Journal: Science Advances

Article Title: TFEB coordinates autophagosome biogenesis and ribophagy during starvation via SQSTM1

doi: 10.1126/sciadv.aea9302

Figure Lengend Snippet: ( A ) Volcano plots of proteins from whole-cell proteomics in TFEB-3xFlag versus wild-type (WT) U2OS cells. Significantly altered proteins: dark [FDR < 0.05, log 2 fold change (FC) > |1|] and light (FDR < 0.05, 0 < |log 2 FC| < |1|) red/blue (FDR-corrected two-sided t test, N = 4). E3 ligases are highlighted in black (table S1). ( B ) Western blot of indicated proteins in control and TFEB-3xFlag U2OS cells ( N = 3) with or without BafA1 (200 nM, 4 hours). Quantification normalized to β-actin; means ± SEM. One-way ANOVA: P = 0.0003. Sidak’s test: ** P < 0.005. M r , relative molecular mass. ( C to E ) Coimmunofluorescence of SQSTM1 (red) with (C) NBR1, (D) TAX1BP1, or (E) LC3B (green) in CTRL and TFEB-GFP (purple) cells. Scale bars, 10 μm (insets, 2 μm). Quantification of puncta per cell; means ± SEM ( N = 3, n = 24 to 30 cells). Student’s unpaired t test: ** P < 0.005 for NBR1, * P < 0.05 for TAX1BP1 and LC3B. ( F ) Electron micrograph of TFEB-GFP U2OS cell labeled for SQSTM1 (nanogold). ( G ) CLEM of TFEB-GFP U2OS cells starved in HBSS (2 hours) and labeled for SQSTM1 (red) and LC3B (green). The arrow indicates membranes surrounding an SQSTM1- and LC3B-positive structure. ( H ) Coimmunofluorescence of SQSTM1 (red) and LC3B (green) in FLCN KO HeLa cells ± siSQSTM1 (100 nM for 48 hours). Scale bars, 10 μm (insets, 5 μm). Quantification of LC3B puncta per cell; means ± SEM ( N = 3, n = 30 cells). Student’s unpaired t test: * P < 0.05.

Article Snippet: Membranes were blocked with 5% BSA for 1 hour and then incubated overnight with the following antibodies diluted 1:1000 in 5% BSA: RPS2 (Bethy, A303-79A), P62 clone 2C11 (Abnova, H00008878-M01), TFEB (Bethyl Laboratories, A303-673A), calnexin (Enzo Life Sciences, ADI-SPA 860), β-actin (Novus Biologicals, NB600-501), and citrate synthase (Abcam, ab96600).

Techniques: Western Blot, Control, Labeling

Journal: Science Advances

Article Title: TFEB coordinates autophagosome biogenesis and ribophagy during starvation via SQSTM1

doi: 10.1126/sciadv.aea9302

Figure Lengend Snippet: ( A ) Schematic representation of the SQSTM1 promoter with putative TFEB binding sites. Regions 1 and 2 were cloned into the pGL3-basic luciferase reporter plasmid, and the luciferase activity was determined. Means ± SEM of N = 3. One-way ANOVA: P = 0.0016. Sidak’s test: ** P < 0.005. ns, not significant. TSS, transcription start site; 5′UTR, 5′ untranslated region; A.U., arbitrary units. ( B ) qRT-PCR analysis of SQSTM1 expression in CTRL and ΔCLEAR HeLa cells with or without TFEB overexpression (TFEB OE). Fold change normalized to HPRT and expressed relative to CTRL. Means ± SEM of N = 3. One-way ANOVA, P < 0.0001. Sidak’s test: *** P < 0.0001. ( C ) Immunofluorescence of SQSTM1 (red) and TFEB-GFP (purple) in WT and ΔCLEAR HeLa cells overexpressing TFEB-GFP. Nuclei stained with DAPI (blue). Scale bars, 10 μm (insets, 2 μm). Quantification of SQSTM1 puncta per cell; means ± SEM ( N = 3, n = 40 cells). Student’s unpaired t test: * P < 0.05. ( D ) Western blot analysis of indicated proteins in WT and ΔCLEAR HeLa cells infected with TFEB3xFlag, with or without BafA1 (200 nM, 4 hours). Quantification of LC3BII normalized to β-actin; means ± SEM ( N = 4). One-way ANOVA: *** P < 0.0001. Sidak’s test: *** P < 0.0005; ** P < 0.005; * P < 0.05. ( E ) Coimmunofluorescence staining of LC3B (green), SQSTM1 (red), and TFEB-GFP (purple) in WT and ΔCLEAR HeLa cells overexpressing TFEB-GFP. Scale bars, 10 μm. Quantification of LC3B puncta per cell; means ± SEM [ N = 3, n = 41 (HeLa) and n = 40 (ΔCLEAR HeLa) cells]. Student’s unpaired t test: *** P = 0.001.

Article Snippet: Membranes were blocked with 5% BSA for 1 hour and then incubated overnight with the following antibodies diluted 1:1000 in 5% BSA: RPS2 (Bethy, A303-79A), P62 clone 2C11 (Abnova, H00008878-M01), TFEB (Bethyl Laboratories, A303-673A), calnexin (Enzo Life Sciences, ADI-SPA 860), β-actin (Novus Biologicals, NB600-501), and citrate synthase (Abcam, ab96600).

Techniques: Binding Assay, Clone Assay, Luciferase, Plasmid Preparation, Activity Assay, Quantitative RT-PCR, Expressing, Over Expression, Immunofluorescence, Staining, Western Blot, Infection

Journal: Science Advances

Article Title: TFEB coordinates autophagosome biogenesis and ribophagy during starvation via SQSTM1

doi: 10.1126/sciadv.aea9302

Figure Lengend Snippet: ( A ) The UBQ-HA interactome in TFEB-3xFlag U2OS cells identified ribosomal proteins as a prominent category (table S2). ( B ) Volcano plot of ubiquitinated peptides by diGly proteomics in TFEB-3xFlag versus WT U2OS; significant changes in red/blue (FDR < 0.05, log 2 FC > 1 or < 1); two-sided t test, N = 4 (table S1). ( C ) Top five significant GO CC terms among 465 up-regulated proteins; enrichment score (ES) shown (FDR = 10% and ES > 1.5; table S5). ( D ) Heatmap of TFEB-regulated E3 ligase (FDR-corrected t test, N = 4; table S1). ( E ) ZNF598 promoter schematic showing putative TFEB binding sites. ( F and G ) qRT-PCR of ZNF598 in mock, TFEB-GFP, or siTFEB-TFE3 U2OS ± HBSS (4 hours); normalized fold change (means ± SEM, N = 3 or 4; ** P < 0.005; ANOVA: * P = 0.031; Sidak’s test: * P < 0.05). ( H ) Western blot of ZNF598 under indicated conditions; quantified versus β-actin (means ± SEM, N = 3), ** P < 0.005. ( I ) Heatmap of significantly HA-ubiquitinated ribosomal proteins (S0 = 0.1, FDR < 0.05, N = 4) under indicated conditions (red: up-regulated; blue: down-regulated; table S3). ( J ) Coimmunofluorescence of SQSTM1 (red) and RPS3 (green) in TFEB-GFP ± siZNF598 ; scale bars, 10 μm (insets, 2 μm). Quantification: SQSTM1-RPS3 colocalization (%) and SQSTM1 puncta per cell (means ± SEM of N = 3, n = 38), * P < 0.05. ( K ) Fluorescence microscopy of the RPS3 reporter in starved (ON) TFEB-3xFlag ± siZNF598 . Scale bars, 10 μm (insets, 2 μm). RFP intensity relative to scramble (means ± SEM of N = 3, n = 30), * P = 0.007. ( L ) FACS of the RPS3 WT or K214R reporter. Red fluorescence shift, means ± SEM [ N = 4 (WT), N = 6 (K214R)]. ANOVA: P = 0.0006 (WT), P = 0.01 (K214R). Sidak’s test: * P < 0.05; ** P < 0.005.

Article Snippet: Membranes were blocked with 5% BSA for 1 hour and then incubated overnight with the following antibodies diluted 1:1000 in 5% BSA: RPS2 (Bethy, A303-79A), P62 clone 2C11 (Abnova, H00008878-M01), TFEB (Bethyl Laboratories, A303-673A), calnexin (Enzo Life Sciences, ADI-SPA 860), β-actin (Novus Biologicals, NB600-501), and citrate synthase (Abcam, ab96600).

Techniques: Ubiquitin Proteomics, Binding Assay, Quantitative RT-PCR, Western Blot, Fluorescence, Microscopy

Journal: Orphanet Journal of Rare Diseases

Article Title: Cryptogenic stroke and small fiber neuropathy of unknown etiology in patients with alpha-galactosidase A -10T genotype

doi: 10.1186/s13023-014-0178-5

Figure Lengend Snippet: GLA promoter constructs are selectively activated by TFEB. (A) Representation of the four putative TFEB binding sites (underlined) in the GLA promoter. (B) . Overexpression of TFEB in EA.hy926 cells (black bar) compared to mock transfected cells (white bar) and mutagenesis of conserved TFEB binding sites. (C) ChIP analysis in IHKE cells demonstrated the binding of TFEB. Input: Extracted chromatin served as positive control for PCR. Data are given as mean ± SEM. LU: light units; Luc : luciferase ; ***p < 0.001.

Article Snippet: ChIP was conducted using 3 μg of TFEB antibody (TFEB-C6, #166736, Santa Cruz Biotechnology).

Techniques: Construct, Binding Assay, Over Expression, Transfection, Mutagenesis, Positive Control, Luciferase

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