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: Autophagy

Article Title: YWHA/14-3-3 proteins recognize phosphorylated TFEB by a noncanonical mode for controlling TFEB cytoplasmic localization

doi: 10.1080/15548627.2019.1569928

Figure Lengend Snippet: TFEB S211 is responsible for phosphorylation-dependent interaction with YWHA/14-3-3 proteins. (a) Domain organizations of YWHA/14-3-3 proteins and TFEB. TFEB contains an N-terminal glutamine-rich domain, a transcriptional activation domain (AD) followed by a bHLH and a leucine zipper (LZ), and a C-terminal proline-rich domain. (b) Sequence alignment of human TFEB, TFE3, MITF and TFEC from the MiT/TFE family. The identical residues are colored in red and the highly conserved residues are colored in green. Notably, the extremely conserved S211 in TFEB is very close to the NLS. (c) Co-IP assay of the interactions between YWHA/14-3-3 proteins and TFEB. Both YWHAB and YWHAG can co-immunoprecipitate with wild-type TFEB. Mutations at S211 abolished the interactions but the same type of mutations at S142 did not significantly impair the binding. (d) The binding affinities between TFEB phosphorylated peptides and YWHA/14-3-3 proteins determined by ITC experiments. YWHAB and YWHAG both interact with the p-S211-peptide but not the p-S142-peptide. The binding affinities are indicated in each panel.

Article Snippet: To check the autophagic flux, cells transfected with a plasmid encoding wild-type TFEB and various mutants were treated with bafilomycin A 1 (MedChemExpress, HY-100558), an inhibitor that blocks the fusion of autophagosomes with lysosomes, at the concentration of 100 nM for 24 h, and then were followed by starvation treatment.

Techniques: Phospho-proteomics, Activation Assay, Sequencing, Co-Immunoprecipitation Assay, Binding Assay

Journal: Autophagy

Article Title: YWHA/14-3-3 proteins recognize phosphorylated TFEB by a noncanonical mode for controlling TFEB cytoplasmic localization

doi: 10.1080/15548627.2019.1569928

Figure Lengend Snippet: The overall structures of YWHA/14-3-3 proteins in complex with the TFEB p-S211-peptide. (a-b) Ribbon diagrams of the YWHAB-p-S211-peptide (a) and YWHAG-p-S211-peptide (b) complexes. YWHAB and YWHAG are colored in cyan and pink, respectively. The sidechains of the residues in the p-S211-peptide are shown as sticks. (c) Superimposition of the structures of the YWHAB-p-S211-peptide and YWHAG-p-S211-peptide complexes. (d-e) The resolved electron density maps of the p-S211-peptide in the structures of the YWHAB-p-S211-peptide (d) and YWHAG-p-S211-peptide (e) complexes. In this drawing, YWHA/14-3-3 proteins are shown as cylinders and colored as those in panel A and B. The omit electron density maps of the p-S211-peptide in the 2 structures are shown and contoured at the 1.5 σ level.

Article Snippet: To check the autophagic flux, cells transfected with a plasmid encoding wild-type TFEB and various mutants were treated with bafilomycin A 1 (MedChemExpress, HY-100558), an inhibitor that blocks the fusion of autophagosomes with lysosomes, at the concentration of 100 nM for 24 h, and then were followed by starvation treatment.

Techniques:

Journal: Autophagy

Article Title: YWHA/14-3-3 proteins recognize phosphorylated TFEB by a noncanonical mode for controlling TFEB cytoplasmic localization

doi: 10.1080/15548627.2019.1569928

Figure Lengend Snippet: YWHA/14-3-3 proteins recognize TFEB p-S211-peptide by a non-canonical mode. (a-c) A combined ribbon-and-stick representation of the ‘mode I’ (PDB: 1QJB) (a), ‘mode II’ (PDB: 1QJA) (b) and ‘mode III’ (PDB: 1Q9D) (c) binding motifs for YWHA/14-3-3 proteins. The consensus binding motifs for each mode are indicated in each panel. Besides the phosphorylated-serine-mediated electrostatic interactions, R(−3) and S(−2) in mode I, R(−4) in mode II and the carboxyl tail of V(+1) in mode III also contribute to the binding. (d) TFEB binds to YWHA/14-3-3 proteins via a noncanonical mode. Compared with the canonical mode I and II binding motifs, the TFEB p-S211-peptide lacks the N-terminal arginine but contains C-terminal hydrophobic residues that bind to the lower part of the target-binding groove, which can be defined as mode IV. (e) The schematic models of the different YWHA/14-3-3-binding modes (mode I-IV). In this drawing, YWHA/14-3-3 proteins are colored in gray and their central target-binding grooves are highlighted in dark gray. The phosphorylated peptides are drawn as black lines and the key residues for binding to YWHA/14-3-3 proteins in different modes are depicted as green dots (S[−2]), blue triangles (R[−3] or R[−4]), red forks (p-S[0]), yellow pentagons (P[+2]), pink squares (V[+1]-COOH) and orange parallelogram (L[+5]). (f-g) GST affinity-isolation analysis (f) and yeast two-hybrid assay (g) of the interactions between the TFEB mutants and YWHAB. As compared to the wild-type protein, the point mutations in TFEB affected its binding to YWHAB. The interaction between TP53/p53 and T-Ag were used as the positive control for yeast two-hybrid assay. T-Ag, SV40 T antigen. (h) The binding affinities between TFEB p-S211-peptide (with mutations) and YWHAB determined by ITC experiments. The T208R mutation can enhance the binding affinity but the S209A and L216Q mutations both reduce it.

Article Snippet: To check the autophagic flux, cells transfected with a plasmid encoding wild-type TFEB and various mutants were treated with bafilomycin A 1 (MedChemExpress, HY-100558), an inhibitor that blocks the fusion of autophagosomes with lysosomes, at the concentration of 100 nM for 24 h, and then were followed by starvation treatment.

Techniques: Binding Assay, Isolation, Y2H Assay, Positive Control, Mutagenesis

Journal: Autophagy

Article Title: YWHA/14-3-3 proteins recognize phosphorylated TFEB by a noncanonical mode for controlling TFEB cytoplasmic localization

doi: 10.1080/15548627.2019.1569928

Figure Lengend Snippet: The interactions between YWHA/14-3-3 proteins and TFEB are essential for TFEB subcellular localization. (a) Subcellular distribution of TFEB and its mutants under nutrient-rich and starvation conditions. Under nutrient-rich conditions, wild-type TFEB is largely distributed in the cytoplasm. The TFEB mutants (S209A, S211A, P213A, A214Q and L216Q) showed increased nuclear localization, whereas the TFEBT208R mutant did not. Upon starvation, wild-type TFEB and the TFEB mutants are mainly localized in the nucleus. Scale bar: 50 μm. (b) Quantification of the subcellular distribution data shown in panel A. The percentage of the fluorescence intensity of each construct in the nucleus was quantified (average of 4 experiments, n > 50 cells for each experiment). Each bar represents the mean ±SD, **p < 0.01, *p < 0.05. (c) A schematic model illustrating the YWHA/14-3-3-mediated regulation of TFEB subcellular localization. Briefly, the binding of YWHA/14-3-3 proteins could induce the conformational changes of the p-S211-site and the region between the NLS and the p-S211-site, which would potentially interfere with the NLS. The structural model was built based on the structures of the bHLH domain of MITF (PDB: 4ATH) and the YWHA-p-S211-peptide complex.

Article Snippet: To check the autophagic flux, cells transfected with a plasmid encoding wild-type TFEB and various mutants were treated with bafilomycin A 1 (MedChemExpress, HY-100558), an inhibitor that blocks the fusion of autophagosomes with lysosomes, at the concentration of 100 nM for 24 h, and then were followed by starvation treatment.

Techniques: Mutagenesis, Fluorescence, Construct, Binding Assay

Journal: Autophagy

Article Title: YWHA/14-3-3 proteins recognize phosphorylated TFEB by a noncanonical mode for controlling TFEB cytoplasmic localization

doi: 10.1080/15548627.2019.1569928

Figure Lengend Snippet: Modulations of the binding of TFEB to YWHA/14-3-3 proteins affect TFEB cellular functions. (a) Immunoblotting analysis of LC3 in TFEB-overexpressing cells (with wild-type TFEB and its various mutants) in the absence and presence of bafilomycin A1 under nutrient-rich and starvation conditions. (b) Western-blot analysis of the expression of TFEB target genes (ATP6V1H, CTSB, CTSD and LAMP1) after transfection with wild-type TFEB and its various mutants. ACTB was used as the loading control.

Article Snippet: To check the autophagic flux, cells transfected with a plasmid encoding wild-type TFEB and various mutants were treated with bafilomycin A 1 (MedChemExpress, HY-100558), an inhibitor that blocks the fusion of autophagosomes with lysosomes, at the concentration of 100 nM for 24 h, and then were followed by starvation treatment.

Techniques: Binding Assay, Western Blot, Expressing, Transfection, Control

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: The Journal of Experimental Medicine

Article Title: Acid sphingomyelinase modulates the autophagic process by controlling lysosomal biogenesis in Alzheimer’s disease

doi: 10.1084/jem.20132451

Figure Lengend Snippet: ASM causes abnormal autophagic protein degradation by altering ALP. (A) Autophagic flux assay. Human fibroblasts were cultured in: (1) complete medium with or without 10 µM ASM in the presence or absence of NH 4 Cl (left), (2) complete medium or starvation condition in the presence or absence of NH 4 Cl (middle), or (3) complete medium or starvation condition with or without 10 µM ASM (right). The LC3-II levels were examined by Western blotting ( n = 6–7 per group). (B) The accumulation of p62 was assessed in the human fibroblast cultured with 10 µM ASM, 20 mM NH 4 Cl, or starvation condition ( n = 4 per group). (C) Western blot analysis of LC3-II levels in controls, PS1-FAD, and ApoE4 fibroblasts in the presence or absence of NH 4 Cl ( n = 6 per group). (D) Western blot analysis for LC3-II levels in fibroblasts derived from WT, APP/PS1, and APP/PS1/ ASM +/− mice in the presence or absence of NH 4 Cl ( n = 6 per group). (E) Effect of ASM on lysosomal pH. FACS and histological analysis of fibroblasts stained with LysoTracker red ( n = 5 per group; bars, 20 µm). H 2 O 2 - and NH 4 Cl-treated cells were used as positive and negative controls, respectively. (F and G) Western blot analyses for TFEB and Lamp1 in human fibroblasts (F; n = 6 per group) and neurons (G; n = 6 per group) after treatment with ASM. (H) Immunocytochemistry of Lamp1 in control and ASM-treated fibroblast ( n = 5 per group; bars, 20 µm). (I) Western blot analysis for nuclear localization of TFEB in ASM-treated cells ( n = 5 per group). (J) Quantitative real-time PCR analysis of TFEB-target gene expression in normal ( n = 6) and ASM-treated ( n = 10) fibroblasts. (K) ASM activity was estimated in the fibroblast with or without NH4Cl ( n = 5 per group). Data are representative of two (E, H, and I) or three (A–D, F, G, J, and K) independent experiments. A, B, and E–G, one-way ANOVA, Tukey’s post hoc test. C, D, and H–K, Student’s t test. *, P < 0.05; **, P < 0.01. All error bars indicate SEM.

Article Snippet: Primary antibodies to the following proteins were used: BACE-1 (mouse, 1:1,000; Millipore), LC3 (rabbit, 1:1,000; Cell Signaling Technology), Beclin-1 (rabbit, 1:1,000; Cell Signaling Technology), p62 (rabbit, 1:1,000; Cell Signaling Technology), rab5 (rabbit, 1:1,000; Cell Signaling Technology), rab7 (rabbit, 1:1,000; Cell Signaling Technology), TFEB (rabbit, 1:1,000; Cell Signaling Technology), Lamp1 (rabbit, 1:1,000; Abcam), TFEB (rabbit, 1:500; Novus Biologicals), cathepsin D (goat, 1:500; R&D Systems), 6E10 (mouse, 1:500; Signet), Histon H3 (rabbit, 1:1,000; Cell Signaling Technology), and β-actin (1:1,000; Santa Cruz Biotechnology, Inc.).

Techniques: Flux Assay, Cell Culture, Western Blot, Derivative Assay, Staining, Immunocytochemistry, Control, Real-time Polymerase Chain Reaction, Targeted Gene Expression, Activity Assay

Journal: The Journal of Experimental Medicine

Article Title: Acid sphingomyelinase modulates the autophagic process by controlling lysosomal biogenesis in Alzheimer’s disease

doi: 10.1084/jem.20132451

Figure Lengend Snippet: ASM causes autophagic dysfunction in vivo by sequestrating ALP function. (A and B) ASM was estimated in the brain and blood plasma of C57BL/6 mice after ASM-CM treatment into the hippocampus (A; i.c., n = 6 per group) or tail vein (B; i.v., n = 6 per group). (C and D) Western blot analyses for LC3, beclin-1, p62, and cathepsin D in the brains of C57BL/6 mice after ASM-CM treatment into the hippocampus (C; n = 5–6 per group) or tail vein (D; n = 4–5 per group). (E) Cathepsin D activity in the brain extracts of C57BL/6 mice after ASM-CM treatment ( n = 4 per group). (F and G) Protein expression of TFEB and Lamp1 in the brains after ASM-CM treatment into the hippocampus (F; n = 5–6 per group) or tail vein (G; n = 5 per group). (H) Protein expression of TFEB and Lamp1 in the brains of 9-mo-old WT, APP/PS1, ASM +/− , and APP/PS1/ ASM +/− mice ( n = 6–7 per group). Data are representative of three independent experiments. A–G, Student’s t test. H, one-way ANOVA, Tukey’s post hoc test. *, P < 0.05. All error bars indicate SEM.

Article Snippet: Primary antibodies to the following proteins were used: BACE-1 (mouse, 1:1,000; Millipore), LC3 (rabbit, 1:1,000; Cell Signaling Technology), Beclin-1 (rabbit, 1:1,000; Cell Signaling Technology), p62 (rabbit, 1:1,000; Cell Signaling Technology), rab5 (rabbit, 1:1,000; Cell Signaling Technology), rab7 (rabbit, 1:1,000; Cell Signaling Technology), TFEB (rabbit, 1:1,000; Cell Signaling Technology), Lamp1 (rabbit, 1:1,000; Abcam), TFEB (rabbit, 1:500; Novus Biologicals), cathepsin D (goat, 1:500; R&D Systems), 6E10 (mouse, 1:500; Signet), Histon H3 (rabbit, 1:1,000; Cell Signaling Technology), and β-actin (1:1,000; Santa Cruz Biotechnology, Inc.).

Techniques: In Vivo, Clinical Proteomics, Western Blot, Activity Assay, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Acid sphingomyelinase modulates the autophagic process by controlling lysosomal biogenesis in Alzheimer’s disease

doi: 10.1084/jem.20132451

Figure Lengend Snippet: Pharmacological restoration of ASM to the normal range improves pathology in AD mice. (A) Protocol of AMI treatment in APP/PS1 mice. (B) ASM was estimated in the blood plasma ( n = 12–14 per group) and brain ( n = 9–10 per group) of APP/PS1 mice after AMI treatment. (C) Sphingomyelin, ceramide, and AC were determined using UPLC based methods in the plasma ( n = 9 per group) and brain ( n = 8 per group). (D) Mice brain sections were stained with thioflavin S to detect Aβ (bars, 200 µm). The relative area occupied by Aβ plaques were determined ( n = 6 per group). (E–G) Aβ40 and Aβ42 in the brains of AMI treated or nontreated APP/PS1 mice were assessed using immunofluorescence staining (E and F; n = 8 per group; bars, 200 µm) and ELISA kits (G; n = 6 per group). (H and I) Western blot analyses and quantification for LC3, Beclin-1, p62, cathepsin D, TFEB, and Lamp1 in the brains of APP/PS1 mice treated with AMI or control ( n = 6–8 per group). (J) Cathepsin D activity in the brain extracts of AMI-treated or nontreated APP/PS1 mice ( n = 4 per group). (K) Escape latencies of APP/PS1 mice treated with AMI or control over 10 d (WT, n = 14; nontreated APP/PS1, n = 10; and AMI-treated APP/PS1, n = 12). (L–O) Probe trial day 11. (L and M) Path length (L) and swim speed (M) were recorded and analyzed. (N) Time spent in target platform and other quadrants was measured. (O) The number of times each animal entered the small target zone during the 60-s probe trial. (P) Representative swimming paths at day 10 of training. Data are representative three independent experiments. B–J and N, Student’s t test; K–M and O, one-way ANOVA, Tukey’s post hoc test. *, P < 0.05; **, P < 0.01. All error bars indicate SEM.

Article Snippet: Primary antibodies to the following proteins were used: BACE-1 (mouse, 1:1,000; Millipore), LC3 (rabbit, 1:1,000; Cell Signaling Technology), Beclin-1 (rabbit, 1:1,000; Cell Signaling Technology), p62 (rabbit, 1:1,000; Cell Signaling Technology), rab5 (rabbit, 1:1,000; Cell Signaling Technology), rab7 (rabbit, 1:1,000; Cell Signaling Technology), TFEB (rabbit, 1:1,000; Cell Signaling Technology), Lamp1 (rabbit, 1:1,000; Abcam), TFEB (rabbit, 1:500; Novus Biologicals), cathepsin D (goat, 1:500; R&D Systems), 6E10 (mouse, 1:500; Signet), Histon H3 (rabbit, 1:1,000; Cell Signaling Technology), and β-actin (1:1,000; Santa Cruz Biotechnology, Inc.).

Techniques: Clinical Proteomics, Staining, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Activity Assay

Journal: The Journal of Experimental Medicine

Article Title: Acid sphingomyelinase modulates the autophagic process by controlling lysosomal biogenesis in Alzheimer’s disease

doi: 10.1084/jem.20132451

Figure Lengend Snippet: Restoration of ASM to the normal level reverses impaired autophagy in the AD patient-specific cells. (A) SMPD1 gene suppression by ASM-siRNA in human fibroblasts. ASM activity was assessed after ASM siRNA treatment in the control and AD fibroblast ( n = 6 per group). (B) LC3-II and p62 levels were examined in human AD fibroblast with or without ASM inhibition. siRNA-mediated suppression of ASM reduced LC3-II and p62 levels in PS1-FAD (left; n = 7 per group) and ApoE4 fibroblast (right; n = 6 per group). (C) Protein expression of TFEB and Lamp1 in the PS1-FAD and ApoE4 fibroblast after ASM inhibition ( n = 5–6 per group). (D–G) Generation of PS1 iPSC lines from patient fibroblast. (D) Established iPSCs showed embryonic stem cell–like morphology (Phase; bar, 1 mm), AP activity (bar, 200 µm), and expressed pluripotent stem cell markers SSEA4 (bar 100 µm), TRA1-60 (bar 100 µm), and TRA1-81 (bar 100 µm). (E) Normal karyotype of PS1 iPSC. (F) Quantitative real-time PCR analysis of hESC marker gene of PS1 iPSC ( n = 3 per group). (G) Gross morphology and hematoxylin-eosin staining of representative teratomas generated from PS1-4 iPSCs (bars, 50 µm). (H) Estimation of neural differentiation from control and PS1-4 iPSCs. Representative images of immunocytochemical staining the β-III tubulin after neural differentiation (bars, 50 µm). (I) The amount of Aβ42 secreted from control iPSC-derived neuron and PS1 iPSC-derived neuron ( n = 5 per group). (J) Characterization of ASM activity in the control and PS1 iPSC and iPSC-derived neurons ( n = 6 per group). (K) Western blot analyses for LC3, beclin-1, p62, TFEB, and Lamp1 in the control and PS1-4 iPSC–derived neuron after ASM siRNA treatment ( n = 5–6 per group). (L) EM images and quantification data of control and PS1 iPSC-derived neurons. Higher magnification of boxed area shows detail of AVs (arrow; n = 4 per group; bars: [low magnification] 1 µm, [high magnification] 500 nm). (M) Quantitative real-time PCR analysis of TFEB-target gene expression in iPSC-derived neurons after ASM siRNA treatment ( n = 5–6 per group). Data are representative of two (A, D–G, I, and L), or three (B, C, H, J, K, and M) independent experiments. A, C, F, I, J, and M, Student’s t test. B, K, and L, one-way ANOVA, Tukey’s post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. A–K, error bars indicate SEM. L and M, Error bars indicate SD.

Article Snippet: Primary antibodies to the following proteins were used: BACE-1 (mouse, 1:1,000; Millipore), LC3 (rabbit, 1:1,000; Cell Signaling Technology), Beclin-1 (rabbit, 1:1,000; Cell Signaling Technology), p62 (rabbit, 1:1,000; Cell Signaling Technology), rab5 (rabbit, 1:1,000; Cell Signaling Technology), rab7 (rabbit, 1:1,000; Cell Signaling Technology), TFEB (rabbit, 1:1,000; Cell Signaling Technology), Lamp1 (rabbit, 1:1,000; Abcam), TFEB (rabbit, 1:500; Novus Biologicals), cathepsin D (goat, 1:500; R&D Systems), 6E10 (mouse, 1:500; Signet), Histon H3 (rabbit, 1:1,000; Cell Signaling Technology), and β-actin (1:1,000; Santa Cruz Biotechnology, Inc.).

Techniques: Activity Assay, Control, Inhibition, Expressing, Real-time Polymerase Chain Reaction, Marker, Staining, Generated, Derivative Assay, Western Blot, Targeted Gene Expression

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