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: Cell Signaling Technology (CST) antibodies used in the study were as follows: TFEB (Cat# 4240), monoclonal TFEB (D2O7D, Cat# 37785), monoclonal Phospho-TFEB S122 (Cat# 86843), Tfe3 (Cat# 14779), FNIP1 (Cat# 36892), Phospho-FNIP1 S220 (Cat# 40812, in development), FLCN (Cat# 3697 (D14G9), RagA (D8B5, Cat# 4357), RagB (D18F3 Cat# 8150), RagC (D8H5, Cat# 9480), IDH2 (D8E3B, Cat# 56439), PDHA1 (C54G1, Cat# 3205), Tricarboxylic Acid Cycle Antibody Sampler Kit (Cat# 47767), monoclonal anti-LAMP2 (H4B4, Abcam Cat# ab25631), monoclonal anti-LAMP1 (D2D11, Cat# 9091), ERRα (Cat# 13826), AMPKα (Cat# 2532), Phospho AMPKα T172 (40H9, Cat# 2535), ACC (Cat# 3662), Phospho-ACC S79 (Cat# 3661), Raptor (Cat# 2280), Phospho-Raptor S792 (Cat# 2083), anti-Ulk1 (D8H5 Cat# 8054), Phospho-S6K T389 (Cat# 9205), 4EBP1(Cat# 9452), S6 (5G10, Cat# 2217), Phospho-S6 S235/236 (Cat# 4858), mTOR (Cat# 2972), LAMTOR1/C11orf59 (D11H6, Cat# 8975), monoclonal Hdac3 (7G6C5 Cat# 3949), Golgin-97 (D8P2K Cat# 13192), monocloncal GAPDH (D16H11, Cat# 8884), GFP (D5.1, Cat# 2956), and DYKDDDDK (FLAG) tag (Cat# 2368).

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: Cell Signaling Technology (CST) antibodies used in the study were as follows: TFEB (Cat# 4240), monoclonal TFEB (D2O7D, Cat# 37785), monoclonal Phospho-TFEB S122 (Cat# 86843), Tfe3 (Cat# 14779), FNIP1 (Cat# 36892), Phospho-FNIP1 S220 (Cat# 40812, in development), FLCN (Cat# 3697 (D14G9), RagA (D8B5, Cat# 4357), RagB (D18F3 Cat# 8150), RagC (D8H5, Cat# 9480), IDH2 (D8E3B, Cat# 56439), PDHA1 (C54G1, Cat# 3205), Tricarboxylic Acid Cycle Antibody Sampler Kit (Cat# 47767), monoclonal anti-LAMP2 (H4B4, Abcam Cat# ab25631), monoclonal anti-LAMP1 (D2D11, Cat# 9091), ERRα (Cat# 13826), AMPKα (Cat# 2532), Phospho AMPKα T172 (40H9, Cat# 2535), ACC (Cat# 3662), Phospho-ACC S79 (Cat# 3661), Raptor (Cat# 2280), Phospho-Raptor S792 (Cat# 2083), anti-Ulk1 (D8H5 Cat# 8054), Phospho-S6K T389 (Cat# 9205), 4EBP1(Cat# 9452), S6 (5G10, Cat# 2217), Phospho-S6 S235/236 (Cat# 4858), mTOR (Cat# 2972), LAMTOR1/C11orf59 (D11H6, Cat# 8975), monoclonal Hdac3 (7G6C5 Cat# 3949), Golgin-97 (D8P2K Cat# 13192), monocloncal GAPDH (D16H11, Cat# 8884), GFP (D5.1, Cat# 2956), and DYKDDDDK (FLAG) tag (Cat# 2368).

Techniques: RNA Sequencing Assay, CRISPR, Activation Assay, Expressing, ChIP-sequencing, 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: Cell Signaling Technology (CST) antibodies used in the study were as follows: TFEB (Cat# 4240), monoclonal TFEB (D2O7D, Cat# 37785), monoclonal Phospho-TFEB S122 (Cat# 86843), Tfe3 (Cat# 14779), FNIP1 (Cat# 36892), Phospho-FNIP1 S220 (Cat# 40812, in development), FLCN (Cat# 3697 (D14G9), RagA (D8B5, Cat# 4357), RagB (D18F3 Cat# 8150), RagC (D8H5, Cat# 9480), IDH2 (D8E3B, Cat# 56439), PDHA1 (C54G1, Cat# 3205), Tricarboxylic Acid Cycle Antibody Sampler Kit (Cat# 47767), monoclonal anti-LAMP2 (H4B4, Abcam Cat# ab25631), monoclonal anti-LAMP1 (D2D11, Cat# 9091), ERRα (Cat# 13826), AMPKα (Cat# 2532), Phospho AMPKα T172 (40H9, Cat# 2535), ACC (Cat# 3662), Phospho-ACC S79 (Cat# 3661), Raptor (Cat# 2280), Phospho-Raptor S792 (Cat# 2083), anti-Ulk1 (D8H5 Cat# 8054), Phospho-S6K T389 (Cat# 9205), 4EBP1(Cat# 9452), S6 (5G10, Cat# 2217), Phospho-S6 S235/236 (Cat# 4858), mTOR (Cat# 2972), LAMTOR1/C11orf59 (D11H6, Cat# 8975), monoclonal Hdac3 (7G6C5 Cat# 3949), Golgin-97 (D8P2K Cat# 13192), monocloncal GAPDH (D16H11, Cat# 8884), GFP (D5.1, Cat# 2956), and DYKDDDDK (FLAG) tag (Cat# 2368).

Techniques: 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: Cell Signaling Technology (CST) antibodies used in the study were as follows: TFEB (Cat# 4240), monoclonal TFEB (D2O7D, Cat# 37785), monoclonal Phospho-TFEB S122 (Cat# 86843), Tfe3 (Cat# 14779), FNIP1 (Cat# 36892), Phospho-FNIP1 S220 (Cat# 40812, in development), FLCN (Cat# 3697 (D14G9), RagA (D8B5, Cat# 4357), RagB (D18F3 Cat# 8150), RagC (D8H5, Cat# 9480), IDH2 (D8E3B, Cat# 56439), PDHA1 (C54G1, Cat# 3205), Tricarboxylic Acid Cycle Antibody Sampler Kit (Cat# 47767), monoclonal anti-LAMP2 (H4B4, Abcam Cat# ab25631), monoclonal anti-LAMP1 (D2D11, Cat# 9091), ERRα (Cat# 13826), AMPKα (Cat# 2532), Phospho AMPKα T172 (40H9, Cat# 2535), ACC (Cat# 3662), Phospho-ACC S79 (Cat# 3661), Raptor (Cat# 2280), Phospho-Raptor S792 (Cat# 2083), anti-Ulk1 (D8H5 Cat# 8054), Phospho-S6K T389 (Cat# 9205), 4EBP1(Cat# 9452), S6 (5G10, Cat# 2217), Phospho-S6 S235/236 (Cat# 4858), mTOR (Cat# 2972), LAMTOR1/C11orf59 (D11H6, Cat# 8975), monoclonal Hdac3 (7G6C5 Cat# 3949), Golgin-97 (D8P2K Cat# 13192), monocloncal GAPDH (D16H11, Cat# 8884), GFP (D5.1, Cat# 2956), and DYKDDDDK (FLAG) tag (Cat# 2368).

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