Journal: Frontiers in Pharmacology

Article Title: Berbamine sensitizes hepatocellular carcinoma to chemotherapy by inhibiting autophagy via modulating SIRT1-mediated acetylation

doi: 10.3389/fphar.2026.1763828

Figure Lengend Snippet: BBM suppresses the SIRT1-TFEB axis at the protein level. (A,B) Western blot analysis of SIRT1 and TFEB protein levels in cells treated with BBM at indicated concentrations (A) or for indicated times (B) . (C) Immunofluorescence analysis of TFEB subcellular localization. Cells were treated with BBM (5 μM) or TFEB activator 1 (0.5 μM) for 24 h and stained for TFEB (red) and DAPI (blue). Scale bar, 5 μm. (D) Intracellular NAD + levels and NAD + /NADH ratio in cells treated with BBM (2.5, 5, 10 μM) for 24 h. All values represent the mean ± SD (n = 3), * p < 0.05, ** p < 0.01. (E) Transcriptomic analysis of differentially expressed genes in SMMC-7721 cells after BBM (5 μM) treatment for 24 h. (F) Reactome pathway enrichment analysis of downregulated gene. (G) Western blot analysis of OXPHOS components (MT-ND4, MT-ND4L) in cells treated with BBM (0-10 μM) for 24 h. (H) Molecular docking analysis showing the predicted binding mode of BBM with ERRα. BBM is shown in green sticks, and hydrogen bonds are indicated as yellow dashed lines.

Article Snippet: Sorafenib (HY-10201), Baflomycin A1 (HY-100558), chloroquine (HY-17589A), rapamycin (AY-22989), resveratrol (HY-16561), and TFEB activator 1 (HY-135825) were purchased from MedChemExpress (Princeton, NJ, USA).

Techniques: Western Blot, Immunofluorescence, Staining, Binding Assay

Journal: Frontiers in Pharmacology

Article Title: Berbamine sensitizes hepatocellular carcinoma to chemotherapy by inhibiting autophagy via modulating SIRT1-mediated acetylation

doi: 10.3389/fphar.2026.1763828

Figure Lengend Snippet: SIRT1 activation rescues BBM-induced autophagy blockade. (A,B) Western blot analysis of SIRT1, TFEB, lysosomal proteins (CTSB, CTSD, RAB7), and autophagy flux markers (p62, LC3) in cells treated with BBM (5 μM) in the presence or absence of the SIRT1 agonist Resveratrol (Res, 10 μM) or the TFEB agonist TFEB activator 1 (0.5 μM) for 24 h. Data are presented as mean ± SD (n = 3). * p < 0.05, ** p < 0.01 vs. control; # p < 0.05, ## p < 0.01 vs. BBM-only group.

Article Snippet: Sorafenib (HY-10201), Baflomycin A1 (HY-100558), chloroquine (HY-17589A), rapamycin (AY-22989), resveratrol (HY-16561), and TFEB activator 1 (HY-135825) were purchased from MedChemExpress (Princeton, NJ, USA).

Techniques: Activation Assay, Western Blot, Control

Journal: Science (New York, N.Y.)

Article Title: Induction of lysosomal and mitochondrial biogenesis by AMPK phosphorylation of FNIP1

doi: 10.1126/science.abj5559

Figure Lengend Snippet: (A) ClustalW alignment of five conserved AMPK phosphorylation sites on FNIP1 matching the AMPK substrate consensus motif. (B) AMPK phosphorylation sites on FNIP1 identified by MS in HEK293T cells, expressing FLAG-tagged FNIP1 cDNA, after treatment with vehicle or 1 hour of phenformin. (C) In vitro AMPK kinase assay. WT FNIP1 or FNIP1 mutants (SA2, SA3, SA4, SA5) were immunoprecipitated and incubated with recombinant active AMPK and [γ32P]-ATP. Kinase reactions were separated by SDS-PAGE and incorporation of [γ32P]-ATP was detected by autoradiography. (D) Immunoblot showing endogenous TFEB and FNIP1 (Ser220) phosphorylation status in WT (+/+) or AMPK DKO (−/−) MEFs after DMSO or a 991 time course, as detected by an antibody to FNIP1 P-Ser220. (E) Immunoblots of murine liver lysates from inducible KO of AMPKα1 and α2 (Alb-CreERT2; Prkaa1fl/fl; Prkaa2fl/fl) mice (AMPK KO) or control mice treated with vehicle or AMPK activator compound MK8722 for 2 hours, showing endogenous FNIP1 and TFEB phosphorylation status. (F) Immunoblots of lysates from primary mouse hepatocytes Prkaa2fl/fl) mice (AMPK liver DKO), treated with vehicle or metformin for 5 hours. (G) Immunoblots of endogenous pFNIP1 Ser220 and TFEB in in FNIP1 KO HEK293T cells stably reconstituted with WT, SA4, or SA5 FNIP1, treated with a 991 (50 μM) time course. (H) Nucleocytoplasmic fractionation of WT or SA5 FNIP1 HEK293T cells after 1-hour DMSO or 991 treatment, followed by immunoblotting. (I) Quantitation of TFEB colocalization with DAPI-stained nuclei in (J). Data are shown as the means ± SEMs of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; unpaired t test. (J) Representative immunofluorescence images of endogenous TFEB stained in WT FNIP1 and SA5-FNIP1 HEK293T cells, treated with 1-hour DMSO or 991. Nuclei are stained with DAPI.

Article Snippet: Confocal imaging of TFEB, TFE3, and RagC For visualization of TFEB, TFE3, and RagC, the following primary rabbit antibodies were used: TFEB (CST no. 37785), TFE3 (CST no.14779), and RagC (CST no. 9480) followed by anti-rabbit secondary antibodies conjugated to Alexa Fluor (AF) 594.

Techniques: Phospho-proteomics, Expressing, In Vitro, Kinase Assay, Immunoprecipitation, Incubation, Recombinant, SDS Page, Autoradiography, Western Blot, Control, Stable Transfection, Fractionation, Quantitation Assay, Staining, Immunofluorescence

Journal: Science (New York, N.Y.)

Article Title: Induction of lysosomal and mitochondrial biogenesis by AMPK phosphorylation of FNIP1

doi: 10.1126/science.abj5559

Figure Lengend Snippet: (A to C) Western blots probing mitochondrial protein expression after a 991 time course ranging from 0 to 30 hours in WT FNIP1 and SA5 FNIP1 HEK293T cells (A), WT and TFEB-TFE3 DKO HEK293T cells (B), and WT and ERRα KO HEK293T cells (C). (D) Mitochondrial DNA content analysis. The ratio of mitochondrial (16S) to nuclear (actin) DNA was determined by qRT-PCR after treatment for 24 hours with 991 or DMSO (vehicle), as indicated. (E) Quantitation of IDH2 staining in (F). (F) Representative Airyscan microscopy images of mitochondrial IDH2 staining in WT FNIP1 and SA5 FNIP1 HEK293T cells treated for 24 hours with 991 or DMSO, as indicated. (G) Representative Airyscan images of Lamp2-stained lysosomes and Cox IV–stained mitochondria in WT FNIP1 and SA5 FNIP1 HEK293T cells treated for 24 hours with DMSO or 991, as indicated. (H) Quantitation of mitochondrial volume in (G). (I) Quantitation of lysosomal volume in (G). (J) Seahorse assay to measure OCR in WT compared with AMPK KO HEK293T cells. (K) Seahorse assays displaying OCR in WT FNIP1 compared with SA5 FNIP1 HEK293T cells. (L) Seahorse assays measuring OCR in WT compared with ERRα KO HEK293T cells. Graphs are shown as the means ± SEMs. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; unpaired t test.

Article Snippet: Confocal imaging of TFEB, TFE3, and RagC For visualization of TFEB, TFE3, and RagC, the following primary rabbit antibodies were used: TFEB (CST no. 37785), TFE3 (CST no.14779), and RagC (CST no. 9480) followed by anti-rabbit secondary antibodies conjugated to Alexa Fluor (AF) 594.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Quantitation Assay, Staining, Microscopy

Journal: Science (New York, N.Y.)

Article Title: Induction of lysosomal and mitochondrial biogenesis by AMPK phosphorylation of FNIP1

doi: 10.1126/science.abj5559

Figure Lengend Snippet: RNA-seq analysis of WT and CRISPR-Cas9–mediated AMPK KO HEK293T cells upon 0- to 16-hour treatment with the mitochondrial poisons CCCP (5 μM), rotenone (100 ng/ml), phenformin (2 mM), and the AMPK-specific activation drug 991 (50 μM). (A) Unbiased heatmap displaying gene expression pattern of all AMPK-dependent, differentially expressed (DE) genes (FC ≥ 1.3, P ≤ 0.05) commonly regulated by all three mitochondrial poisons and 991. (B) Stacked Venn diagram showing the proportion of DE CCCP-induced genes that require AMPK. (C) GSEA analysis shows significantly up-regulated GTRD (ChIP-seq–based Gene Transcription Regulation Database) transcription factor targets upon CCCP treatment. (D) Gene clustering analysis and heat-map displaying the expression pattern of all mitochondria-specific genes as defined by the Mitocarta 3.0 inventory. Right heatmap is a zoomed-in view of the AMPK-dependent mitochondrial genes induced by the four drugs. (E) Overlap in regulation of AMPK-dependent mitochondrial genes in (D) by CCCP, rotenone, phenformin, and 991. (F) Volcano plot depicting DE mitochondrial genes from (D) after 991 compared with DMSO. Red dots represent genes significantly induced by 991 compared with DMSO. The y axis denotes −log10 P values, and the x axis shows log2 FC values. (G) Volcano plot denoting differential expression of mitochondrial genes between WT 16-hour 991-treated cells compared with AMPK KO 16-hour 991-treated cells. Blue dots represent genes significantly down-regulated by AMPK deletion compared with WT AMPK condition. The y axis denotes −log10 P values, and the x axis shows log2 FC values. (H to J) Quantitative RT-PCR (qRT-PCR) for lysosomal gene Lamp2 (H), mitochondrial genes IDH2 (I), and Cox6A1 (J) in WT and AMPK KO HEK293T cells after CCCP. (K to M) qRT-PCR for lysosomal gene Lamp2 (K) and mitochondrial genes IDH2 (L) and ACO2 (M) in WT and AMPK KO HEK293T cells after 991 treatment. All qRT-PCR graphs are shown as the means ± SEMs. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; unpaired t test. (N) Analysis of AMPK signaling and TFEB protein immunoblotting of WT and AMPK KO HEK293T cells treated with DMSO, 991, or phenformin for 1 hour. (O) Analysis of AMPK, TFEB, and mitochondrial protein immunoblotting of WT and AMPK KO HEK293T cells treated with a rotenone (100 ng/ml) time course, ranging from 0 to 24 hours. (P) Analysis of AMPK, TFEB, and mitochondrial protein immunoblotting of WT and AMPK KO HEK293T cells treated with a 991 (50 μM) time course, ranging from 0 to 24 hours. (Q) Analysis of TFEB and TFE3 protein cytoplasm-to-nuclear shuttling by fractionation of WT and AMPK KO HEK293T cells with or without 1-hour 991 treatment, followed by immunoblotting. (R) Quantification of TFEB colocalization with DAPI-stained nuclei. Data are shown as the means ± SEMs of three independent experiments. *P < 0.05; **P < 0.01; unpaired t test. (S) Representative images from immunofluorescence microscopy of endogenous TFEB stained in WT and AMPK KO HEK293T cells, pretreated with 1 hour of DMSO or 991. Nuclei are stained with DAPI. (T) Model. After ETC poisons or the direct small-molecule activator 991, AMPK becomes activated and, through an unknown mechanism, triggers TFEB translocation to the nucleus, where TFEB induces expression of lysosomal and mitochondrial genes.

Article Snippet: Confocal imaging of TFEB, TFE3, and RagC For visualization of TFEB, TFE3, and RagC, the following primary rabbit antibodies were used: TFEB (CST no. 37785), TFE3 (CST no.14779), and RagC (CST no. 9480) followed by anti-rabbit secondary antibodies conjugated to Alexa Fluor (AF) 594.

Techniques: RNA Sequencing, CRISPR, Activation Assay, Gene Expression, ChIP-sequencing, Expressing, Quantitative Proteomics, Quantitative RT-PCR, Western Blot, Fractionation, Staining, Immunofluorescence, Microscopy, Translocation Assay

Journal: Science (New York, N.Y.)

Article Title: Induction of lysosomal and mitochondrial biogenesis by AMPK phosphorylation of FNIP1

doi: 10.1126/science.abj5559

Figure Lengend Snippet: (A) WT and AMPK KO HEK293T cells were subjected to a short 991 (50 μM) time course, and lysates were immunoblotted with the indicated antibodies to probe for mTORC1 signaling. (B) WT FNIP1 and SA5 FNIP1 HEK293T cells were treated as in (A), and lysates were immunoblotted to probe for mTORC1 signaling. (C) WT parental or AMPK-null HEK29T cells were AA starved for the indicated times, and lysates were immunoblotted to examine mTORC1 signaling. (D) WT FNIP1 or SA5 FNIP1 HEK293T cells were administered with the mTOR inhibitors AZD8055 or Torin1 either individually or in combination with 991, as indicated. Lysates were subsequently immunoblotted to examine TFEB phosphorylation status. (E) WT or SA5 FNIP1 cells, stably expressing GFP-TFEB cDNA were treated with or without 50-μM 991 for 1 hour. GFP-TFEB was immunoprecipitated from the lysates, and immunoprecipitates were analyzed by Western blotting. (F) Immunoprecipitates of GFP-TFEB, stably expressed in WT FNIP1 and SA5 FNIP1 HEK293T cells, were subjected to immunoblotting to probe interactions with the Rag GTPases. (G) Quantitation of RagC colocalization with Lamp2 in (H). Data are shown as the means ± SEMs of three independent experiments. *P < 0.05; **P < 0.01; unpaired t test. (H) Representative immunofluorescence images of endogenous RagC costained with Lamp2 in WT FNIP1 and SA5 FNIP1 HEK293T cells treated with 1 hour of DMSO or 991 (50 μM). (I) WT FNIP1 or SA5 FNIP1 cells were transiently transfected with HA-RagC mutants locked in either the GTP-bound state (Q120L) or the GDP-bound state (S75N) and subsequently treated with or without 1-hour 991. Lysates were immunoblotted with the indicated antibodies. (J) HA-RagC mutants from (I) were immunoprecipitated from lysates using HA magnetic beads and immunoprecipitates analyzed by Western blotting.

Article Snippet: Confocal imaging of TFEB, TFE3, and RagC For visualization of TFEB, TFE3, and RagC, the following primary rabbit antibodies were used: TFEB (CST no. 37785), TFE3 (CST no.14779), and RagC (CST no. 9480) followed by anti-rabbit secondary antibodies conjugated to Alexa Fluor (AF) 594.

Techniques: Phospho-proteomics, Stable Transfection, Expressing, Immunoprecipitation, Western Blot, Quantitation Assay, Immunofluorescence, Transfection, Magnetic Beads

Journal: Science (New York, N.Y.)

Article Title: Induction of lysosomal and mitochondrial biogenesis by AMPK phosphorylation of FNIP1

doi: 10.1126/science.abj5559

Figure Lengend Snippet: (A) GSEA plot for the “KEGG Lysosome” gene set, which was enriched in WT FNIP1 16-hour 991-treated but not SA5 conditions. (B) RNA-seq analysis of WT FNIP1 and SA5 FNIP1 cells subjected to a 0- to 16-hour 991 (50 μM) time course. Clusteringanalysis and heatmap displaysexpression patterns of AMPK-FNIP1–dependent CLEAR network genes that have been previously validated or GSEA defined. (C) Volcano plot depicting differential expression of CLEAR network genes after 4-hour 991 in WT FNIP1 versus SA5 FNIP1 conditions. Blue dots represent genes significantly down-regulated by mutation of AMPK sites on FNIP1. The y axis denotes −log10 P values, and the x axis shows log2 FC values. (D to I) qRT-PCR of CLEAR network genes SESN (C), Hex A (D), Neu1 (E), Lamp1 (F), FNIP2 (G), and ULK1 (H) in WT FNIP1 and SA5 FNIP1 HEK293T cells subjected to a 0- to 30-hour 991 (50 μM) time course. Graphs are shown as means ± SEMs. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001; unpaired t test. (J) RNA-seq analysis of WT parental and CRISPR-Cas9–mediated TFEB-TFE3 DKO HEK293T cells treated with a 0- to 24-hour 991 time course. Heatmap shows AMPK-FNIP1–dependent genes whose expression is reduced by loss of TFEB-TFE3. (K) Volcano plot denoting CLEAR network DE genes after 16 hours of 991 in WT versus TFEB-TFE3 DKO FNIP1 cells. Blue dots represent genes significantly reduced by deletion of TFEB-TFE3. The y axis denotes −log10 P values, and the x axis shows log2 FC values. (L) Immunoblotting of lysosomal proteins in WT FNIP1 and SA5 FNIP1 HEK 293 cells after a 0- to 30-hour 991 time course. (M) Representative immunofluorescence images of lysosome structures stained with Lamp2 antibody after DMSO or 4-hour 991 treatment of WT FNIP1 and SA5 FNIP1 cells. (N) Quantitation of Lamp2 lysosomal structures from (M) and at the time points indicated, showing percentage of lysosome structures with volume greater than 0.1 μm3 after a 0- to 30-hour 991 time course in WT FNIP1 and SA5 FNIP1 cells. (O) Quantitation of Lamp2 sum intensity per lysosome in (M) and other time points from the same experiment. (P) Model. AMPK phosphorylation of FNIP1, induced by 991 or energetic stress, triggers TFEB entry into the nucleus, where it binds to CLEAR elements on lysosomal gene promoters, inducing lysosomal gene transcription, enhancing lysosomal protein expression and thereby lysosome biogenesis.

Article Snippet: Confocal imaging of TFEB, TFE3, and RagC For visualization of TFEB, TFE3, and RagC, the following primary rabbit antibodies were used: TFEB (CST no. 37785), TFE3 (CST no.14779), and RagC (CST no. 9480) followed by anti-rabbit secondary antibodies conjugated to Alexa Fluor (AF) 594.

Techniques: RNA Sequencing, Quantitative Proteomics, Mutagenesis, Quantitative RT-PCR, CRISPR, Expressing, Western Blot, Immunofluorescence, Staining, Quantitation Assay, Phospho-proteomics

Journal: Science (New York, N.Y.)

Article Title: Induction of lysosomal and mitochondrial biogenesis by AMPK phosphorylation of FNIP1

doi: 10.1126/science.abj5559

Figure Lengend Snippet: (A) qRT-PCR showing expression of total PPARGC1A compared with expression of the shorter NT-PPARGC1A splice isoform. Graphs are shown as means ± SEMs. n = 3. *P < 0.05; **P < 0.01; unpaired t test. (B) Schematic of the two predominant splice isoforms of PPARGC1A induced in this cell type in our conditions. (C) Immunoblots reflecting changes in expression of NT-PGC1α protein levels in WT FNIP1 versus SA5 FNIP1 cells in the presence or absence of a 991 time course. Molecular weights are indicated on the right. (D) Densitometry analysis of NT-PGC1α immunoblots. (E) qRT-PCR comparing expression of total PPARGC1A compared with that of shorter NT-PPARGC1A splice isoform in WT and AMPK KO HEK293T after a 0- to 16-hour CCCP time course. (F) qRT-PCR comparing expression of NT-PPARGC1A splice isoform in WT and AMPK KO HEK293T after a 0- to 16-hour 991 time course. For (D) to (F), all values are shown as means ± SEMs. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001; unpaired t test. (G) Gene clustering analysis of RNA-seq data from WT FNIP1 and SA5 FNIP1 cells subjected to 991 time course treatments. Heatmap displays expression pattern of AMPK- and FNIP1-dependent mitochondria specific genes, as defined by the Mitocarta 3.0 inventory. (H) RNA-seq analysis of HEK293T cells transfected with control siRNA (siCont) or siRNA targeting PGC1α (siPGC1α) and treated with 0 to 24 hours of 991, as indicated. Heatmap displays AMPK-FNIP1–dependent Mitocarta 3.0 genes whose expression is lost with PGC1α knockdown. (I) RNA-seq analysis of WT and TFEB-TFE3 DKO HEK293T cells treated with 0 to 24 hours of 991. Heatmap displays expression pattern of AMPK-FNIP1–dependent mitochondrial genes, as defined by Mitocarta 3.0, that are lost upon TFEB-TFE3 deletion. (J) RNA-seq analysis of WT versus ERRα KO HEK293T cells subjected to a 991 time course, as indicated. Heatmap visualizes the expression pattern of AMPK-FNIP1–dependent mitochondrial genes in WT and ERRα KO HEK293T cells. (K) Volcano plot displaying Mitocarta 3.0 DE genes after 16-hour 991 in WT versus SA5 FNIP1 cells. Blue dots represent genes significantly down-regulated by mutation of AMPK sites on FNIP1. (L) Volcano plot depicting DE Mitocarta 3.0 genes in 16-hour 991-treated WT versus 16-hour 991-treated TFEB-TFE3 DKO RNA-seq dataset. Blue dots represent genes significantly down-regulated by CRISPR deletion of TFEB-TFE3. (M) Volcano plot depicting DE Mitocarta 3.0 genes in 16-hour 991 siCont versus 16-hour 991 siPGC1α RNA-seq dataset. Blue dots represent genes significantly down-regulated by knockdown of PGC1α. (N) RNA-seq analysis of WT versus ERRα KO HEK293T cells. Volcano plot displaying DE Mitocarta 3.0 genes after 16-hour 991 in WT versus ERRα null cells. Blue dots represent genes significantly down-regulated by deletion of ERRα. (O) Four-way Venn diagram showing overlap of gene sets controlled by AMPK-FNIP1, TFEB-TFE3, PGC1α, and ERRα. (P) qRT-PCR of mitochondrial genes including IDH2, Cox IV, CytoC, UCP2, and SOD2 in WT FNIP1 and SA5 FNIP1 HEK293T cells subjected to a 0- to 30-hour 991 time course. All data are shown as means ± SEMs. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; unpaired t test. (Q) Model. AMPK phosphorylation of FNIP1 after energy stress or 991 facilitates TFEB nuclear entry where it binds to CLEAR network gene promoters, including the PPARGC1A promoter, which induces expression of the short ~35-kDa transcriptional coactivator NT-PGC1α isoform. In turn NT-PGC1α transactivates the ERRα transcription factor for induction of mitochondrial genes.

Article Snippet: Confocal imaging of TFEB, TFE3, and RagC For visualization of TFEB, TFE3, and RagC, the following primary rabbit antibodies were used: TFEB (CST no. 37785), TFE3 (CST no.14779), and RagC (CST no. 9480) followed by anti-rabbit secondary antibodies conjugated to Alexa Fluor (AF) 594.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, RNA Sequencing, Transfection, Control, Knockdown, Mutagenesis, CRISPR, Phospho-proteomics

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: Aging (Albany NY)

Article Title: PERK activation by SB202190 ameliorates amyloidogenesis via the TFEB-induced autophagy-lysosomal pathway

doi: 10.18632/aging.203899

Figure Lengend Snippet: SB202190 activates the PERK/eIF2α/ATF4 pathway. ( A ) HEK293 cells were treated with SB202190 (0, 5, 10, and 20 μM) for 6 h. PERK phosphorylation was determined by western blotting. Quantification of p-PERK is shown in the right panel. ( B ) HEK293 cells were incubated with SB202190 (10 and 20 μM) for 6 h after pretreatment with or without the PERK inhibitor, GSK2606414 (1 μM) for 1 h. Thapsigargin (Tg, 2 μM) was used as a positive control. Cell lysates were used for western blotting analysis for p-PERK, PERK, p-eIF2α, eIF2α, and ATF4. ( C ) For knockdown of Perk , SH-SY5Y cells were transfected with control siRNA (scRNA) or si Perk for 48 h and then treated with different doses (10 and 20 μM) of SB202190 (SB) or Tg (2 μM) for 6 h. Cell lysates were measured for PERK activation by western blot using the indicated antibodies. ( D ) Perk +/+ and Perk -/- MEFs were treated with SB202190 (10 and 20 μM) for 6 h or Tg (2 μM). The levels of p-PERK, PERK, p-eIF2α, eIF2α, and ATF4 were measured by western blotting. ( E ) SH-SY5Y cells were treated with SB202190 (10 and 20 μM) or Tg (2 μM) for 6 h. Xbp-1 splicing and Grp78 expression were detected by RT-PCR. ( F , G ) Hepatocytes isolated from Ire1 α +/+ , Ire1 α -/- ( F ) or Atf6 α +/+ , and Atf6 α -/- ( G ) mice were treated with SB202190 (10 and 20 μM) for 6 h to assess the levels of PERK phosphorylation and ATF4 expression by western blotting. Quantification of p-PERK is shown in the right panel ( A – D , F , G ). Data are mean ± SD ( n =3); * p <0.05, ** p <0.01, and *** p <0.001.

Article Snippet: To knockdown the genes of p38 , Perk and Tfeb , SH-SY5Y cells were transfected with scramble siRNA (scRNA) (Ambion, Austin, TX, USA), as the control siRNA, p38 (Cell Signaling), Perk and Tfeb siRNA (Santa Cruz Biotechnology, CA, USA) using LipofectamineTM 2000 (Invitrogen) method according to the manufacturer’s protocol.

Techniques: Phospho-proteomics, Western Blot, Incubation, Positive Control, Knockdown, Transfection, Control, Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation

Journal: Aging (Albany NY)

Article Title: PERK activation by SB202190 ameliorates amyloidogenesis via the TFEB-induced autophagy-lysosomal pathway

doi: 10.18632/aging.203899

Figure Lengend Snippet: PERK is required for TFEB nuclear translocation by SB202190. ( A , B ) SH-SY5Y cells were treated with SB202190 at the indicated concentrations (5, 10, and 20 μM) for 4 h and subjected to nuclear and cytosolic fractionation. ( A ) Resulting fractions were then detected with antibody against TFEB. Starvation (STV) was used as positive control. PARP and α-tubulin were used as nuclear and cytosolic markers, respectively. Quantification of TFEB translocation is shown at the right. Data are mean ± SD ( n =3), * p <0.05; ** p <0.01; *** p <0.001. ( B ) The translocation of TFE3 was analyzed by western blotting. ( C ) The human microglial HMC3 cells were treated with SB202190 (0, 5, 10, and 20 μM) for 6 h. Torin-1 (2μM) treatment was used as a positive control. The fractionated HMC3 cells were evaluated for TFEB translocation by western blotting (left). Quantification of TFEB translocation was analyzed (right). Data are mean ± SD ( n =3), *** p <0.001. ( D ) TFEB-EGFP-transfected SH-SY5Y cells were treated with 20 μM SB202190 in the presence or absence of the PERK inhibitor (GSK2606414, GSK). Torin-1, an mTOR inhibitor, was used as a positive control. The fluorescence of TFEB was visualized by confocal microscopy (left). Scale bar: 10 μm. Cells were evaluated to calculate the percentage of cells showing nuclear TFEB localization. n > 20 cells per condition (right). Data are mean ± SD; ** p <0.01 and *** p <0.001. ( E ) Perk +/+ and Perk -/- MEFs were treated with SB202190 (10 and 20 μM) for 6 h or Tg (2 μM). Torin-1 (2μM) treatment was used as a positive control. Cells were detected with TFEB antibody in the nuclear and cytosol fraction by western blotting (left). Quantification of TFEB translocation was analyzed (right). Data are mean ± SD ( n =3), * p <0.05; ** p <0.01; *** p <0.001; NS, not significant.

Article Snippet: To knockdown the genes of p38 , Perk and Tfeb , SH-SY5Y cells were transfected with scramble siRNA (scRNA) (Ambion, Austin, TX, USA), as the control siRNA, p38 (Cell Signaling), Perk and Tfeb siRNA (Santa Cruz Biotechnology, CA, USA) using LipofectamineTM 2000 (Invitrogen) method according to the manufacturer’s protocol.

Techniques: Translocation Assay, Fractionation, Positive Control, Western Blot, Transfection, Fluorescence, Confocal Microscopy

Journal: Aging (Albany NY)

Article Title: PERK activation by SB202190 ameliorates amyloidogenesis via the TFEB-induced autophagy-lysosomal pathway

doi: 10.18632/aging.203899

Figure Lengend Snippet: PERK-Ca 2+ -calcineurin pathway is required for SB202190-induced TFEB nuclear translocation. ( A – C ) The change of [Ca 2+ ] in MEF cells was measured using confocal microscopy after loading with Fluo-4 AM. ( A ) Arrow indicates the time point at which SB202190 (SB) was added. The data represent mean ± SD from 3 independent experiments. ( B , C ) MEF cells were preincubated for 30 min with the PERK inhibitor GSK2606414 (1 μM). Arrow indicates the time point at which 20 μM SB202190 was added. The data represent mean ± SD from 3 independent experiments. ( D ) SH-SY5Y cells were pretreated with FK506 (10 μM) for 30 min and then treated with SB202190 (20 μM) for another 3 h. Measurement of TFEB activation was performed by western blotting of nuclear and cytoplasmic extracts. TFEB expression in the nucleus and cytoplasm was normalized to PARP and α-tubulin, respectively (left). Quantification of TFEB translocation is shown in the right panel. Data represent mean ± SD, ** p <0.01; *** p <0.001. ( E ) TFEB-GFP-transfected SH-SY5Y cells were treated with SB202190 (20 μM) for 6 h in the presence or absence of calcineurin inhibitors, FK506 (10 μM) and Cyclosporin A (CsA, 20μM). Representative images were detected by confocal microscopy (left). Quantification of nuclear translocation of TFEB-GFP (right). n > 20 cells per condition. Data represent mean ± SD; ** p <0.01 and *** p <0.001.

Article Snippet: To knockdown the genes of p38 , Perk and Tfeb , SH-SY5Y cells were transfected with scramble siRNA (scRNA) (Ambion, Austin, TX, USA), as the control siRNA, p38 (Cell Signaling), Perk and Tfeb siRNA (Santa Cruz Biotechnology, CA, USA) using LipofectamineTM 2000 (Invitrogen) method according to the manufacturer’s protocol.

Techniques: Translocation Assay, Confocal Microscopy, Activation Assay, Western Blot, Expressing, Transfection

Journal: Aging (Albany NY)

Article Title: PERK activation by SB202190 ameliorates amyloidogenesis via the TFEB-induced autophagy-lysosomal pathway

doi: 10.18632/aging.203899

Figure Lengend Snippet: PERK activation by SB202190 facilitates autophagy and lysosome biogenesis via TFEB activation. ( A ) SH-SY5Y cells were treated for 6 h with SB202190 (5, 10, and 20 μM) and subjected to western blotting by antibodies against p62 and LC3B. ( B ) SH-SY5Y cells were transiently transfected with mCherry-GFP-LC3 for 48 h and subsequently pretreated with chloroquine (CQ, 10 μM) for 1 h, and then treated with SB (20 μM) or Torin1 (2 μM) for 6 h. Cells were observed for fluorescence of both GFP and mCherry using confocal microscopy. The number of autolysosomes (GFP - RFP + ) and autophagosomes (GFP + RFP + ) per cell in each condition were quantified. Data represent mean ± SD; *** p <0.001. ( C ) SH-SY5Y cells were treated with CQ (10 μM) for 1 h before SB202190 (20 μM) for 6 h. The levels of LC3B-II conversion were analyzed by western blotting. ( D ) To check of PERK dependence in ALP-related genes, Perk +/+ and Perk -/- MEFs were treated with SB202190 (20 μM) for 6 h. Lysosomal genes (LAMP1, MCOLN1, TPP1, and CTSD) and autophagy gene (p62) measured by qRT-PCR. Data represent mean ± SD; *** p <0.001, NS, not significant. ( E – H ) SH-SY5Y cells were transfected with siTfeb for 48 h and then treated with SB202190 (20 μM) for 6 h. ( E ) Cells were subjected to western blotting by using antibodies against TFEB upper, LAMP1, p62, and LC3B lower. ( F ) lysosomal genes, LAMP1, MCOLN1, and TPP1, were measured by qRT-PCR. Data are represented as mean ± SD; * p <0.05, *** p <0.001, NS, not significant. ( G ) Samples were stained with Lysotracker Red. Representative image was obtained by confocal microscopy (left). Scale bar, 10 μm. Quantification of lysosome intensity was determined by counting red puncta (r ight ). Data represent mean ± SD; *** p <0.001, NS, not significant. ( H ) LysoTracker fluorescence was measured by flow cytometry. Fold changes in LysoTracker intensity are indicated at the right and are presented as mean ± SD (n=3); ** p <0.01, NS, not significant.

Article Snippet: To knockdown the genes of p38 , Perk and Tfeb , SH-SY5Y cells were transfected with scramble siRNA (scRNA) (Ambion, Austin, TX, USA), as the control siRNA, p38 (Cell Signaling), Perk and Tfeb siRNA (Santa Cruz Biotechnology, CA, USA) using LipofectamineTM 2000 (Invitrogen) method according to the manufacturer’s protocol.

Techniques: Activation Assay, Western Blot, Transfection, Fluorescence, Confocal Microscopy, Quantitative RT-PCR, Staining, Flow Cytometry

Journal: Aging (Albany NY)

Article Title: PERK activation by SB202190 ameliorates amyloidogenesis via the TFEB-induced autophagy-lysosomal pathway

doi: 10.18632/aging.203899

Figure Lengend Snippet: PERK activation by SB202190 reduces the aggregation of APP accumulation through TFEB-ALP activation in SH-SY5Y cells. ( A , B ) SH-SY5Y cells were transfected with pCAX-APP-Swe/Ind (APP swe/ind ) for 48 h. Under this condition, cells were treated with the PERK inhibitor, GSK2606414 (1 μM, 1 h), before SB202190 (20 μM, 12 h) treatment. ( A ) Cells were stained with DAPI and immunostained with anti-FL-APP antibody. Representative image of FL-APP was observed by confocal microscopy (left) and quantification of FL-APP intensity ( right ). Data represent mean ± SD; *** p <0.001. ( B ) Western blotting was performed to detect the expression levels of FL-APP. Quantification of FL-APP expression was shown in the upper panel. ( C ) The levels of intracellular Aβ and secreted Aβ were detected by western blotting. ( D , E ) The cells were subjected to western blotting by antibodies against α/β-CTF ( D ) and LC3B ( E ). Quantification of α/β-CTF and LC3B-II conversion was analyzed upper. Data are mean ± SD ( n =3), ** p <0.01; *** p <0.001. ( F , G ) HMC3 cells were transfected with pCAX-APP-Swe/Ind (APP swe/ind ) for 48 h and then treated with SB202190 (20 μM, 12 h) in the presence or absence of PERK inhibitor, GSK2606414 (1 μM, 1 h). ( F ) Cells were detected to antibodies against APP and LC3B by western blotting. Quantification of FL-APP and LC3B-II conversion was analyzed. Data are mean ± SD ( n =3), ** p <0.01; *** p <0.001. ( G ) Lysosomal genes, LAMP1, MCOLN1, TPP1, CTSB, and CTSD were measured by qRT-PCR. Data are represented as mean ± SD, *** p <0.001. ( H , I ) SH-SY5Y cells were co-transfected with pCAX-APP-Swe/Ind (APP swe/ind ) and si Tfeb for 48 h and were treated with SB202190 (20 μM, 12 h). ( H ) Cells were detected to antibody against FL-APP by western blotting. ( I ) Cells were stained with antibody against FL-APP and DAPI. Representative image of FL-APP was obtained by confocal microscopy (left) and quantification of FL-APP intensity (right). Data represent as mean ± SD; *** p <0.001 and not significant (NS).

Article Snippet: To knockdown the genes of p38 , Perk and Tfeb , SH-SY5Y cells were transfected with scramble siRNA (scRNA) (Ambion, Austin, TX, USA), as the control siRNA, p38 (Cell Signaling), Perk and Tfeb siRNA (Santa Cruz Biotechnology, CA, USA) using LipofectamineTM 2000 (Invitrogen) method according to the manufacturer’s protocol.

Techniques: Activation Assay, Transfection, Staining, Confocal Microscopy, Western Blot, Expressing, Quantitative RT-PCR

Journal: Aging (Albany NY)

Article Title: PERK activation by SB202190 ameliorates amyloidogenesis via the TFEB-induced autophagy-lysosomal pathway

doi: 10.18632/aging.203899

Figure Lengend Snippet: Schematic overview of the mechanisms by which SB202190 ameliorates amyloidogenesis via PERK activation. PERK, a tethering molecule of the mitochondrial-associated ER membrane (MAM), is activated by mitochondrial ROS (mtROS) in response to treatment with the PERK activator (SB202190). Activated PERK leads to increase in cytosolic Ca 2+ levels and subsequently promotes the translocation of TEFB into the nucleus via the calcineurin-dependent dephosphorylation TFEB, which culminates in the increased transcription of autophagy-lysosome related genes. The increase of autophagy-lysosomal pathway (ALP) by PERK activation enhances the degradation of misfolded proteins that accumulate in neurodegenerative disorders. Therefore, the PERK-TFEB-ALP pathway activated by SB202190 suggests the novel target for ameliorating amyloidogenesis.

Article Snippet: To knockdown the genes of p38 , Perk and Tfeb , SH-SY5Y cells were transfected with scramble siRNA (scRNA) (Ambion, Austin, TX, USA), as the control siRNA, p38 (Cell Signaling), Perk and Tfeb siRNA (Santa Cruz Biotechnology, CA, USA) using LipofectamineTM 2000 (Invitrogen) method according to the manufacturer’s protocol.

Techniques: Activation Assay, Membrane, Translocation Assay, De-Phosphorylation Assay