, Uvrag, Lamp1, Ctsb, and Wipi1). All data are presented as mean ± SD. n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cells were fixed with 4% paraformaldehyde and immunostained with primary antibodies against TFEB (Novus, USA), followed by secondary antibodies incubation for 1 h. DAPI Staining Solution (Beyotime, China) was used to achieve nuclear counterstaining.

Techniques: Translocation Assay, Activity Assay, Control, Immunofluorescence, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Ecotoxicology and environmental safety

Article Title: Graphene oxide induced dynamic changes of autophagy-lysosome pathway and cell apoptosis via TFEB dysregulation in F98 cells.

doi: 10.1016/j.ecoenv.2022.114172

Figure Lengend Snippet: Fig. 5. STUB1 participated in the downregulation of TFEB activity induced by GO. (A) Western blots of STUB1 expression after exposure to PBS (control) or 30 μg/ mL GO from 6 to 24 h. The right bar graphs show quantified data (n = 3). (B) Cells were transfected with empty expression plasmid (control) and STUB1-pcDNA (STUB1) before exposure to 30 μg/mL GO. After GO exposure, the expression of nuclear TFEB in the STUB1 overexpression experiment was detected using west ern blotting. Quantification of western blotting data was presented in the right histogram. (C) TFEB nuclear translocation was observed by immunofluorescence using a TFEB antibody in the STUB1 overexpression experiment. Scale bar: 10 µm. Nuclear TFEB intensity was quantified in the right histogram. (D)Western blotting for STUB1, LC3, SQSTM1, LAMP1, and CTSB expression. The bar graphs at the bottom show the relative optical densities reflecting protein expression levels. (E) Fluorescence microscopy of cells stained with Lysotracker Red. The right bar graphs show the quantified intracellular fluorescence intensities. Scale bar: 30 µm. (F) Cell apoptosis rate in the STUB1 overexpression experiment was investigated using Annexin V-FTIC-PI apoptosis kit with flow cytometry (right). (G) The relative levels of cleaved Caspase 3 in the STUB1 overexpression experiment were detected by western blotting and quantified in the right histogram. All data are expressed as mean ± SD of three independent tests. * P < 0.05, ** P < 0.01, ***P < 0.001.

Article Snippet: Cells were fixed with 4% paraformaldehyde and immunostained with primary antibodies against TFEB (Novus, USA), followed by secondary antibodies incubation for 1 h. DAPI Staining Solution (Beyotime, China) was used to achieve nuclear counterstaining.

Techniques: Activity Assay, Western Blot, Expressing, Control, Transfection, Plasmid Preparation, Over Expression, Translocation Assay, Immunofluorescence, Fluorescence, Microscopy, Staining, Flow Cytometry

Journal: Frontiers in cell and developmental biology

Article Title: Liraglutide Alleviates Hepatic Steatosis by Activating the TFEB-Regulated Autophagy-Lysosomal Pathway.

doi: 10.3389/fcell.2020.602574

Figure Lengend Snippet: FIGURE 6 | Liraglutide stimulates lysosome biogenesis by inducing TFEB nuclear translocation. (A) Expression of nuclear TFEB, CTSB, and LAMP1 in the livers of HFD-fed mice treated with or without liraglutide. Relative expression levels were normalized to β-actin and Histone 3 levels. (B) HepG2 cells were co-incubated with or without PA and liraglutide for 24 h. After fixation, immunofluorescence staining was performed for TFEB localization (scale bars = 50 µm). (C) Western blot detection of cytoplasm and nuclear TFEB expression in HepG2 cells treated with or without liraglutide. Relative expression levels were normalized to GAPDH and Histone 3 levels, respectively. (D) Western blot detection of CTSB and LAMP1 expression in HepG2 cells. The data are expressed as mean ± SEM; n = 3. *P < 0.01, **P < 0.05 vs. BSA group; #P < 0.01, ##P < 0.05 vs. PA group.

Article Snippet: After fixation, the cells were permeabilized with 0.25% Triton X-100 for 15 min, blocked with 2% BSA for 30 min, and then incubated with an anti-TFEB antibody (1: 100, MAB9170-100, R&D Systems, United States) overnight at 4◦C.

Techniques: Translocation Assay, Expressing, Incubation, Staining, Western Blot

Journal: Frontiers in cell and developmental biology

Article Title: Liraglutide Alleviates Hepatic Steatosis by Activating the TFEB-Regulated Autophagy-Lysosomal Pathway.

doi: 10.3389/fcell.2020.602574

Figure Lengend Snippet: FIGURE 8 | Liraglutide activates TFEB and its downstream targets through GLP-1R. (A) Western blot analysis of GLP-1R protein levels in the liver of HFD-fed mice treated with or without liraglutide. Relative expression levels were normalized to β-actin levels. (B,C) Western blot detection of GLP-1R in primary mouse hepatocytes and HepG2 cells treated with or without liraglutide. (D) Western blot detection of GLP-1R, TFEB, and its downstream targets CTSB and LAMP1 in HepG2 cells transfected with TFEB-siRNA or control-siRNA. (E) Western blot detection of GLP-1R, TFEB, and its downstream targets CTSB and LAMP1 in HepG2 cells with or without GLP-1R overexpression. The data are expressed as the mean ± SEM; n = 3. *P < 0.01, **P < 0.05 vs. Chow + saline group or BSA group or siCON + PA group or Vector-PA group; #P < 0.01,##P < 0.05 vs. HFD + saline group or PA group or siCON + PA group or Vector-PA group; &P < 0.01, &&P < 0.05 vs. siCON + PA + LG group or Vector-PA + LG group.

Article Snippet: After fixation, the cells were permeabilized with 0.25% Triton X-100 for 15 min, blocked with 2% BSA for 30 min, and then incubated with an anti-TFEB antibody (1: 100, MAB9170-100, R&D Systems, United States) overnight at 4◦C.

Techniques: Western Blot, Expressing, Transfection, Control, Over Expression, Saline, Plasmid Preparation

Journal: Autophagy

Article Title: TMBIM6 (transmembrane BAX inhibitor motif containing 6) enhances autophagy through regulation of lysosomal calcium

doi: 10.1080/15548627.2020.1732161

Figure Lengend Snippet: TMBIM6 enhances TFEB nuclear localization independent of MTORC1 activity. (A) Proximity ligation assay (PLA) between TFEB and PPP3CA (red dots) in TMBIM6- or TMBIM6 D213A expressing HT1080 cells and vector cells under starvation or torin or PP242-treatment. Scale bar: 15 µm. The data are represented as mean ± SEM from n = 3 independent experiments (right). (B) Fluorescence images of endogenous TFEB after 3 h of starvation or torin or PP242-treatment TFEB nuclear translocation. Scale bar: 15 µm. The data are represented as the mean ± SEM from n = 3 independent experiments. (C) Fluorescence images of endogenous TFEB in siRNA of TMBIM6 and MCOLN1 -pretreated cells under starvation or torin or PP242-treatment after 3 h. Scale bar: 15 µm. (D) qPCR analysis of TMBIM6 and MCOLN1 was performed to confirm the efficacy of siRNA-mediated silencing. Asterisks indicate significant differences from vector or scramble siRNA treatments. The hash indicates significant differences between TMBIM6 and TMBIM6 D213A

Article Snippet: Antibodies used in this study were from the following sources: mouse anti-TFEB antibody (MBS120432; 1:50) from MyBioSource and rabbit anti-PPP3CA/calcineurin A antibody (2614; 1:50) from Cell Signaling Technology.

Techniques: Activity Assay, Proximity Ligation Assay, Expressing, Plasmid Preparation, Fluorescence, Translocation Assay