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  • 99
    ATCC hek 293t cells
    Hek 293t Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 5177 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hek293t cells
    Interactions of FIGLA, LHX8 and SOHLH1. ( A ) Representative protein domains (bHLH, LIM, homeobox) of FIGLA, SOHLH1 and LHX8. ( B ) FIGLA HA and LHX8 FLAG expression vectors were co-transfected into <t>HEK-293T</t> cells. Cell lysates were probed with HA and FLAG antibodies to detect input protein FIGLA and LHX8, respectively. After immunoprecipitation with HA and FLAG antibody, immunoblots were performed to detect FIGLA and associated LHX8 protein or LHX8 and associated FIGLA protein, respectively. ( C ) Same as (B) except that FIGLA HA and SOHLH1 MYC were co-transfected into HEK-293T cells. Antibodies to HA and MYC were used to detect input proteins and immunoprecipitate FIGLA and SOHLH1, respectively, and their associated proteins. ( D ) Same as (B) except that LHX8 FLAG and SOHLH1 MYC were co-transfected into HEK-293T cells. Antibodies to FLAG and MYC were used to detect input proteins and immunoprecipitate LHX8 and SOHLH1, respectively, and their associated proteins. ( E ) Co-expression of FIGLA, LHX8 and SOHLH1 in P0 ovaries from Figla FLAG mice. The dashed circles indicate co-expression of FIGLA, LHX8 and SOHLH1 in the same oocytes. Scale bar, 20 μm. ( F ) FIGLA, LHX8 and SOHLH1 appear to form a nuclear complex in oocytes. Representative of n = 3 (B–E) independent biological replicates with similar results per condition.
    Hek293t Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 35647 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 293t cells
    Interactions of FIGLA, LHX8 and SOHLH1. ( A ) Representative protein domains (bHLH, LIM, homeobox) of FIGLA, SOHLH1 and LHX8. ( B ) FIGLA HA and LHX8 FLAG expression vectors were co-transfected into <t>HEK-293T</t> cells. Cell lysates were probed with HA and FLAG antibodies to detect input protein FIGLA and LHX8, respectively. After immunoprecipitation with HA and FLAG antibody, immunoblots were performed to detect FIGLA and associated LHX8 protein or LHX8 and associated FIGLA protein, respectively. ( C ) Same as (B) except that FIGLA HA and SOHLH1 MYC were co-transfected into HEK-293T cells. Antibodies to HA and MYC were used to detect input proteins and immunoprecipitate FIGLA and SOHLH1, respectively, and their associated proteins. ( D ) Same as (B) except that LHX8 FLAG and SOHLH1 MYC were co-transfected into HEK-293T cells. Antibodies to FLAG and MYC were used to detect input proteins and immunoprecipitate LHX8 and SOHLH1, respectively, and their associated proteins. ( E ) Co-expression of FIGLA, LHX8 and SOHLH1 in P0 ovaries from Figla FLAG mice. The dashed circles indicate co-expression of FIGLA, LHX8 and SOHLH1 in the same oocytes. Scale bar, 20 μm. ( F ) FIGLA, LHX8 and SOHLH1 appear to form a nuclear complex in oocytes. Representative of n = 3 (B–E) independent biological replicates with similar results per condition.
    293t Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 37214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC hek293t cells
    Interactions of FIGLA, LHX8 and SOHLH1. ( A ) Representative protein domains (bHLH, LIM, homeobox) of FIGLA, SOHLH1 and LHX8. ( B ) FIGLA HA and LHX8 FLAG expression vectors were co-transfected into <t>HEK-293T</t> cells. Cell lysates were probed with HA and FLAG antibodies to detect input protein FIGLA and LHX8, respectively. After immunoprecipitation with HA and FLAG antibody, immunoblots were performed to detect FIGLA and associated LHX8 protein or LHX8 and associated FIGLA protein, respectively. ( C ) Same as (B) except that FIGLA HA and SOHLH1 MYC were co-transfected into HEK-293T cells. Antibodies to HA and MYC were used to detect input proteins and immunoprecipitate FIGLA and SOHLH1, respectively, and their associated proteins. ( D ) Same as (B) except that LHX8 FLAG and SOHLH1 MYC were co-transfected into HEK-293T cells. Antibodies to FLAG and MYC were used to detect input proteins and immunoprecipitate LHX8 and SOHLH1, respectively, and their associated proteins. ( E ) Co-expression of FIGLA, LHX8 and SOHLH1 in P0 ovaries from Figla FLAG mice. The dashed circles indicate co-expression of FIGLA, LHX8 and SOHLH1 in the same oocytes. Scale bar, 20 μm. ( F ) FIGLA, LHX8 and SOHLH1 appear to form a nuclear complex in oocytes. Representative of n = 3 (B–E) independent biological replicates with similar results per condition.
    Hek293t Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 13948 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Addgene inc hek293t cells
    FRMD8 stabilises endogenous iRhom2. ( A, B ) Levels of endogenously 3xHA tagged iRhom2 were analysed in <t>HEK293T-iRhom2-3xHA</t> cells transfected with FRMD8-V5 plasmid, siRNAs targeting iRhom2, non-targeting siRNA control pool (c trl) or FRMD8 SMARTpool siRNA. Cell lysates were anti-HA immunoprecipitated (HA-IP) to detect endogenous iRhom2-3xHA levels and immunoblotted using anti-HA antibody. Cell lysates were immunoblotted for ADAM17, V5, and actin. ( C ) FRMD8 and iRhom2 mRNA levels relative to actin mRNA levels were determined by TaqMan PCR in cells used for the experiment shown in ( B ) to demonstrate that the destabilisation of endogenous iRhom2 was not induced by a change in iRhom2 mRNA levels.
    Hek293t Cells, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 4756 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Mirus Bio hek293t cells
    Hsp70 interacts with PB2, PB1 monomers, and their dimers, but not with PB2/PB1/PA heterotrimer. A and B , effects of addition of HA and FLAG tags on the interaction of Hsp70 with PB2 of HK483 influenza virus. <t>HEK293T</t> cells were transfected with indicated
    Hek293t Cells, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 92/100, based on 1871 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher geneblazer cckbr nfat bla hek293t cells
    Hsp70 interacts with PB2, PB1 monomers, and their dimers, but not with PB2/PB1/PA heterotrimer. A and B , effects of addition of HA and FLAG tags on the interaction of Hsp70 with PB2 of HK483 influenza virus. <t>HEK293T</t> cells were transfected with indicated
    Geneblazer Cckbr Nfat Bla Hek293t Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC hek293t 17 cells
    Tat interacts with CypA and DHHC-20.  a  HEK 293 T cells were transfected with an empty vector or Tat-FLAG (WT, 31 S, or 11Y). Cells were lysed 48 h after transfection before anti-FLAG immunoprecipitation and western blots against CypA, DHHC-5 and DHHC-20.  b  Cells were transfected with an empty (pCi) or Tat vector. GST or GST-CypA was added to cell extracts for GST pull-down before western blots. The graph shows the quantification of the DHHC pulled-down/input intensity ratio, setting the empty vector ratio to 100%. Representative data (mean ± SEM,  n  = 3 independent experiments) are shown.*** p
    Hek293t 17 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 975 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Polyplus Transfection hek293t cells
    The VEDEC motif is sufficient to prevent Slo1 from being expressed on the cell surface. <t>HEK293T</t> cells were transiently cotransfected with Myc-tagged Short-QEERL and HA-tagged Short-VEDEC. The amounts of plasmids used are indicated (in micrograms). A, results from representative cell-surface biotinylation assays as well as analyses of total expression of the HA and Myc tags as indicated. Note that total expression of each splice variant is closely related to the amount of each plasmid used in transfection. B, quantification (mean ± S.E.M.) of densitometric analyses of three repetitions of this experiment. Note the reduction of surface expression of Slo1 when even small amounts of Short-VEDEC are present.
    Hek293t Cells, supplied by Polyplus Transfection, used in various techniques. Bioz Stars score: 92/100, based on 1097 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hek293t
    The VEDEC motif is sufficient to prevent Slo1 from being expressed on the cell surface. <t>HEK293T</t> cells were transiently cotransfected with Myc-tagged Short-QEERL and HA-tagged Short-VEDEC. The amounts of plasmids used are indicated (in micrograms). A, results from representative cell-surface biotinylation assays as well as analyses of total expression of the HA and Myc tags as indicated. Note that total expression of each splice variant is closely related to the amount of each plasmid used in transfection. B, quantification (mean ± S.E.M.) of densitometric analyses of three repetitions of this experiment. Note the reduction of surface expression of Slo1 when even small amounts of Short-VEDEC are present.
    Hek293t, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5734 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Corning Life Sciences hek293t cells
    Schematic of SILAC-based proteomic mapping of KEAP1 modifications in response to CBR-470-1 and NMR characterization of CR-MGx peptide. a, Stable isotope-labeled cells (stable isotope labeling with amino acids in cell culture, SILAC) expressing FLAG-tagged KEAP1 were treated with vehicle (‘light’) and CBR-470-1 or MGx (‘heavy’), respectively. Subsequent mixing of the cell lysates, anti-FLAG enrichment, tryptic digestion and LC-MS/MS analysis permitted detection of unmodified portions of KEAP1, which retained ∼1:1 SILAC ratios relative to the median ratios for all detected KEAP1 peptides. In contrast, peptides that are modified under one condition will no longer match tryptic MS/MS searches, resulting skewed SILAC ratios that “drop out” (bottom). b, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched DMSO treated ‘light’ cells and CBR-470-1 treated ‘heavy’ cells, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 3- to 4-fold upon relative to the KEAP1 median, indicative of structural modification ( n =8). c, Structural depiction of potentially modified stretches of human KEAP1 (red) using published x-ray crystal structure of the BTB (PDB: 4CXI) and KELCH (PDB: 1U6D) domains. Intervening protein stretches are depicted as unstructured loops in green. d, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched MGx treated ‘heavy’ cell lysates and no treated ‘light’ cell lysates, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 2- to 2.5- fold upon relative to the KEAP1 median, indicative of structural modification ( n =12). e, Representative Western blotting analysis of FLAG-KEAP1 dimerization from <t>HEK293T</t> cells pre-treated with Bardoxolone methyl followed by CBR-470-1 treatment for 4 hours ( n =3). f, 1 H-NMR of CR-MGx peptide (isolated product of MGx incubated with Ac-NH-VVCGGGRGG-C(O)NH 2 peptide). 1 H NMR (500MHz, d6-DMSO) δ 12.17 (s, 1H), 12.02 (s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 8.32-8.29 (m, 2H), 8.23 (t, J = 5.6 Hz, 1H), 8.14 (t, J = 5.9 Hz, 1H), 8.05 (t, J = 5.9 Hz, 1H), 8.01 (t, J = 5.9 Hz, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.26 (s, 1H), 7.09 (s, 1H), 4.33-4.28 (m, 1H), 4.25-4.16 (m, 3H), 3.83 (dd, J = 6.9 Hz, J = 16.2 Hz, 1H), 3.79-3.67 (m, 6H), 3.63 (d, J = 5.7 Hz, 2H), 3.54 (dd, J = 4.9 Hz, J = 16.2 Hz, 1H), 3.18-3.13 (m, 2H), 3.04 (dd, J = 4.9 Hz, J = 13.9 Hz, 1H), 2.88 (dd, J = 8.6 Hz, J = 13.6 Hz, 1H), 2.04 (s, 3H), 1.96 (sep, J = 6.8 Hz, 2H), 1.87 (s, 3H), 1.80-1.75 (m, 1H), 1.56-1.47 (m, 3H), .87-.82 (m, 12H). g, 1 H-NMR of CR peptide (Ac-NH-VVCGGGRGG-C(O)NH 2 ). 1 H NMR (500MHz, d6-DMSO) δ 8.27-8.24 (m, 2H), 8.18 (t, J = 5.7 Hz, 1H), 8.13-8.08 (m, 3H), 8.04 (t, J = 5.7 Hz, 1H), 7.91 (d, J = 8.8 Hz), 7.86 (d, J = 8.8 Hz, 1H), 7.43 (t, J = 5.4 Hz, 1H), 7.28 (s, 1H), 7.10 (s, 1H), 4.39 (dt, J = 5.6 Hz, J = 7.4 Hz, 1H), 4.28 (dt, J = 5.7 Hz, J = 7.2 Hz, 1H), 4.21-4.13 (m, 2H), 3.82-3.70 (m, 8H), 3.64 (d, J = 5.8, 2H), 3.08 (dt, J = 6.5 Hz, J = 6.5 Hz, 2H), 2.80-2.67 (m, 2H), 2.43 (t, J = 8.6 Hz, 1H), 1.94 (sep, J = 6.8 Hz, 2H), 1.85 (s, 3H), 1.75-1.68 (m, 1H), 1.54-1.42 (m, 3H), .85-.81 (m, 12H) h, 1 H- 1 H TOCSY of CR-MGx peptide. i, Peak assignment for CR-MGx peptide TOCSY spectrum. Data are mean ± SEM of biologically independent samples.
    Hek293t Cells, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 92/100, based on 850 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson hek293t cells
    Effect of nucleotide substitution on RNA replication and translational capacity mediated by CA16 5′UTR. ( A ) Construction of reporter plasmid to determine translational activity mediated by CA16 5′UTR. ( B ) Effect of base 104 of 5′UTR on RNA replication in cells. WT or M2 vector expressing luciferase were transfected into <t>HEK293T</t> cells, and cells were harvested at 12 h, 24 h, 36 h and 48 h after transfection. RNA was extracted from a portion of each cell sample and analyzed by RT-qPCR with primers specific for GAPDH RNA or CA16 5′UTR RNA. GAPDH was used as a control. The RNA level obtained from transfection with WT 5′UTR was normalized to 100%. ( C ) Effect of base 104 of 5′UTR on translational activity in cells. Luciferase activiy was detected in a portion of each cell sample.and the viral replication rate was expressed as a fold increase in luciferase activity. * P
    Hek293t Cells, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 999 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SignaGen hek293t cells
    ALKBH5 demethylates m 6 A but not m 6 A m in mRNA in <t>HEK293T</t> cells a , ALKBH5 expression does not decrease m 6 A m in HEK293T cells. The relative abundance of modified adenosines in mRNA caps of HEK293T cells expressing GST vector (Ctrl) or ALKBH5 with an N-terminal GST tag (GST–ALKBH5) was determined by 2D TLC. When determining the ratio of m 6 A m to A m , we did not observe a significant decrease of m 6 A m in ALKBH5-overexpressing cells, indicating that ALKBH5 does not convert m 6 A m to A m in vivo (representative images show n; n = 3 biologic al replic ates; me an ± s.e.m.). b , ALKBH5 knockdown does not increase m 6 A m in HEK293T cells. The relative abundance of modified adenosines in mRNA caps of HEK293T cells transfected with scrambled siRNA (siCtrl) or siRNA directed against ALKBH5 (siALKBH5) was determined by 2D TLC. When determining the ratio of m 6 A m to A m , we did not observe a significant increase of m 6 A m in ALKBH5-expressing cells, indicating that ALKBH5 does not convert m 6 A m to A m in vivo (repres entative images shown; n = 3 biological replicates; mean ± s.e.m.). c , ALKBH5 knockdown increases m 6 A in HEK293T cells. The relative abundance of m 6 A versus (A + C + U) in mRNA of HEK293T cells transfected with scrambled siRNA (siCtrl) or siRNA directed against ALKBH5 (siALKBH5) was determined by 2D TLC. We observed an approximately 30% increase of m 6 A upon ALKBH5 knockdown, indicating that ALKBH5 readily influences the levels of m 6 A in vivo (representative images shown; n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t -test, * P ≤ 0.05). d , ALKBH5 expression decreases m 6 A in HEK293T cells. The relative abundance of m 6 A versus (A + C + U) in mRNA of HEK293T cells expressing GST vector (Ctrl) or ALKBH5 with an N-terminal GST tag (GST-ALKBH5) was determined by 2D TLC. We observed a significant decrease of m 6 A upon ALKBH5 expression, indicating that SLKBH5 readily influences levels of m 6 A in vivo (representative images shown; n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t -test, ** P ≤ 0.01).
    Hek293t Cells, supplied by SignaGen, used in various techniques. Bioz Stars score: 93/100, based on 613 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alexa Fluor 594 anti SATB1 O96C6 Isotype Mouse IgG1 Reactivity Human Apps IF Size 100 μg
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    Purified anti CD36L1 SCARB1 SR BI O91E1 Isotype Mouse IgG2b Reactivity Human Apps WB Size 100 μg
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    Mouse monoclonal antibody to MDM4 Isotype Note IgG2b Host Note Mouse Reactivity Note Human Application Note WB IHC P IF ICC
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    Purified anti SATB1 O96C6 Isotype Mouse IgG1 Reactivity Human Apps WB IF Size 100 μg
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    Image Search Results


    Interactions of FIGLA, LHX8 and SOHLH1. ( A ) Representative protein domains (bHLH, LIM, homeobox) of FIGLA, SOHLH1 and LHX8. ( B ) FIGLA HA and LHX8 FLAG expression vectors were co-transfected into HEK-293T cells. Cell lysates were probed with HA and FLAG antibodies to detect input protein FIGLA and LHX8, respectively. After immunoprecipitation with HA and FLAG antibody, immunoblots were performed to detect FIGLA and associated LHX8 protein or LHX8 and associated FIGLA protein, respectively. ( C ) Same as (B) except that FIGLA HA and SOHLH1 MYC were co-transfected into HEK-293T cells. Antibodies to HA and MYC were used to detect input proteins and immunoprecipitate FIGLA and SOHLH1, respectively, and their associated proteins. ( D ) Same as (B) except that LHX8 FLAG and SOHLH1 MYC were co-transfected into HEK-293T cells. Antibodies to FLAG and MYC were used to detect input proteins and immunoprecipitate LHX8 and SOHLH1, respectively, and their associated proteins. ( E ) Co-expression of FIGLA, LHX8 and SOHLH1 in P0 ovaries from Figla FLAG mice. The dashed circles indicate co-expression of FIGLA, LHX8 and SOHLH1 in the same oocytes. Scale bar, 20 μm. ( F ) FIGLA, LHX8 and SOHLH1 appear to form a nuclear complex in oocytes. Representative of n = 3 (B–E) independent biological replicates with similar results per condition.

    Journal: Nucleic Acids Research

    Article Title: FIGLA, LHX8 and SOHLH1 transcription factor networks regulate mouse oocyte growth and differentiation

    doi: 10.1093/nar/gkaa101

    Figure Lengend Snippet: Interactions of FIGLA, LHX8 and SOHLH1. ( A ) Representative protein domains (bHLH, LIM, homeobox) of FIGLA, SOHLH1 and LHX8. ( B ) FIGLA HA and LHX8 FLAG expression vectors were co-transfected into HEK-293T cells. Cell lysates were probed with HA and FLAG antibodies to detect input protein FIGLA and LHX8, respectively. After immunoprecipitation with HA and FLAG antibody, immunoblots were performed to detect FIGLA and associated LHX8 protein or LHX8 and associated FIGLA protein, respectively. ( C ) Same as (B) except that FIGLA HA and SOHLH1 MYC were co-transfected into HEK-293T cells. Antibodies to HA and MYC were used to detect input proteins and immunoprecipitate FIGLA and SOHLH1, respectively, and their associated proteins. ( D ) Same as (B) except that LHX8 FLAG and SOHLH1 MYC were co-transfected into HEK-293T cells. Antibodies to FLAG and MYC were used to detect input proteins and immunoprecipitate LHX8 and SOHLH1, respectively, and their associated proteins. ( E ) Co-expression of FIGLA, LHX8 and SOHLH1 in P0 ovaries from Figla FLAG mice. The dashed circles indicate co-expression of FIGLA, LHX8 and SOHLH1 in the same oocytes. Scale bar, 20 μm. ( F ) FIGLA, LHX8 and SOHLH1 appear to form a nuclear complex in oocytes. Representative of n = 3 (B–E) independent biological replicates with similar results per condition.

    Article Snippet: HEK-293T cells were cultured in DMEM (Gibco) supplement with 10% fetal bovine serum at 37°C with 5% CO2.

    Techniques: Expressing, Transfection, Immunoprecipitation, Western Blot, Mouse Assay

    Inhibition of UCHL3 weakened cancer stem cell properties, as noted in a schematic model of the effects of UCHL3 on tumorigenesis and stem-like properties. a TCID decreased AhR ubiquitination in HEK293T cells by inhibiting UCHL3 activity. AhR-Flag and Ub-His were coexpressed with vectors in HEK293T cells, and cells were treated with DMSO and TCID (10 μM) for 24 h. Cell lysates were harvested after 72 h. The AhR protein was immunoprecipitated, and polyubiquitinated AhR protein was detected by WB using anti-Ub antibody. b Western blot analysis was used to detect stemness-associated markers in UCHL3-overexpressing A549 cells treated with DMSO or TCID (10 μM). c RT-qPCR was used to detect AhR mRNA levels in UCHL3-overexpressing A549 cells treated with DMSO or TCID (10 μM) ( n = 3). Data are shown as the mean ± SD; ns indicates nonsignificant ( p > 0.05). d TCID accelerated AhR protein degradation. After cotreatment of A549 cells overexpressing UCHL3 with DMSO/TCID (10 μM) for 24 h and cycloheximide (CHX, 10 μg/ml) for the indicated duration, AhR protein expression was analyzed by WB. e Flow cytometry analysis showing side populations among A549 cells stably overexpressing UCHL3 treated with DMSO or TCID (10 μM) for 24 h, with the results shown as a bar graph ( n = 3). Data are shown as the mean ± SD; * p

    Journal: Signal Transduction and Targeted Therapy

    Article Title: The deubiquitylase UCHL3 maintains cancer stem-like properties by stabilizing the aryl hydrocarbon receptor

    doi: 10.1038/s41392-020-0181-3

    Figure Lengend Snippet: Inhibition of UCHL3 weakened cancer stem cell properties, as noted in a schematic model of the effects of UCHL3 on tumorigenesis and stem-like properties. a TCID decreased AhR ubiquitination in HEK293T cells by inhibiting UCHL3 activity. AhR-Flag and Ub-His were coexpressed with vectors in HEK293T cells, and cells were treated with DMSO and TCID (10 μM) for 24 h. Cell lysates were harvested after 72 h. The AhR protein was immunoprecipitated, and polyubiquitinated AhR protein was detected by WB using anti-Ub antibody. b Western blot analysis was used to detect stemness-associated markers in UCHL3-overexpressing A549 cells treated with DMSO or TCID (10 μM). c RT-qPCR was used to detect AhR mRNA levels in UCHL3-overexpressing A549 cells treated with DMSO or TCID (10 μM) ( n = 3). Data are shown as the mean ± SD; ns indicates nonsignificant ( p > 0.05). d TCID accelerated AhR protein degradation. After cotreatment of A549 cells overexpressing UCHL3 with DMSO/TCID (10 μM) for 24 h and cycloheximide (CHX, 10 μg/ml) for the indicated duration, AhR protein expression was analyzed by WB. e Flow cytometry analysis showing side populations among A549 cells stably overexpressing UCHL3 treated with DMSO or TCID (10 μM) for 24 h, with the results shown as a bar graph ( n = 3). Data are shown as the mean ± SD; * p

    Article Snippet: The following media were required for cell culture: 1:1 DME/F12 (HyClone, UT, USA) for the A549 cell line, DMEM (Gibco, NY, USA) for the HEK293T cell line, and RPMI1640 (Gibco) for other cell lines.

    Techniques: Inhibition, Activity Assay, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Expressing, Flow Cytometry, Stable Transfection

    UCHL3 interacts with AhR and stabilizes the AhR protein through deubiquitination. a–c Western blot analysis was used to detect the expression level of AhR in A549 ( a ), H1299 ( b ) and H358 ( c ) cells after overexpression or depletion of UCHL3. d Exogenous UCHL3 and AhR proteins interacted in HEK293T cells. AhR and UCHL3 were coexpressed in HEK293T cells, and the AhR protein was immunoprecipitated with anti-AhR antibody. IgG served as a negative control, and exogenous UCHL3 was detected by WB. e , f UCHL3 overexpression delayed AhR protein degradation. After the treatment of UCHL3-overexpressing A549 ( e ) and H1299 ( f ) cells with cycloheximide (CHX, 10 μg/ml) for the indicated durations, AhR protein expression was analyzed by WB. Quantification of the AhR protein band was performed using ImageJ software. g UCHL3 knockdown enhanced AhR protein degradation. After UCHL3 knockdown, H358 cells were treated with cycloheximide (CHX, 10 μg/ml) for the indicated duration, and AhR protein expression was analyzed by WB. Quantification of the AhR protein band was performed using ImageJ software. h, i The lysates of A549 ( h ) and H1299 ( i ) cells stably overexpressing UCHL3 and vector-transfected cells containing 1 mg of total protein for each panel were immunoprecipitated with 2 μg of anti-AhR antibody, following which AhR ubiquitination was examined using anti-Ub antibody. j The lysates of stable UCHL3-knockdown H358 cells and shCtrl-transfected cells containing 1 mg of total protein were immunoprecipitated with 2 μg of anti-AhR antibody, and AhR ubiquitination was examined using anti-Ub antibody

    Journal: Signal Transduction and Targeted Therapy

    Article Title: The deubiquitylase UCHL3 maintains cancer stem-like properties by stabilizing the aryl hydrocarbon receptor

    doi: 10.1038/s41392-020-0181-3

    Figure Lengend Snippet: UCHL3 interacts with AhR and stabilizes the AhR protein through deubiquitination. a–c Western blot analysis was used to detect the expression level of AhR in A549 ( a ), H1299 ( b ) and H358 ( c ) cells after overexpression or depletion of UCHL3. d Exogenous UCHL3 and AhR proteins interacted in HEK293T cells. AhR and UCHL3 were coexpressed in HEK293T cells, and the AhR protein was immunoprecipitated with anti-AhR antibody. IgG served as a negative control, and exogenous UCHL3 was detected by WB. e , f UCHL3 overexpression delayed AhR protein degradation. After the treatment of UCHL3-overexpressing A549 ( e ) and H1299 ( f ) cells with cycloheximide (CHX, 10 μg/ml) for the indicated durations, AhR protein expression was analyzed by WB. Quantification of the AhR protein band was performed using ImageJ software. g UCHL3 knockdown enhanced AhR protein degradation. After UCHL3 knockdown, H358 cells were treated with cycloheximide (CHX, 10 μg/ml) for the indicated duration, and AhR protein expression was analyzed by WB. Quantification of the AhR protein band was performed using ImageJ software. h, i The lysates of A549 ( h ) and H1299 ( i ) cells stably overexpressing UCHL3 and vector-transfected cells containing 1 mg of total protein for each panel were immunoprecipitated with 2 μg of anti-AhR antibody, following which AhR ubiquitination was examined using anti-Ub antibody. j The lysates of stable UCHL3-knockdown H358 cells and shCtrl-transfected cells containing 1 mg of total protein were immunoprecipitated with 2 μg of anti-AhR antibody, and AhR ubiquitination was examined using anti-Ub antibody

    Article Snippet: The following media were required for cell culture: 1:1 DME/F12 (HyClone, UT, USA) for the A549 cell line, DMEM (Gibco, NY, USA) for the HEK293T cell line, and RPMI1640 (Gibco) for other cell lines.

    Techniques: Western Blot, Expressing, Over Expression, Immunoprecipitation, Negative Control, Software, Stable Transfection, Plasmid Preparation, Transfection

    FRMD8 stabilises endogenous iRhom2. ( A, B ) Levels of endogenously 3xHA tagged iRhom2 were analysed in HEK293T-iRhom2-3xHA cells transfected with FRMD8-V5 plasmid, siRNAs targeting iRhom2, non-targeting siRNA control pool (c trl) or FRMD8 SMARTpool siRNA. Cell lysates were anti-HA immunoprecipitated (HA-IP) to detect endogenous iRhom2-3xHA levels and immunoblotted using anti-HA antibody. Cell lysates were immunoblotted for ADAM17, V5, and actin. ( C ) FRMD8 and iRhom2 mRNA levels relative to actin mRNA levels were determined by TaqMan PCR in cells used for the experiment shown in ( B ) to demonstrate that the destabilisation of endogenous iRhom2 was not induced by a change in iRhom2 mRNA levels.

    Journal: eLife

    Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

    doi: 10.7554/eLife.35012

    Figure Lengend Snippet: FRMD8 stabilises endogenous iRhom2. ( A, B ) Levels of endogenously 3xHA tagged iRhom2 were analysed in HEK293T-iRhom2-3xHA cells transfected with FRMD8-V5 plasmid, siRNAs targeting iRhom2, non-targeting siRNA control pool (c trl) or FRMD8 SMARTpool siRNA. Cell lysates were anti-HA immunoprecipitated (HA-IP) to detect endogenous iRhom2-3xHA levels and immunoblotted using anti-HA antibody. Cell lysates were immunoblotted for ADAM17, V5, and actin. ( C ) FRMD8 and iRhom2 mRNA levels relative to actin mRNA levels were determined by TaqMan PCR in cells used for the experiment shown in ( B ) to demonstrate that the destabilisation of endogenous iRhom2 was not induced by a change in iRhom2 mRNA levels.

    Article Snippet: CRISPR/Cas9 genome editing in HEK293T cells For CRISPR/Cas9-mediated knockout of FRMD8 the plasmid pSpCas9(BB)−2A-Puro (pX459; Addgene plasmid #48139) co-expressing the wild-type Streptococcus pyogenes Cas9 and the guide RNA (gRNA) was used.

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Polymerase Chain Reaction

    FRMD8 loss reduces mature ADAM17 levels and impairs ADAM17-dependent shedding activity. ( A ) ADAM17 levels were analysed in HEK293T cells transfected with non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA after western blotting with anti-ADAM17 and anti-actin staining. In this and subsequent figures, pro- and mature form of ADAM17 are indicated with black and white arrowheads, respectively. Lower panel: Knockdown efficiency of FRMD8 was analysed by TaqMan PCR. ( B, C ) Lysates from wild-type (WT) and FRMD8 knockout (KO) HEK293T cells, transiently transfected with FRMD8-V5 for 72 hr (where indicated) and immunoblotted for endogenous ADAM17, ADAM10, FRMD8 and actin using western blotting. Nonspecific bands are marked with an asterisk. ( D ) Cell surface levels of endogenous ADAM10 and ADAM17 were analysed in WT and FRMD8 KO HEK293T cells after stimulation with 200 nM PMA for 5 min. Unpermeabilised cells were stained on ice with ADAM10 and ADAM17 antibodies, or only with the secondary antibody as a control (grey). The immunostaining was analysed by flow cytometry. The graph shown is one representative experiment out of four biological replicates. The geometric mean fluorescence was calculated for each experiment using FlowJo software. Statistical analysis was performed using an unpaired t-test. ( E, F ) WT and FRMD8 KO HEK293T cells were transiently transfected with alkaline phosphatase (AP)-tagged AREG, HB-EGF or TGFα, and then either incubated with 200 nM PMA, with 200 nM PMA and 1 µM GW (ADAM10/ADAM17 inhibitor), or with DMSO for 30 min. In addition, cells transfected with AP-TGFα were either left unstimulated for 20 hr or incubated with GW for 20 hr. AP activity was measured in supernatants and cell lysates. Each experiment was performed in biological triplicates. The results of three independent shedding experiments are shown. Statistical analysis was performed of using a Mann-Whitney test. ns = p value > 0.05; *=p value

    Journal: eLife

    Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

    doi: 10.7554/eLife.35012

    Figure Lengend Snippet: FRMD8 loss reduces mature ADAM17 levels and impairs ADAM17-dependent shedding activity. ( A ) ADAM17 levels were analysed in HEK293T cells transfected with non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA after western blotting with anti-ADAM17 and anti-actin staining. In this and subsequent figures, pro- and mature form of ADAM17 are indicated with black and white arrowheads, respectively. Lower panel: Knockdown efficiency of FRMD8 was analysed by TaqMan PCR. ( B, C ) Lysates from wild-type (WT) and FRMD8 knockout (KO) HEK293T cells, transiently transfected with FRMD8-V5 for 72 hr (where indicated) and immunoblotted for endogenous ADAM17, ADAM10, FRMD8 and actin using western blotting. Nonspecific bands are marked with an asterisk. ( D ) Cell surface levels of endogenous ADAM10 and ADAM17 were analysed in WT and FRMD8 KO HEK293T cells after stimulation with 200 nM PMA for 5 min. Unpermeabilised cells were stained on ice with ADAM10 and ADAM17 antibodies, or only with the secondary antibody as a control (grey). The immunostaining was analysed by flow cytometry. The graph shown is one representative experiment out of four biological replicates. The geometric mean fluorescence was calculated for each experiment using FlowJo software. Statistical analysis was performed using an unpaired t-test. ( E, F ) WT and FRMD8 KO HEK293T cells were transiently transfected with alkaline phosphatase (AP)-tagged AREG, HB-EGF or TGFα, and then either incubated with 200 nM PMA, with 200 nM PMA and 1 µM GW (ADAM10/ADAM17 inhibitor), or with DMSO for 30 min. In addition, cells transfected with AP-TGFα were either left unstimulated for 20 hr or incubated with GW for 20 hr. AP activity was measured in supernatants and cell lysates. Each experiment was performed in biological triplicates. The results of three independent shedding experiments are shown. Statistical analysis was performed of using a Mann-Whitney test. ns = p value > 0.05; *=p value

    Article Snippet: CRISPR/Cas9 genome editing in HEK293T cells For CRISPR/Cas9-mediated knockout of FRMD8 the plasmid pSpCas9(BB)−2A-Puro (pX459; Addgene plasmid #48139) co-expressing the wild-type Streptococcus pyogenes Cas9 and the guide RNA (gRNA) was used.

    Techniques: Activity Assay, Transfection, Western Blot, Staining, Polymerase Chain Reaction, Knock-Out, Immunostaining, Flow Cytometry, Cytometry, Fluorescence, Software, Incubation, MANN-WHITNEY

    Setup and confirmation of the mass spectrometry screen. ( A ) HEK293T cells transiently transfected with human iRhom2-3xHA or UNC93B1-3xHA were stained with DAPI (blue) to label nuclei, anti-HA to label iRhom2-HA (red), and anti-calnexin to label the ER (green). Scale bar = 10 μm. ( B ) Lysates and anti-HA immunoprecipitation (HA-IP) from wild-type (WT) and FRMD8 knockout (KO) HEK293T cells stably expressing iRhom2-3xHA (where indicated) were immunoblotted for HA and FRMD8. Nonspecific bands are marked with an asterisk.

    Journal: eLife

    Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

    doi: 10.7554/eLife.35012

    Figure Lengend Snippet: Setup and confirmation of the mass spectrometry screen. ( A ) HEK293T cells transiently transfected with human iRhom2-3xHA or UNC93B1-3xHA were stained with DAPI (blue) to label nuclei, anti-HA to label iRhom2-HA (red), and anti-calnexin to label the ER (green). Scale bar = 10 μm. ( B ) Lysates and anti-HA immunoprecipitation (HA-IP) from wild-type (WT) and FRMD8 knockout (KO) HEK293T cells stably expressing iRhom2-3xHA (where indicated) were immunoblotted for HA and FRMD8. Nonspecific bands are marked with an asterisk.

    Article Snippet: CRISPR/Cas9 genome editing in HEK293T cells For CRISPR/Cas9-mediated knockout of FRMD8 the plasmid pSpCas9(BB)−2A-Puro (pX459; Addgene plasmid #48139) co-expressing the wild-type Streptococcus pyogenes Cas9 and the guide RNA (gRNA) was used.

    Techniques: Mass Spectrometry, Transfection, Staining, Immunoprecipitation, Knock-Out, Stable Transfection, Expressing

    FRMD8 promotes cell surface localisation of iRhom2. ( A, B ) Immunofluorescence of iRhom1/2 double knockout HEK293T cells stably expressing iRhom2-3xHA or iRhom2 Δ300 -3xHA and transiently transfected with FRMD8-V5 for 72 hr. Cells were stained for HA (red), V5 (green) and DAPI for DNA (blue). Single confocal sections are shown, taken through the centre of the nucleus. ( C ) Schematic model of the FRMD8-iRhom2 Δ300 construct used in ( E ). ( D, E ) Immunofluorescence of iRhom1/2 double knockout HEK293T cells stably expressing iRhom2 Δ300 -3xHA or FRMD8-iRhom2 Δ300 -3xHA and transiently transfected with ADAM17-V5 for 72 hr. Cells were stained for HA (green), V5 (red) and DAPI for DNA (blue). Single confocal sections are shown, taken either through the centre of the nucleus (MEDIAL), or at basal regions close to the coverslip (BASAL). In all images the scale bar = 10 µm.

    Journal: eLife

    Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

    doi: 10.7554/eLife.35012

    Figure Lengend Snippet: FRMD8 promotes cell surface localisation of iRhom2. ( A, B ) Immunofluorescence of iRhom1/2 double knockout HEK293T cells stably expressing iRhom2-3xHA or iRhom2 Δ300 -3xHA and transiently transfected with FRMD8-V5 for 72 hr. Cells were stained for HA (red), V5 (green) and DAPI for DNA (blue). Single confocal sections are shown, taken through the centre of the nucleus. ( C ) Schematic model of the FRMD8-iRhom2 Δ300 construct used in ( E ). ( D, E ) Immunofluorescence of iRhom1/2 double knockout HEK293T cells stably expressing iRhom2 Δ300 -3xHA or FRMD8-iRhom2 Δ300 -3xHA and transiently transfected with ADAM17-V5 for 72 hr. Cells were stained for HA (green), V5 (red) and DAPI for DNA (blue). Single confocal sections are shown, taken either through the centre of the nucleus (MEDIAL), or at basal regions close to the coverslip (BASAL). In all images the scale bar = 10 µm.

    Article Snippet: CRISPR/Cas9 genome editing in HEK293T cells For CRISPR/Cas9-mediated knockout of FRMD8 the plasmid pSpCas9(BB)−2A-Puro (pX459; Addgene plasmid #48139) co-expressing the wild-type Streptococcus pyogenes Cas9 and the guide RNA (gRNA) was used.

    Techniques: Immunofluorescence, Double Knockout, Stable Transfection, Expressing, Transfection, Staining, Construct

    iRhom2 binds to FRMD8 and ADAM17 simultaneously. ( A ) Lysates, anti-HA and anti-V5 immunoprecipitations (HA-IP, V5–IP) of HEK293T cells co-expressing human iRhom2-3xHA and human FRMD8-V5 were immunoblotted for ADAM17, HA and V5. ( B ) Lysates of wild-type (WT) and ADAM17 knockout (KO) HEK293T cells were transiently transfected with human iRhom2-3xHA and FRMD8-V5 (where indicated), anti-HA and anti-V5 immunoprecipitated (HA-IP; V5–IP) and immunoblotted for ADAM17, HA, and V5. ( C ) Lysates of WT and FRMD8 KO HEK293T cells stably expressing human iRhom2-3xHA were anti-HA immunoprecipitated (HA-IP) and stained for ADAM17 and HA. Nonspecific bands are indicated by an asterisk. ( D ) Lysates of WT and iRhom1/2 double knockout (DKO) HEK293T cells stably expressing human iRhom2 WT -3xHA or iRhom2 Δ201-300 -3xHA were anti-V5 immunoprecipitated (V5–IP) and immunoblotted for ADAM17, HA and V5.

    Journal: eLife

    Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

    doi: 10.7554/eLife.35012

    Figure Lengend Snippet: iRhom2 binds to FRMD8 and ADAM17 simultaneously. ( A ) Lysates, anti-HA and anti-V5 immunoprecipitations (HA-IP, V5–IP) of HEK293T cells co-expressing human iRhom2-3xHA and human FRMD8-V5 were immunoblotted for ADAM17, HA and V5. ( B ) Lysates of wild-type (WT) and ADAM17 knockout (KO) HEK293T cells were transiently transfected with human iRhom2-3xHA and FRMD8-V5 (where indicated), anti-HA and anti-V5 immunoprecipitated (HA-IP; V5–IP) and immunoblotted for ADAM17, HA, and V5. ( C ) Lysates of WT and FRMD8 KO HEK293T cells stably expressing human iRhom2-3xHA were anti-HA immunoprecipitated (HA-IP) and stained for ADAM17 and HA. Nonspecific bands are indicated by an asterisk. ( D ) Lysates of WT and iRhom1/2 double knockout (DKO) HEK293T cells stably expressing human iRhom2 WT -3xHA or iRhom2 Δ201-300 -3xHA were anti-V5 immunoprecipitated (V5–IP) and immunoblotted for ADAM17, HA and V5.

    Article Snippet: CRISPR/Cas9 genome editing in HEK293T cells For CRISPR/Cas9-mediated knockout of FRMD8 the plasmid pSpCas9(BB)−2A-Puro (pX459; Addgene plasmid #48139) co-expressing the wild-type Streptococcus pyogenes Cas9 and the guide RNA (gRNA) was used.

    Techniques: Expressing, Knock-Out, Transfection, Immunoprecipitation, Stable Transfection, Staining, Double Knockout

    A ) Amino acid sequence alignment of human and mouse iRhom2 N-terminal region using Clustal Omega. The region required for FRMD8 binding is highlighted in red. Conserved phosphorylation sites that have been mutated to alanine in the iRhom2 pDEAD ( Figure 10—figure supplement 1 ) are marked in yellow. Grey residues indicate additional phosphorylation sites that have been reported on PhosphoSitePlus ( www.phosphosite.org ). An asterisk (*) indicates positions which have a fully conserved residue, a colon (:) indicates strongly similar properties of the amino acids, and a period (.) indicates weakly similar properties according to the Clustal Omega tool. ( B ) Lysates and anti-HA immunoprecipitation (HA-IP) from HEK293T cells transiently transfected with FRMD8-V5 and either empty vector (vect), mouse iRhom2 WT (WT) or Rhom2 cub (Δ268) were immunoblotted for V5 and HA.

    Journal: eLife

    Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

    doi: 10.7554/eLife.35012

    Figure Lengend Snippet: A ) Amino acid sequence alignment of human and mouse iRhom2 N-terminal region using Clustal Omega. The region required for FRMD8 binding is highlighted in red. Conserved phosphorylation sites that have been mutated to alanine in the iRhom2 pDEAD ( Figure 10—figure supplement 1 ) are marked in yellow. Grey residues indicate additional phosphorylation sites that have been reported on PhosphoSitePlus ( www.phosphosite.org ). An asterisk (*) indicates positions which have a fully conserved residue, a colon (:) indicates strongly similar properties of the amino acids, and a period (.) indicates weakly similar properties according to the Clustal Omega tool. ( B ) Lysates and anti-HA immunoprecipitation (HA-IP) from HEK293T cells transiently transfected with FRMD8-V5 and either empty vector (vect), mouse iRhom2 WT (WT) or Rhom2 cub (Δ268) were immunoblotted for V5 and HA.

    Article Snippet: CRISPR/Cas9 genome editing in HEK293T cells For CRISPR/Cas9-mediated knockout of FRMD8 the plasmid pSpCas9(BB)−2A-Puro (pX459; Addgene plasmid #48139) co-expressing the wild-type Streptococcus pyogenes Cas9 and the guide RNA (gRNA) was used.

    Techniques: Sequencing, Binding Assay, Immunoprecipitation, Transfection, Plasmid Preparation

    FRMD8 stabilises iRhom levels by preventing its lysosomal degradation. ( A ) iRhom1/2 double knockout HEK293T cells stably expressing iRhom2 WT -3xHA, iRhom2 Δ300 -3xHA or FRMD8-iRhom2 Δ300 -3xHA were treated with 100 µg/ml cycloheximide (CHX) for the indicated time (0–8 hr) to block protein synthesis. Cell lysates were immunoblotted for HA and actin. ( B ) Cell lysates of wild-type (WT) and FRMD8 knockout (KO) HEK293T cells treated with 10 µM MG-132 (MG), 200 nM bafilomycin A1 (Baf) or 50 mM ammonium chloride (NH 4 Cl) for 16 hr were immunoblotted for ADAM17, FRMD8, and actin. An asterisk marks a nonspecific band. ( C ) N-glycosylation of iRhom2 was analysed using EndoH and PNGase to distinguish ER/ cis- Golgi (EndoH sensitive) and late Golgi localisation (EndoH resistant). Lysates of WT and FRMD8 KO HEK293T cells transiently transfected with mouse iRhom2-3xHA were deglycosylated with EndoH or PNGase and then immunoblotted for mouse iRhom2, human FRMD8 and actin. An asterisk marks a nonspecific band. ( D ) Lysates of HEK293T cells stably expressing human iRhom1-3xHA and transfected with FRMD8-V5 (where indicated) were immunoblotted for HA, V5, and actin. ( E ) Levels of ADAM17 were analysed in HEK293T-iRhom2-3xHA and HEK293T WT cells transfected with siRNAs targeting iRhom2 where indicated. Cell lysates were immunoblotted using an anti-ADAM17 or anti-actin antibody. An asterisk marks a nonspecific band.

    Journal: eLife

    Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

    doi: 10.7554/eLife.35012

    Figure Lengend Snippet: FRMD8 stabilises iRhom levels by preventing its lysosomal degradation. ( A ) iRhom1/2 double knockout HEK293T cells stably expressing iRhom2 WT -3xHA, iRhom2 Δ300 -3xHA or FRMD8-iRhom2 Δ300 -3xHA were treated with 100 µg/ml cycloheximide (CHX) for the indicated time (0–8 hr) to block protein synthesis. Cell lysates were immunoblotted for HA and actin. ( B ) Cell lysates of wild-type (WT) and FRMD8 knockout (KO) HEK293T cells treated with 10 µM MG-132 (MG), 200 nM bafilomycin A1 (Baf) or 50 mM ammonium chloride (NH 4 Cl) for 16 hr were immunoblotted for ADAM17, FRMD8, and actin. An asterisk marks a nonspecific band. ( C ) N-glycosylation of iRhom2 was analysed using EndoH and PNGase to distinguish ER/ cis- Golgi (EndoH sensitive) and late Golgi localisation (EndoH resistant). Lysates of WT and FRMD8 KO HEK293T cells transiently transfected with mouse iRhom2-3xHA were deglycosylated with EndoH or PNGase and then immunoblotted for mouse iRhom2, human FRMD8 and actin. An asterisk marks a nonspecific band. ( D ) Lysates of HEK293T cells stably expressing human iRhom1-3xHA and transfected with FRMD8-V5 (where indicated) were immunoblotted for HA, V5, and actin. ( E ) Levels of ADAM17 were analysed in HEK293T-iRhom2-3xHA and HEK293T WT cells transfected with siRNAs targeting iRhom2 where indicated. Cell lysates were immunoblotted using an anti-ADAM17 or anti-actin antibody. An asterisk marks a nonspecific band.

    Article Snippet: CRISPR/Cas9 genome editing in HEK293T cells For CRISPR/Cas9-mediated knockout of FRMD8 the plasmid pSpCas9(BB)−2A-Puro (pX459; Addgene plasmid #48139) co-expressing the wild-type Streptococcus pyogenes Cas9 and the guide RNA (gRNA) was used.

    Techniques: Double Knockout, Stable Transfection, Expressing, Blocking Assay, Knock-Out, Transfection

    Lysates and anti-HA immunoprecipitation (HA-IP) from HEK293T cells transiently transfected with human FRMD8-V5 and mouse iRhom2 WT (WT) or iRhom2 pDEAD (pDEAD) were immunoblotted for V5 and HA. Where indicated cells have been stimulated with 200 nM PMA for 30 min.

    Journal: eLife

    Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

    doi: 10.7554/eLife.35012

    Figure Lengend Snippet: Lysates and anti-HA immunoprecipitation (HA-IP) from HEK293T cells transiently transfected with human FRMD8-V5 and mouse iRhom2 WT (WT) or iRhom2 pDEAD (pDEAD) were immunoblotted for V5 and HA. Where indicated cells have been stimulated with 200 nM PMA for 30 min.

    Article Snippet: CRISPR/Cas9 genome editing in HEK293T cells For CRISPR/Cas9-mediated knockout of FRMD8 the plasmid pSpCas9(BB)−2A-Puro (pX459; Addgene plasmid #48139) co-expressing the wild-type Streptococcus pyogenes Cas9 and the guide RNA (gRNA) was used.

    Techniques: Immunoprecipitation, Transfection

    FRMD8 loss leads to degradation of iRhoms and mature ADAM17 through the lysosomal pathway. ( A–D ) Immunofluorescence of iRhom1/2 double knockout HEK293T cells stably expressing iRhom2-3xHA or iRhom2 Δ300 -3xHA treated with DMSO (CON) or 100 nM bafilomycin A1 (BAF) for 16 hr prior to fixation. Cells were stained for HA (green), the lysosomal marker LAMP1 (red) and DAPI for DNA (blue). LAMP1-labelled regions (within white boxes) have been magnified. Scale bar = 10 µm. ( E, F ) iRhom2 Δ300 -3xHA cells were treated as in ( A–D ), but with 72 hr expression of ADAM17-V5 and labelling of HA (green), V5 (red) and DAPI for DNA (blue). Arrows indicate colocalising puncta. Single confocal sections are shown, taken through the centre of the nucleus. HA- and V5-labelled regions (within white boxes) have been magnified. Scale bar = 10 µm. ( G ) Cell lysates of wild-type (WT) and FRMD8 knockout (KO) HEK293T cells treated with the solvent DMSO (–), 10 µM MG-132 (MG) or 200 nM bafilomycin A1 (Baf) for 16 hr were enriched for glycosylated proteins using concanavalin A (conA) beads and immunoblotted for ADAM17 and transferrin receptor 1 (TfR). TfR was used as a loading control although it is also susceptible to bafilomycin treatment. Mature ADAM17 levels from three experiments were quantified relative to TfR levels using ImageJ.

    Journal: eLife

    Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

    doi: 10.7554/eLife.35012

    Figure Lengend Snippet: FRMD8 loss leads to degradation of iRhoms and mature ADAM17 through the lysosomal pathway. ( A–D ) Immunofluorescence of iRhom1/2 double knockout HEK293T cells stably expressing iRhom2-3xHA or iRhom2 Δ300 -3xHA treated with DMSO (CON) or 100 nM bafilomycin A1 (BAF) for 16 hr prior to fixation. Cells were stained for HA (green), the lysosomal marker LAMP1 (red) and DAPI for DNA (blue). LAMP1-labelled regions (within white boxes) have been magnified. Scale bar = 10 µm. ( E, F ) iRhom2 Δ300 -3xHA cells were treated as in ( A–D ), but with 72 hr expression of ADAM17-V5 and labelling of HA (green), V5 (red) and DAPI for DNA (blue). Arrows indicate colocalising puncta. Single confocal sections are shown, taken through the centre of the nucleus. HA- and V5-labelled regions (within white boxes) have been magnified. Scale bar = 10 µm. ( G ) Cell lysates of wild-type (WT) and FRMD8 knockout (KO) HEK293T cells treated with the solvent DMSO (–), 10 µM MG-132 (MG) or 200 nM bafilomycin A1 (Baf) for 16 hr were enriched for glycosylated proteins using concanavalin A (conA) beads and immunoblotted for ADAM17 and transferrin receptor 1 (TfR). TfR was used as a loading control although it is also susceptible to bafilomycin treatment. Mature ADAM17 levels from three experiments were quantified relative to TfR levels using ImageJ.

    Article Snippet: CRISPR/Cas9 genome editing in HEK293T cells For CRISPR/Cas9-mediated knockout of FRMD8 the plasmid pSpCas9(BB)−2A-Puro (pX459; Addgene plasmid #48139) co-expressing the wild-type Streptococcus pyogenes Cas9 and the guide RNA (gRNA) was used.

    Techniques: Immunofluorescence, Double Knockout, Stable Transfection, Expressing, Staining, Marker, Knock-Out

    TACI co-localizes with MyD88 and TRAF6 in the late endosomal compartment. (A) mCherry labeled TACI-S, TACI-L or TACI-S194X isoform transfected HEK-293T cells, were stained with rabbit Ab to MyD88 or TRAF6; nuclei were stained with DAPI. Merged images show that each TACI isoform co-stains with MyD88 (A) and TRAF6 (B) , but colocalization is absent for the S194X mutant. (C) HEK-293T cells transfected with mCherry labeled TACI-L and TACI-S were incubated with Alexa Fluor 647-conjugated transferrin (40 μg/ml Tfn-647) at 37°C for 5 min fixed with 4% paraformaldehyde. Merged images show that both TACI-L and TACI-S co-localize with Tfn-647 (white arrows). (D) In other experiments, transfected HEK-293T cells were stained with mAb Rab7 as a marker of late endosomes, also showing co-localization. Samples were examined by Leica SP5 DMI confocal microscopy, acquiring 3 different xy planes with 63×/1.4 NA objective lenses (Carl Zeiss) with optimal z spacing (~0.016 μm). Images were processed using Adobe Photoshop.

    Journal: Frontiers in Immunology

    Article Title: TACI Isoforms Regulate Ligand Binding and Receptor Function

    doi: 10.3389/fimmu.2018.02125

    Figure Lengend Snippet: TACI co-localizes with MyD88 and TRAF6 in the late endosomal compartment. (A) mCherry labeled TACI-S, TACI-L or TACI-S194X isoform transfected HEK-293T cells, were stained with rabbit Ab to MyD88 or TRAF6; nuclei were stained with DAPI. Merged images show that each TACI isoform co-stains with MyD88 (A) and TRAF6 (B) , but colocalization is absent for the S194X mutant. (C) HEK-293T cells transfected with mCherry labeled TACI-L and TACI-S were incubated with Alexa Fluor 647-conjugated transferrin (40 μg/ml Tfn-647) at 37°C for 5 min fixed with 4% paraformaldehyde. Merged images show that both TACI-L and TACI-S co-localize with Tfn-647 (white arrows). (D) In other experiments, transfected HEK-293T cells were stained with mAb Rab7 as a marker of late endosomes, also showing co-localization. Samples were examined by Leica SP5 DMI confocal microscopy, acquiring 3 different xy planes with 63×/1.4 NA objective lenses (Carl Zeiss) with optimal z spacing (~0.016 μm). Images were processed using Adobe Photoshop.

    Article Snippet: To examine the cytoplasmic intersection of TACI isoforms with TLR9 by confocal microscopy, we transfected HEK-293T cells with mCherry labeled TACI-S or TACI-L, or for comparison the S194X mutant, along with TLR9-YFP (pcDNA3-TLR9-YFP was a gift from Doug Golenbock; Addgene plasmid # 13642).

    Techniques: Labeling, Transfection, Staining, Mutagenesis, Incubation, Marker, Confocal Microscopy

    TACI variants with missense mutations bind un-mutated isoforms but lack signaling function. (A) TACI mCherry labeled mutants, C104R, A181E, and S194X were generated in the TACI-L and TACI-S isoforms, and co-transfected into HEK-293T cells with WT TACI-YFP. These were examined in FRET experiments by FACS (LSRII) to judge complex formation using CD40-eYFP as a control. (B) Frequency of FRET positive cells in the double positive YFP and mCherry gate. Data shows average ± SD from 6 independent experiments. In other experiments, ligands, APRIL, or BAFF (0, 5, 20, or 50 ng/ml) were added to judge the effects on FRET signal, showing no alteration in the signal (not shown). (C) For validation of complexes found in FRET, complexes forming with FLAG-TACI were precipitated with anti-FLAG sepharose beads and run on 10% PAGE gels; immunoblots were developed with an anti-HA antibody. Lower panel shows FLAG expression control. (D) To examine NF-kB luciferase induction, TACI-S, TACI-L or mutant C104R, A181E and S194X constructs were transfected into HEK-293T cells, along with NF-kB–luc reporter and control pRL-null plasmids and cultured for 48hrs; TACI expression was confirmed by western blot. (E) These cells were cultured with or without activation for 6h with 100 ng/ml APRIL or BAFF. Reporter gene activity was determined and NF-kB luciferase induction normalized to Renilla luciferase. Values reported are represented as Relative Luciferase Units (RLU) and are the mean ± SD from 5 to 7 independent experiments. * p

    Journal: Frontiers in Immunology

    Article Title: TACI Isoforms Regulate Ligand Binding and Receptor Function

    doi: 10.3389/fimmu.2018.02125

    Figure Lengend Snippet: TACI variants with missense mutations bind un-mutated isoforms but lack signaling function. (A) TACI mCherry labeled mutants, C104R, A181E, and S194X were generated in the TACI-L and TACI-S isoforms, and co-transfected into HEK-293T cells with WT TACI-YFP. These were examined in FRET experiments by FACS (LSRII) to judge complex formation using CD40-eYFP as a control. (B) Frequency of FRET positive cells in the double positive YFP and mCherry gate. Data shows average ± SD from 6 independent experiments. In other experiments, ligands, APRIL, or BAFF (0, 5, 20, or 50 ng/ml) were added to judge the effects on FRET signal, showing no alteration in the signal (not shown). (C) For validation of complexes found in FRET, complexes forming with FLAG-TACI were precipitated with anti-FLAG sepharose beads and run on 10% PAGE gels; immunoblots were developed with an anti-HA antibody. Lower panel shows FLAG expression control. (D) To examine NF-kB luciferase induction, TACI-S, TACI-L or mutant C104R, A181E and S194X constructs were transfected into HEK-293T cells, along with NF-kB–luc reporter and control pRL-null plasmids and cultured for 48hrs; TACI expression was confirmed by western blot. (E) These cells were cultured with or without activation for 6h with 100 ng/ml APRIL or BAFF. Reporter gene activity was determined and NF-kB luciferase induction normalized to Renilla luciferase. Values reported are represented as Relative Luciferase Units (RLU) and are the mean ± SD from 5 to 7 independent experiments. * p

    Article Snippet: To examine the cytoplasmic intersection of TACI isoforms with TLR9 by confocal microscopy, we transfected HEK-293T cells with mCherry labeled TACI-S or TACI-L, or for comparison the S194X mutant, along with TLR9-YFP (pcDNA3-TLR9-YFP was a gift from Doug Golenbock; Addgene plasmid # 13642).

    Techniques: Labeling, Generated, Transfection, FACS, Polyacrylamide Gel Electrophoresis, Western Blot, Expressing, Luciferase, Mutagenesis, Construct, Cell Culture, Activation Assay, Activity Assay

    (A) TACI variants with diverse deleted extracellular domains retain capacity for complex assembly. Upper panel shows HA labeled-TACI deletion mutant products: Full length TACI-L, TACI-S (minus CRD1), exon 1 and CRD1 (ΔE1-CRD1), CRD2 (ΔCDR2), minus the remaining extracellular 59 amino acids (Δaa105-164) and excision of both CRD1 and CRD2 (Δ21-104) were constructed. (B) Constructs were transfected into HEK-293T cells along with TACI-L-FLAG; after cell lysis, precipitates were harvested with anti-FLAG sepharose beads, and complexes in lysates examined after PAGE and immunoblotting using an anti-HA antibody. FLAG expression was tested as control (lower panel). Data are representative of 3 independent experiments.

    Journal: Frontiers in Immunology

    Article Title: TACI Isoforms Regulate Ligand Binding and Receptor Function

    doi: 10.3389/fimmu.2018.02125

    Figure Lengend Snippet: (A) TACI variants with diverse deleted extracellular domains retain capacity for complex assembly. Upper panel shows HA labeled-TACI deletion mutant products: Full length TACI-L, TACI-S (minus CRD1), exon 1 and CRD1 (ΔE1-CRD1), CRD2 (ΔCDR2), minus the remaining extracellular 59 amino acids (Δaa105-164) and excision of both CRD1 and CRD2 (Δ21-104) were constructed. (B) Constructs were transfected into HEK-293T cells along with TACI-L-FLAG; after cell lysis, precipitates were harvested with anti-FLAG sepharose beads, and complexes in lysates examined after PAGE and immunoblotting using an anti-HA antibody. FLAG expression was tested as control (lower panel). Data are representative of 3 independent experiments.

    Article Snippet: To examine the cytoplasmic intersection of TACI isoforms with TLR9 by confocal microscopy, we transfected HEK-293T cells with mCherry labeled TACI-S or TACI-L, or for comparison the S194X mutant, along with TLR9-YFP (pcDNA3-TLR9-YFP was a gift from Doug Golenbock; Addgene plasmid # 13642).

    Techniques: Labeling, Mutagenesis, Construct, Transfection, Lysis, Polyacrylamide Gel Electrophoresis, Expressing

    TACI-S display increased ligand binding. (A) To compare ligand binding capacities of isoforms, varying concentrations of FLAG-APRIL (0–50 ng/ml) were incubated with TACI-L or TACI-S transduced BJAB cells. Ligand binding was determined by FACS (LSRII) on washed cells, after incubation with anti-FLAG-PE. Comparable expression of TACI was determined by FACS (LSRII) using GFP expression. (B,C) To compare ligand binding in TACI-L or TACI-S transfected HEK-293T cells, cells were incubated with increasing amounts (0-800 ng) of either FLAG-BAFF (B) or FLAG-APRIL (C) . Comparable expression of TACI isoforms in each case was determined by FACS (LSRII) using anti-TACI antibody. (D) The binding affinity of TACI-L and TACI-S to ligands BAFF and APRIL was determined by Microscale thermophoresis (MST). The change in the thermophoretic signal produces these K d values (nM). Data represent the mean and SD of three independent thermophoresis measurements. (E) BJAB cells transduced with TACI-S and TACI-L, displayed binding of FLAG-APRIL intermediate between the TACI-S and TACI-L. (F) Diminishing ligand binding was also found for TACI-S HEK-293T cells also transfected with increasing ratios of TACI-L (ratios 1:1, 5:1, or 10:1) and incubated with to 0–500 ng of FLAG-APRIL. (G) HEK-293T cells transfected with both TACI-S and TACI-L, demonstrated intermediate NF-kB luciferase induction as compared to cells with either isoform. Values reported are represented as Mean of Fluorescence Intensity (MFI) (A-C,E,F) and as Relative Luciferase Units (RLU) (G) and are the mean ± SEM from 5 to 7 independent experiments. * p

    Journal: Frontiers in Immunology

    Article Title: TACI Isoforms Regulate Ligand Binding and Receptor Function

    doi: 10.3389/fimmu.2018.02125

    Figure Lengend Snippet: TACI-S display increased ligand binding. (A) To compare ligand binding capacities of isoforms, varying concentrations of FLAG-APRIL (0–50 ng/ml) were incubated with TACI-L or TACI-S transduced BJAB cells. Ligand binding was determined by FACS (LSRII) on washed cells, after incubation with anti-FLAG-PE. Comparable expression of TACI was determined by FACS (LSRII) using GFP expression. (B,C) To compare ligand binding in TACI-L or TACI-S transfected HEK-293T cells, cells were incubated with increasing amounts (0-800 ng) of either FLAG-BAFF (B) or FLAG-APRIL (C) . Comparable expression of TACI isoforms in each case was determined by FACS (LSRII) using anti-TACI antibody. (D) The binding affinity of TACI-L and TACI-S to ligands BAFF and APRIL was determined by Microscale thermophoresis (MST). The change in the thermophoretic signal produces these K d values (nM). Data represent the mean and SD of three independent thermophoresis measurements. (E) BJAB cells transduced with TACI-S and TACI-L, displayed binding of FLAG-APRIL intermediate between the TACI-S and TACI-L. (F) Diminishing ligand binding was also found for TACI-S HEK-293T cells also transfected with increasing ratios of TACI-L (ratios 1:1, 5:1, or 10:1) and incubated with to 0–500 ng of FLAG-APRIL. (G) HEK-293T cells transfected with both TACI-S and TACI-L, demonstrated intermediate NF-kB luciferase induction as compared to cells with either isoform. Values reported are represented as Mean of Fluorescence Intensity (MFI) (A-C,E,F) and as Relative Luciferase Units (RLU) (G) and are the mean ± SEM from 5 to 7 independent experiments. * p

    Article Snippet: To examine the cytoplasmic intersection of TACI isoforms with TLR9 by confocal microscopy, we transfected HEK-293T cells with mCherry labeled TACI-S or TACI-L, or for comparison the S194X mutant, along with TLR9-YFP (pcDNA3-TLR9-YFP was a gift from Doug Golenbock; Addgene plasmid # 13642).

    Techniques: Ligand Binding Assay, Incubation, FACS, Expressing, Transfection, Binding Assay, Microscale Thermophoresis, Transduction, Luciferase, Fluorescence

    TACI isoforms are associated with activated cleaved TLR9. (A) HEK-293T cells were transfected with either mCherry labeled TACI-S or TACI-L or the S194X mutant (red) along with TLR9-YFP (green); cells were examined by confocal microscopy showing co-localization (yellow) in cells expressing TACI-L or –S, but absent in cells with the S194X mutant. (B) HEK-293T cells were transfected with either TACI-S, TACI-L, or the S194X mutant with TLR9-YFP, to determine complex formation using FRET using FACS (LSRFortessa) as described above. Data is representative of 10 experiments. Right panel shows the % of FRET positive cells in the double positive YFP and mCherry gate in these experiments. Scatter-plot graph shows the mean ± SD. *** p

    Journal: Frontiers in Immunology

    Article Title: TACI Isoforms Regulate Ligand Binding and Receptor Function

    doi: 10.3389/fimmu.2018.02125

    Figure Lengend Snippet: TACI isoforms are associated with activated cleaved TLR9. (A) HEK-293T cells were transfected with either mCherry labeled TACI-S or TACI-L or the S194X mutant (red) along with TLR9-YFP (green); cells were examined by confocal microscopy showing co-localization (yellow) in cells expressing TACI-L or –S, but absent in cells with the S194X mutant. (B) HEK-293T cells were transfected with either TACI-S, TACI-L, or the S194X mutant with TLR9-YFP, to determine complex formation using FRET using FACS (LSRFortessa) as described above. Data is representative of 10 experiments. Right panel shows the % of FRET positive cells in the double positive YFP and mCherry gate in these experiments. Scatter-plot graph shows the mean ± SD. *** p

    Article Snippet: To examine the cytoplasmic intersection of TACI isoforms with TLR9 by confocal microscopy, we transfected HEK-293T cells with mCherry labeled TACI-S or TACI-L, or for comparison the S194X mutant, along with TLR9-YFP (pcDNA3-TLR9-YFP was a gift from Doug Golenbock; Addgene plasmid # 13642).

    Techniques: Transfection, Labeling, Mutagenesis, Confocal Microscopy, Expressing, FACS

    (A) TACI isoforms form hybrid complexes detected by FRET. To examine isoform complexes, YFP and mCherry labeled TACI-L, TACI-S, and/or CD40-eYFP as a control, were co-transfected into HEK-293T cells. After 48 h, the molecular association was analyzed by FRET using FACS (LSRII). A minimum of 50,000 positive cells were examined in all experiments. Data are representative of 6 experiments. (B) Frequency of FRET positive cells in the double positive YFP and mCherry gate. Graph shows the mean ± SD. *** p

    Journal: Frontiers in Immunology

    Article Title: TACI Isoforms Regulate Ligand Binding and Receptor Function

    doi: 10.3389/fimmu.2018.02125

    Figure Lengend Snippet: (A) TACI isoforms form hybrid complexes detected by FRET. To examine isoform complexes, YFP and mCherry labeled TACI-L, TACI-S, and/or CD40-eYFP as a control, were co-transfected into HEK-293T cells. After 48 h, the molecular association was analyzed by FRET using FACS (LSRII). A minimum of 50,000 positive cells were examined in all experiments. Data are representative of 6 experiments. (B) Frequency of FRET positive cells in the double positive YFP and mCherry gate. Graph shows the mean ± SD. *** p

    Article Snippet: To examine the cytoplasmic intersection of TACI isoforms with TLR9 by confocal microscopy, we transfected HEK-293T cells with mCherry labeled TACI-S or TACI-L, or for comparison the S194X mutant, along with TLR9-YFP (pcDNA3-TLR9-YFP was a gift from Doug Golenbock; Addgene plasmid # 13642).

    Techniques: Labeling, Transfection, FACS

    Hsp70 interacts with PB2, PB1 monomers, and their dimers, but not with PB2/PB1/PA heterotrimer. A and B , effects of addition of HA and FLAG tags on the interaction of Hsp70 with PB2 of HK483 influenza virus. HEK293T cells were transfected with indicated

    Journal: The Journal of Biological Chemistry

    Article Title: Heat Shock Protein 70 Modulates Influenza A Virus Polymerase Activity *

    doi: 10.1074/jbc.M113.507798

    Figure Lengend Snippet: Hsp70 interacts with PB2, PB1 monomers, and their dimers, but not with PB2/PB1/PA heterotrimer. A and B , effects of addition of HA and FLAG tags on the interaction of Hsp70 with PB2 of HK483 influenza virus. HEK293T cells were transfected with indicated

    Article Snippet: HEK293T cells, grown in 10-cm tissue culture plates, were transfected with the plasmids indicated in the figures, using TransIT®-LT1 (Mirus).

    Techniques: Transfection

    Subcellular localization of Hsp70 during different phases of the heat shock response. HEK293T ( A ) and HeLa ( B ) cells were subjected to heat shock or allowed to recover as shown in A . Treated cells were fixed, blocked, and stained with mouse anti-Hsp70

    Journal: The Journal of Biological Chemistry

    Article Title: Heat Shock Protein 70 Modulates Influenza A Virus Polymerase Activity *

    doi: 10.1074/jbc.M113.507798

    Figure Lengend Snippet: Subcellular localization of Hsp70 during different phases of the heat shock response. HEK293T ( A ) and HeLa ( B ) cells were subjected to heat shock or allowed to recover as shown in A . Treated cells were fixed, blocked, and stained with mouse anti-Hsp70

    Article Snippet: HEK293T cells, grown in 10-cm tissue culture plates, were transfected with the plasmids indicated in the figures, using TransIT®-LT1 (Mirus).

    Techniques: Staining

    Heat shock, PGA1, and plasmid-mediated overexpressed Hsp70 reduces NF-κB promoter activity. A–C , HEK293T (A) and HeLa ( B and C ) cells were transfected with pNFκB-Luc (1 μg) carrying an NF-κB promoter-dependent luciferase

    Journal: The Journal of Biological Chemistry

    Article Title: Heat Shock Protein 70 Modulates Influenza A Virus Polymerase Activity *

    doi: 10.1074/jbc.M113.507798

    Figure Lengend Snippet: Heat shock, PGA1, and plasmid-mediated overexpressed Hsp70 reduces NF-κB promoter activity. A–C , HEK293T (A) and HeLa ( B and C ) cells were transfected with pNFκB-Luc (1 μg) carrying an NF-κB promoter-dependent luciferase

    Article Snippet: HEK293T cells, grown in 10-cm tissue culture plates, were transfected with the plasmids indicated in the figures, using TransIT®-LT1 (Mirus).

    Techniques: Plasmid Preparation, Activity Assay, Transfection, Luciferase

    PGA1 reduces viral polymerase activity in cells constitutively expressing Hsp70. A and B , HEK293T ( A ) and HeLa ( B ) cells were transfected with HK483 and PR8 RNP expression plasmids and reporter plasmids. After 24 h, cells were treated with PGA1 (20 μg/ml)

    Journal: The Journal of Biological Chemistry

    Article Title: Heat Shock Protein 70 Modulates Influenza A Virus Polymerase Activity *

    doi: 10.1074/jbc.M113.507798

    Figure Lengend Snippet: PGA1 reduces viral polymerase activity in cells constitutively expressing Hsp70. A and B , HEK293T ( A ) and HeLa ( B ) cells were transfected with HK483 and PR8 RNP expression plasmids and reporter plasmids. After 24 h, cells were treated with PGA1 (20 μg/ml)

    Article Snippet: HEK293T cells, grown in 10-cm tissue culture plates, were transfected with the plasmids indicated in the figures, using TransIT®-LT1 (Mirus).

    Techniques: Activity Assay, Expressing, Transfection

    Relations between m-, c-, and vF.LucRNA levels in HeLa and HEK293T cells during the pre-heat shock, heat shock, and recovery phases. HeLa ( A ) and HEK293T ( B ) were transfected with PR8 RNP expression plasmids along with reporter plasmids. After treating

    Journal: The Journal of Biological Chemistry

    Article Title: Heat Shock Protein 70 Modulates Influenza A Virus Polymerase Activity *

    doi: 10.1074/jbc.M113.507798

    Figure Lengend Snippet: Relations between m-, c-, and vF.LucRNA levels in HeLa and HEK293T cells during the pre-heat shock, heat shock, and recovery phases. HeLa ( A ) and HEK293T ( B ) were transfected with PR8 RNP expression plasmids along with reporter plasmids. After treating

    Article Snippet: HEK293T cells, grown in 10-cm tissue culture plates, were transfected with the plasmids indicated in the figures, using TransIT®-LT1 (Mirus).

    Techniques: Transfection, Expressing

    Knocking down Hsp70 decreases the virus transcription and replication. A and C , HEK293T ( A ) and HeLa ( C ) cells were transfected twice on alternate days with transfection reagent only ( Mock ), control siRNA, or Hsp70-specific siRNA (siHsp70-1). Twenty-four

    Journal: The Journal of Biological Chemistry

    Article Title: Heat Shock Protein 70 Modulates Influenza A Virus Polymerase Activity *

    doi: 10.1074/jbc.M113.507798

    Figure Lengend Snippet: Knocking down Hsp70 decreases the virus transcription and replication. A and C , HEK293T ( A ) and HeLa ( C ) cells were transfected twice on alternate days with transfection reagent only ( Mock ), control siRNA, or Hsp70-specific siRNA (siHsp70-1). Twenty-four

    Article Snippet: HEK293T cells, grown in 10-cm tissue culture plates, were transfected with the plasmids indicated in the figures, using TransIT®-LT1 (Mirus).

    Techniques: Transfection

    Hsp70 translocates into the nucleus with PB2 monomer or PB2/PB1 heterodimer. Subcellular localization of Hsp70 with viral polymerase subunits was analyzed by confocal laser-scanning microscopy. A , HEK293T cells were transfected with the indicated plasmids

    Journal: The Journal of Biological Chemistry

    Article Title: Heat Shock Protein 70 Modulates Influenza A Virus Polymerase Activity *

    doi: 10.1074/jbc.M113.507798

    Figure Lengend Snippet: Hsp70 translocates into the nucleus with PB2 monomer or PB2/PB1 heterodimer. Subcellular localization of Hsp70 with viral polymerase subunits was analyzed by confocal laser-scanning microscopy. A , HEK293T cells were transfected with the indicated plasmids

    Article Snippet: HEK293T cells, grown in 10-cm tissue culture plates, were transfected with the plasmids indicated in the figures, using TransIT®-LT1 (Mirus).

    Techniques: Confocal Laser Scanning Microscopy, Transfection

    Correlation between nuclear-cytoplasmic shuttling of Hsp70 and viral polymerase protein levels in subcellular fractions. A , HEK293T cells were infected with PR8 influenza virus (MOI 1) or mock infected. At 12 h post-infection, cells were treated as in

    Journal: The Journal of Biological Chemistry

    Article Title: Heat Shock Protein 70 Modulates Influenza A Virus Polymerase Activity *

    doi: 10.1074/jbc.M113.507798

    Figure Lengend Snippet: Correlation between nuclear-cytoplasmic shuttling of Hsp70 and viral polymerase protein levels in subcellular fractions. A , HEK293T cells were infected with PR8 influenza virus (MOI 1) or mock infected. At 12 h post-infection, cells were treated as in

    Article Snippet: HEK293T cells, grown in 10-cm tissue culture plates, were transfected with the plasmids indicated in the figures, using TransIT®-LT1 (Mirus).

    Techniques: Infection

    Hsp70 enhances the viral polymerase activity during the heat shock phase. A , schematic diagram illustrating the experiment layout. B–H , HEK293T ( B–D ) and HeLa ( F–H ) cells were transfected with PR8 ( B , C , F , and G ) and HK483 ( D

    Journal: The Journal of Biological Chemistry

    Article Title: Heat Shock Protein 70 Modulates Influenza A Virus Polymerase Activity *

    doi: 10.1074/jbc.M113.507798

    Figure Lengend Snippet: Hsp70 enhances the viral polymerase activity during the heat shock phase. A , schematic diagram illustrating the experiment layout. B–H , HEK293T ( B–D ) and HeLa ( F–H ) cells were transfected with PR8 ( B , C , F , and G ) and HK483 ( D

    Article Snippet: HEK293T cells, grown in 10-cm tissue culture plates, were transfected with the plasmids indicated in the figures, using TransIT®-LT1 (Mirus).

    Techniques: Activity Assay, Transfection

    Plasmid-mediated Hsp70 overexpression decreases the influenza virus polymerase activity. A , HEK293T and HeLa cells were transfected with 100, 200, 400, and 800 ng of the HA-Hsp70 expression plasmid or empty vector ( Mock ) along with HK483 RNP expression

    Journal: The Journal of Biological Chemistry

    Article Title: Heat Shock Protein 70 Modulates Influenza A Virus Polymerase Activity *

    doi: 10.1074/jbc.M113.507798

    Figure Lengend Snippet: Plasmid-mediated Hsp70 overexpression decreases the influenza virus polymerase activity. A , HEK293T and HeLa cells were transfected with 100, 200, 400, and 800 ng of the HA-Hsp70 expression plasmid or empty vector ( Mock ) along with HK483 RNP expression

    Article Snippet: HEK293T cells, grown in 10-cm tissue culture plates, were transfected with the plasmids indicated in the figures, using TransIT®-LT1 (Mirus).

    Techniques: Plasmid Preparation, Over Expression, Activity Assay, Transfection, Expressing

    Tat interacts with CypA and DHHC-20.  a  HEK 293 T cells were transfected with an empty vector or Tat-FLAG (WT, 31 S, or 11Y). Cells were lysed 48 h after transfection before anti-FLAG immunoprecipitation and western blots against CypA, DHHC-5 and DHHC-20.  b  Cells were transfected with an empty (pCi) or Tat vector. GST or GST-CypA was added to cell extracts for GST pull-down before western blots. The graph shows the quantification of the DHHC pulled-down/input intensity ratio, setting the empty vector ratio to 100%. Representative data (mean ± SEM,  n  = 3 independent experiments) are shown.*** p

    Journal: Nature Communications

    Article Title: Cyclophilin A enables specific HIV-1 Tat palmitoylation and accumulation in uninfected cells

    doi: 10.1038/s41467-018-04674-y

    Figure Lengend Snippet: Tat interacts with CypA and DHHC-20. a HEK 293 T cells were transfected with an empty vector or Tat-FLAG (WT, 31 S, or 11Y). Cells were lysed 48 h after transfection before anti-FLAG immunoprecipitation and western blots against CypA, DHHC-5 and DHHC-20. b Cells were transfected with an empty (pCi) or Tat vector. GST or GST-CypA was added to cell extracts for GST pull-down before western blots. The graph shows the quantification of the DHHC pulled-down/input intensity ratio, setting the empty vector ratio to 100%. Representative data (mean ± SEM, n  = 3 independent experiments) are shown.*** p

    Article Snippet: HEK 293 T cells (ATCC CRL-11268) were transfected using PEImax as described .

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

    Tat palmitoylation is performed by DHHC-20.  a  HEK 293 T cells were cotransfected (1/5) with the indicated myc-tagged human DHHC and Tat. Tat palmitoylation was then assessed using the acyl-RAC technique, UC unbound control, BC bound control, UH unbound hydroxylamine, BH bound hydroxylamine. Palmitoylated Tat is present in the BH fraction. Palmitoylation was calculated as BH/(BH + UH)-BC/(BC + UC), and flotillin-2 was used as a positive control. The graph shows mean ± SEM of  n  = 3 independent experiments, *** p

    Journal: Nature Communications

    Article Title: Cyclophilin A enables specific HIV-1 Tat palmitoylation and accumulation in uninfected cells

    doi: 10.1038/s41467-018-04674-y

    Figure Lengend Snippet: Tat palmitoylation is performed by DHHC-20. a HEK 293 T cells were cotransfected (1/5) with the indicated myc-tagged human DHHC and Tat. Tat palmitoylation was then assessed using the acyl-RAC technique, UC unbound control, BC bound control, UH unbound hydroxylamine, BH bound hydroxylamine. Palmitoylated Tat is present in the BH fraction. Palmitoylation was calculated as BH/(BH + UH)-BC/(BC + UC), and flotillin-2 was used as a positive control. The graph shows mean ± SEM of n  = 3 independent experiments, *** p

    Article Snippet: HEK 293 T cells (ATCC CRL-11268) were transfected using PEImax as described .

    Techniques: Positive Control

    5′ UTR regulated HBoV1 capsid mRNA abundance and protein translation. (A) Diagram of HBoV1 capsid expression constructs with the T7 promoter for the in vitro assay. (B) In vitro coupled transcription/translation assay. In vitro assays were performed according to the manufacturer's instructions. Expressed proteins were run on a 15% SDS-PAGE gel, and the signal was detected with a Cyclone Plus system (PerkinElmer) and analyzed using OptiQuant software. The ratio of VP1 to VP3 is presented at the bottom of the gel. The experiment was repeated at least three times. (C) Diagram of HBoV1 VP cDNA constructs with the cytomegalovirus (CMV) promoter. (D) Northern blot. Ten micrograms of total RNAs prepared from transfected cells were resolved on 1.5% agarose gels, transferred to Hybond-N + membranes, and hybridized with probes spanning nt 349 to 5167. The signal was detected using a ChemiDoc MP imaging system (Bio-Rad). Ethidium bromide (EB)-stained 18S RNA bands are shown as the loading control. (E) Western blot (WB). The lysates of HEK293T cells transfected with the plasmids described in panel C were analyzed using an anti-Flag antibody to detect capsid expression. β-Actin served as the loading control.

    Journal: Journal of Virology

    Article Title: The 5′ Untranslated Region of Human Bocavirus Capsid Transcripts Regulates Viral mRNA Biogenesis and Alternative Translation

    doi: 10.1128/JVI.00443-18

    Figure Lengend Snippet: 5′ UTR regulated HBoV1 capsid mRNA abundance and protein translation. (A) Diagram of HBoV1 capsid expression constructs with the T7 promoter for the in vitro assay. (B) In vitro coupled transcription/translation assay. In vitro assays were performed according to the manufacturer's instructions. Expressed proteins were run on a 15% SDS-PAGE gel, and the signal was detected with a Cyclone Plus system (PerkinElmer) and analyzed using OptiQuant software. The ratio of VP1 to VP3 is presented at the bottom of the gel. The experiment was repeated at least three times. (C) Diagram of HBoV1 VP cDNA constructs with the cytomegalovirus (CMV) promoter. (D) Northern blot. Ten micrograms of total RNAs prepared from transfected cells were resolved on 1.5% agarose gels, transferred to Hybond-N + membranes, and hybridized with probes spanning nt 349 to 5167. The signal was detected using a ChemiDoc MP imaging system (Bio-Rad). Ethidium bromide (EB)-stained 18S RNA bands are shown as the loading control. (E) Western blot (WB). The lysates of HEK293T cells transfected with the plasmids described in panel C were analyzed using an anti-Flag antibody to detect capsid expression. β-Actin served as the loading control.

    Article Snippet: The human embryonic kidney cell line HEK293T (ATCC, CRL-11268) was maintained in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO2 .

    Techniques: Expressing, Construct, In Vitro, SDS Page, Software, Northern Blot, Transfection, Imaging, Staining, Western Blot

    uATG mutations in an HBoV1 infectious clone altered viral RNA processing. RNase protection assay (RPA) with analysis of viral RNA polyadenylation at the (pA)p site. Ten micrograms of total RNAs prepared from HEK293T cells transfected with plasmids, as indicated, was protected by the (pA)p site-specific probe. RT, read through RNAs. The ratios of RNA polyadenylated at the (pA)p site versus read-through RNA are indicated as the mean and standard deviation. The numbers on the left are molecular size markers.

    Journal: Journal of Virology

    Article Title: The 5′ Untranslated Region of Human Bocavirus Capsid Transcripts Regulates Viral mRNA Biogenesis and Alternative Translation

    doi: 10.1128/JVI.00443-18

    Figure Lengend Snippet: uATG mutations in an HBoV1 infectious clone altered viral RNA processing. RNase protection assay (RPA) with analysis of viral RNA polyadenylation at the (pA)p site. Ten micrograms of total RNAs prepared from HEK293T cells transfected with plasmids, as indicated, was protected by the (pA)p site-specific probe. RT, read through RNAs. The ratios of RNA polyadenylated at the (pA)p site versus read-through RNA are indicated as the mean and standard deviation. The numbers on the left are molecular size markers.

    Article Snippet: The human embryonic kidney cell line HEK293T (ATCC, CRL-11268) was maintained in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO2 .

    Techniques: Rnase Protection Assay, Recombinase Polymerase Amplification, Transfection, Standard Deviation

    The VEDEC motif is sufficient to prevent Slo1 from being expressed on the cell surface. HEK293T cells were transiently cotransfected with Myc-tagged Short-QEERL and HA-tagged Short-VEDEC. The amounts of plasmids used are indicated (in micrograms). A, results from representative cell-surface biotinylation assays as well as analyses of total expression of the HA and Myc tags as indicated. Note that total expression of each splice variant is closely related to the amount of each plasmid used in transfection. B, quantification (mean ± S.E.M.) of densitometric analyses of three repetitions of this experiment. Note the reduction of surface expression of Slo1 when even small amounts of Short-VEDEC are present.

    Journal: Molecular Pharmacology

    Article Title: Dominant-Negative Regulation of Cell Surface Expression by a Pentapeptide Motif at the Extreme COOH Terminus of an Slo1 Calcium-Activated Potassium Channel Splice Variant

    doi: 10.1124/mol.109.061929

    Figure Lengend Snippet: The VEDEC motif is sufficient to prevent Slo1 from being expressed on the cell surface. HEK293T cells were transiently cotransfected with Myc-tagged Short-QEERL and HA-tagged Short-VEDEC. The amounts of plasmids used are indicated (in micrograms). A, results from representative cell-surface biotinylation assays as well as analyses of total expression of the HA and Myc tags as indicated. Note that total expression of each splice variant is closely related to the amount of each plasmid used in transfection. B, quantification (mean ± S.E.M.) of densitometric analyses of three repetitions of this experiment. Note the reduction of surface expression of Slo1 when even small amounts of Short-VEDEC are present.

    Article Snippet: In the first set of experiments, we synthesized the octopeptide IREVEDEC and delivered it into HEK293T cells expressing full-length Slo1VEDEC or Slo1QEERL using a commercially available proprietary reagent called PULSin (PolyPlus Transfection).

    Techniques: Expressing, Variant Assay, Plasmid Preparation, Transfection

    Long-QEERL has higher surface expression than Long-VEDEC. A, representative cell-surface biotinylation assays performed in HEK293T cells heterologously expressing Long-VEDEC or Long-QEERL, as in the previous figure. B, summary of densitometric analysis of surface and total expression of Slo1 presented as mean ± S.E.M. from three repetitions of the experiment shown in A. C, representative traces of families of currents obtained by whole-cell recording are shown to the right of a bar graph summarizing the mean ± S.E.M. of whole-cell current densities evoked by step pulses to +60 mV ( n = 34 cells). ∗, P

    Journal: Molecular Pharmacology

    Article Title: Dominant-Negative Regulation of Cell Surface Expression by a Pentapeptide Motif at the Extreme COOH Terminus of an Slo1 Calcium-Activated Potassium Channel Splice Variant

    doi: 10.1124/mol.109.061929

    Figure Lengend Snippet: Long-QEERL has higher surface expression than Long-VEDEC. A, representative cell-surface biotinylation assays performed in HEK293T cells heterologously expressing Long-VEDEC or Long-QEERL, as in the previous figure. B, summary of densitometric analysis of surface and total expression of Slo1 presented as mean ± S.E.M. from three repetitions of the experiment shown in A. C, representative traces of families of currents obtained by whole-cell recording are shown to the right of a bar graph summarizing the mean ± S.E.M. of whole-cell current densities evoked by step pulses to +60 mV ( n = 34 cells). ∗, P

    Article Snippet: In the first set of experiments, we synthesized the octopeptide IREVEDEC and delivered it into HEK293T cells expressing full-length Slo1VEDEC or Slo1QEERL using a commercially available proprietary reagent called PULSin (PolyPlus Transfection).

    Techniques: Expressing

    Coexpression of Slo1 VEDEC reduces the surface Slo1 QEERL and Slo1 in a dose-dependent manner. HEK293T cells were transiently cotransfected with Myc-tagged Slo1 QEERL and HA-tagged Slo1 VEDEC . The amounts of plasmids used are shown (in micrograms). A, results from representative cell-surface biotinylation assays. B, the total and surface expressions of Slo1 were quantified by densitometry and plotted as mean ± S.E.M. of three experiments.

    Journal: Molecular Pharmacology

    Article Title: Dominant-Negative Regulation of Cell Surface Expression by a Pentapeptide Motif at the Extreme COOH Terminus of an Slo1 Calcium-Activated Potassium Channel Splice Variant

    doi: 10.1124/mol.109.061929

    Figure Lengend Snippet: Coexpression of Slo1 VEDEC reduces the surface Slo1 QEERL and Slo1 in a dose-dependent manner. HEK293T cells were transiently cotransfected with Myc-tagged Slo1 QEERL and HA-tagged Slo1 VEDEC . The amounts of plasmids used are shown (in micrograms). A, results from representative cell-surface biotinylation assays. B, the total and surface expressions of Slo1 were quantified by densitometry and plotted as mean ± S.E.M. of three experiments.

    Article Snippet: In the first set of experiments, we synthesized the octopeptide IREVEDEC and delivered it into HEK293T cells expressing full-length Slo1VEDEC or Slo1QEERL using a commercially available proprietary reagent called PULSin (PolyPlus Transfection).

    Techniques:

    Time course of Slo1 VEDEC and Slo1 QEERL removal from the cell surface. Endocytosis assays were carried out by using HEK293T cells heterologously expressing either Slo1 VEDEC or Slo1 QEERL . The expressed channels bear an Myc tag at the extracellular NH 2 terminus, which allowed surface Slo1 on the surface of intact cells to be labeled by anti-Myc at 4°C. Cells were then placed at 37°C for various times to allow trafficking to resume, at which time they were fixed. The amounts of anti-Myc remaining on the cell surface were determined by HRP-conjugated anti-mouse with colorimetric assays at OD 492 . Data show the time course of OD 492 (Mean ± S.E.M.) from three different experiments. ■, Slo1 VEDEC ; ●, Slo1 QEERL . Data are fitted with single-exponential decay functions with time constants of 15.1 ± 0.9 (for Slo1 QEERL ) and 14.4 ± 3.4 min (for Slo1 VEDEC ).

    Journal: Molecular Pharmacology

    Article Title: Dominant-Negative Regulation of Cell Surface Expression by a Pentapeptide Motif at the Extreme COOH Terminus of an Slo1 Calcium-Activated Potassium Channel Splice Variant

    doi: 10.1124/mol.109.061929

    Figure Lengend Snippet: Time course of Slo1 VEDEC and Slo1 QEERL removal from the cell surface. Endocytosis assays were carried out by using HEK293T cells heterologously expressing either Slo1 VEDEC or Slo1 QEERL . The expressed channels bear an Myc tag at the extracellular NH 2 terminus, which allowed surface Slo1 on the surface of intact cells to be labeled by anti-Myc at 4°C. Cells were then placed at 37°C for various times to allow trafficking to resume, at which time they were fixed. The amounts of anti-Myc remaining on the cell surface were determined by HRP-conjugated anti-mouse with colorimetric assays at OD 492 . Data show the time course of OD 492 (Mean ± S.E.M.) from three different experiments. ■, Slo1 VEDEC ; ●, Slo1 QEERL . Data are fitted with single-exponential decay functions with time constants of 15.1 ± 0.9 (for Slo1 QEERL ) and 14.4 ± 3.4 min (for Slo1 VEDEC ).

    Article Snippet: In the first set of experiments, we synthesized the octopeptide IREVEDEC and delivered it into HEK293T cells expressing full-length Slo1VEDEC or Slo1QEERL using a commercially available proprietary reagent called PULSin (PolyPlus Transfection).

    Techniques: Expressing, Labeling

    Coimmunoprecipitation and colocalization of Slo1 VEDEC and Slo1 QEERL in HEK293T cells. Lysates of HEK293T cells heterologously expressing Myc-tagged Slo1 VEDEC and HA-tagged Slo1 QEERL were immunoprecipitated with either mouse anti-Myc (A) or anti-HA antibodies (B). Normal mouse IgG served as a negative control. The immunoprecipitated Slo1 proteins were detected with anti-HA or anti-Myc as indicated. C, colocalization of HA-tagged Slo1 VEDEC and Myc-tagged Slo1 QEERL in HEK293T cells visualized by confocal microscopy. Slo1 VEDEC was detected with anti-HA (green, a and b), and Slo1 QEERL was detected with anti-Myc (red, c and d). Merged signals of Slo1 VEDEC and Slo1 QEERL are shown in e to h. Boxed regions in e and f are magnified in g and h, respectively. Colocalization in merged images appear as a yellow signal.

    Journal: Molecular Pharmacology

    Article Title: Dominant-Negative Regulation of Cell Surface Expression by a Pentapeptide Motif at the Extreme COOH Terminus of an Slo1 Calcium-Activated Potassium Channel Splice Variant

    doi: 10.1124/mol.109.061929

    Figure Lengend Snippet: Coimmunoprecipitation and colocalization of Slo1 VEDEC and Slo1 QEERL in HEK293T cells. Lysates of HEK293T cells heterologously expressing Myc-tagged Slo1 VEDEC and HA-tagged Slo1 QEERL were immunoprecipitated with either mouse anti-Myc (A) or anti-HA antibodies (B). Normal mouse IgG served as a negative control. The immunoprecipitated Slo1 proteins were detected with anti-HA or anti-Myc as indicated. C, colocalization of HA-tagged Slo1 VEDEC and Myc-tagged Slo1 QEERL in HEK293T cells visualized by confocal microscopy. Slo1 VEDEC was detected with anti-HA (green, a and b), and Slo1 QEERL was detected with anti-Myc (red, c and d). Merged signals of Slo1 VEDEC and Slo1 QEERL are shown in e to h. Boxed regions in e and f are magnified in g and h, respectively. Colocalization in merged images appear as a yellow signal.

    Article Snippet: In the first set of experiments, we synthesized the octopeptide IREVEDEC and delivered it into HEK293T cells expressing full-length Slo1VEDEC or Slo1QEERL using a commercially available proprietary reagent called PULSin (PolyPlus Transfection).

    Techniques: Expressing, Immunoprecipitation, Negative Control, Confocal Microscopy

    Motif-swapped constructs of Slo1. A, schematic drawing of motif-swapped Slo1 constructs (not to scale). Top to bottom: Long-VEDEC, Long-QEERL, Short-QEERL, and Short-VEDEC. All of the constructs encode channels with an HA-tag at the NH 2 termini. White boxes represent the identical amino acid sequences in all Slo1 constructs. Gray boxes indicate the VEDEC-specific regions, and the black boxes indicate the QEERL-specific regions. The last five amino acids swapped are shown by letters. B, expression of motif-swapped Slo1 constructs after transient transfection of HEK293T cells determined by immunoblot analysis using the antibodies indicated.

    Journal: Molecular Pharmacology

    Article Title: Dominant-Negative Regulation of Cell Surface Expression by a Pentapeptide Motif at the Extreme COOH Terminus of an Slo1 Calcium-Activated Potassium Channel Splice Variant

    doi: 10.1124/mol.109.061929

    Figure Lengend Snippet: Motif-swapped constructs of Slo1. A, schematic drawing of motif-swapped Slo1 constructs (not to scale). Top to bottom: Long-VEDEC, Long-QEERL, Short-QEERL, and Short-VEDEC. All of the constructs encode channels with an HA-tag at the NH 2 termini. White boxes represent the identical amino acid sequences in all Slo1 constructs. Gray boxes indicate the VEDEC-specific regions, and the black boxes indicate the QEERL-specific regions. The last five amino acids swapped are shown by letters. B, expression of motif-swapped Slo1 constructs after transient transfection of HEK293T cells determined by immunoblot analysis using the antibodies indicated.

    Article Snippet: In the first set of experiments, we synthesized the octopeptide IREVEDEC and delivered it into HEK293T cells expressing full-length Slo1VEDEC or Slo1QEERL using a commercially available proprietary reagent called PULSin (PolyPlus Transfection).

    Techniques: Construct, Expressing, Transfection

    Short-VEDEC has lower steady-state surface expression than Short-QEERL. A, representative cell-surface biotinylation assays performed in HEK293T cells heterologously expressing Short-QEERL or Short-VEDEC, as indicated. Top, cell surface Slo1; bottom, expression of total Slo1. Signals were obtained by immunoblot analysis using antibodies against the Myc tags. B, summary of densitometric analysis of surface and total expression of Slo1 presented as mean ± S.E.M. from three repetitions of the experiment shown in A. Top, normalized surface Slo1; bottom, total expression of Slo1. C, representative traces of families of currents obtained by whole-cell recording from HEK293T cells expressing Short-QEERL or Short-VEDEC are shown to the right of a bar graph summarizing mean ± S.E.M. of whole-cell current densities evoked by step pulses to +60 mV ( n = 34 cells). ∗, P

    Journal: Molecular Pharmacology

    Article Title: Dominant-Negative Regulation of Cell Surface Expression by a Pentapeptide Motif at the Extreme COOH Terminus of an Slo1 Calcium-Activated Potassium Channel Splice Variant

    doi: 10.1124/mol.109.061929

    Figure Lengend Snippet: Short-VEDEC has lower steady-state surface expression than Short-QEERL. A, representative cell-surface biotinylation assays performed in HEK293T cells heterologously expressing Short-QEERL or Short-VEDEC, as indicated. Top, cell surface Slo1; bottom, expression of total Slo1. Signals were obtained by immunoblot analysis using antibodies against the Myc tags. B, summary of densitometric analysis of surface and total expression of Slo1 presented as mean ± S.E.M. from three repetitions of the experiment shown in A. Top, normalized surface Slo1; bottom, total expression of Slo1. C, representative traces of families of currents obtained by whole-cell recording from HEK293T cells expressing Short-QEERL or Short-VEDEC are shown to the right of a bar graph summarizing mean ± S.E.M. of whole-cell current densities evoked by step pulses to +60 mV ( n = 34 cells). ∗, P

    Article Snippet: In the first set of experiments, we synthesized the octopeptide IREVEDEC and delivered it into HEK293T cells expressing full-length Slo1VEDEC or Slo1QEERL using a commercially available proprietary reagent called PULSin (PolyPlus Transfection).

    Techniques: Expressing

    IREVEDEC peptides increase the surface expression of Slo1 VEDEC in HEK293T cells. In these experiments, 1 μg R-PE (Ctrl) or IREVEDEC peptide was delivered into HEK293T cells transiently expressing Slo1 VEDEC or Slo1 QEERL using PULSin reagent, and cell-surface biotinylation assays were performed 12 h later. Surface and total Slo1 VEDEC and Slo1 QEERL were detected with anti-Slo1 antibodies. A and C, results from representative cell-surface biotinylation assays. B and D, surface (top) and total (bottom) expression of Slo1 quantified by densitometry and plotted as mean ± S.E.M. from three different experiments compared with R-PE controls.

    Journal: Molecular Pharmacology

    Article Title: Dominant-Negative Regulation of Cell Surface Expression by a Pentapeptide Motif at the Extreme COOH Terminus of an Slo1 Calcium-Activated Potassium Channel Splice Variant

    doi: 10.1124/mol.109.061929

    Figure Lengend Snippet: IREVEDEC peptides increase the surface expression of Slo1 VEDEC in HEK293T cells. In these experiments, 1 μg R-PE (Ctrl) or IREVEDEC peptide was delivered into HEK293T cells transiently expressing Slo1 VEDEC or Slo1 QEERL using PULSin reagent, and cell-surface biotinylation assays were performed 12 h later. Surface and total Slo1 VEDEC and Slo1 QEERL were detected with anti-Slo1 antibodies. A and C, results from representative cell-surface biotinylation assays. B and D, surface (top) and total (bottom) expression of Slo1 quantified by densitometry and plotted as mean ± S.E.M. from three different experiments compared with R-PE controls.

    Article Snippet: In the first set of experiments, we synthesized the octopeptide IREVEDEC and delivered it into HEK293T cells expressing full-length Slo1VEDEC or Slo1QEERL using a commercially available proprietary reagent called PULSin (PolyPlus Transfection).

    Techniques: Expressing

    Schematic of SILAC-based proteomic mapping of KEAP1 modifications in response to CBR-470-1 and NMR characterization of CR-MGx peptide. a, Stable isotope-labeled cells (stable isotope labeling with amino acids in cell culture, SILAC) expressing FLAG-tagged KEAP1 were treated with vehicle (‘light’) and CBR-470-1 or MGx (‘heavy’), respectively. Subsequent mixing of the cell lysates, anti-FLAG enrichment, tryptic digestion and LC-MS/MS analysis permitted detection of unmodified portions of KEAP1, which retained ∼1:1 SILAC ratios relative to the median ratios for all detected KEAP1 peptides. In contrast, peptides that are modified under one condition will no longer match tryptic MS/MS searches, resulting skewed SILAC ratios that “drop out” (bottom). b, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched DMSO treated ‘light’ cells and CBR-470-1 treated ‘heavy’ cells, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 3- to 4-fold upon relative to the KEAP1 median, indicative of structural modification ( n =8). c, Structural depiction of potentially modified stretches of human KEAP1 (red) using published x-ray crystal structure of the BTB (PDB: 4CXI) and KELCH (PDB: 1U6D) domains. Intervening protein stretches are depicted as unstructured loops in green. d, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched MGx treated ‘heavy’ cell lysates and no treated ‘light’ cell lysates, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 2- to 2.5- fold upon relative to the KEAP1 median, indicative of structural modification ( n =12). e, Representative Western blotting analysis of FLAG-KEAP1 dimerization from HEK293T cells pre-treated with Bardoxolone methyl followed by CBR-470-1 treatment for 4 hours ( n =3). f, 1 H-NMR of CR-MGx peptide (isolated product of MGx incubated with Ac-NH-VVCGGGRGG-C(O)NH 2 peptide). 1 H NMR (500MHz, d6-DMSO) δ 12.17 (s, 1H), 12.02 (s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 8.32-8.29 (m, 2H), 8.23 (t, J = 5.6 Hz, 1H), 8.14 (t, J = 5.9 Hz, 1H), 8.05 (t, J = 5.9 Hz, 1H), 8.01 (t, J = 5.9 Hz, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.26 (s, 1H), 7.09 (s, 1H), 4.33-4.28 (m, 1H), 4.25-4.16 (m, 3H), 3.83 (dd, J = 6.9 Hz, J = 16.2 Hz, 1H), 3.79-3.67 (m, 6H), 3.63 (d, J = 5.7 Hz, 2H), 3.54 (dd, J = 4.9 Hz, J = 16.2 Hz, 1H), 3.18-3.13 (m, 2H), 3.04 (dd, J = 4.9 Hz, J = 13.9 Hz, 1H), 2.88 (dd, J = 8.6 Hz, J = 13.6 Hz, 1H), 2.04 (s, 3H), 1.96 (sep, J = 6.8 Hz, 2H), 1.87 (s, 3H), 1.80-1.75 (m, 1H), 1.56-1.47 (m, 3H), .87-.82 (m, 12H). g, 1 H-NMR of CR peptide (Ac-NH-VVCGGGRGG-C(O)NH 2 ). 1 H NMR (500MHz, d6-DMSO) δ 8.27-8.24 (m, 2H), 8.18 (t, J = 5.7 Hz, 1H), 8.13-8.08 (m, 3H), 8.04 (t, J = 5.7 Hz, 1H), 7.91 (d, J = 8.8 Hz), 7.86 (d, J = 8.8 Hz, 1H), 7.43 (t, J = 5.4 Hz, 1H), 7.28 (s, 1H), 7.10 (s, 1H), 4.39 (dt, J = 5.6 Hz, J = 7.4 Hz, 1H), 4.28 (dt, J = 5.7 Hz, J = 7.2 Hz, 1H), 4.21-4.13 (m, 2H), 3.82-3.70 (m, 8H), 3.64 (d, J = 5.8, 2H), 3.08 (dt, J = 6.5 Hz, J = 6.5 Hz, 2H), 2.80-2.67 (m, 2H), 2.43 (t, J = 8.6 Hz, 1H), 1.94 (sep, J = 6.8 Hz, 2H), 1.85 (s, 3H), 1.75-1.68 (m, 1H), 1.54-1.42 (m, 3H), .85-.81 (m, 12H) h, 1 H- 1 H TOCSY of CR-MGx peptide. i, Peak assignment for CR-MGx peptide TOCSY spectrum. Data are mean ± SEM of biologically independent samples.

    Journal: Nature

    Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling

    doi: 10.1038/s41586-018-0622-0

    Figure Lengend Snippet: Schematic of SILAC-based proteomic mapping of KEAP1 modifications in response to CBR-470-1 and NMR characterization of CR-MGx peptide. a, Stable isotope-labeled cells (stable isotope labeling with amino acids in cell culture, SILAC) expressing FLAG-tagged KEAP1 were treated with vehicle (‘light’) and CBR-470-1 or MGx (‘heavy’), respectively. Subsequent mixing of the cell lysates, anti-FLAG enrichment, tryptic digestion and LC-MS/MS analysis permitted detection of unmodified portions of KEAP1, which retained ∼1:1 SILAC ratios relative to the median ratios for all detected KEAP1 peptides. In contrast, peptides that are modified under one condition will no longer match tryptic MS/MS searches, resulting skewed SILAC ratios that “drop out” (bottom). b, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched DMSO treated ‘light’ cells and CBR-470-1 treated ‘heavy’ cells, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 3- to 4-fold upon relative to the KEAP1 median, indicative of structural modification ( n =8). c, Structural depiction of potentially modified stretches of human KEAP1 (red) using published x-ray crystal structure of the BTB (PDB: 4CXI) and KELCH (PDB: 1U6D) domains. Intervening protein stretches are depicted as unstructured loops in green. d, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched MGx treated ‘heavy’ cell lysates and no treated ‘light’ cell lysates, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 2- to 2.5- fold upon relative to the KEAP1 median, indicative of structural modification ( n =12). e, Representative Western blotting analysis of FLAG-KEAP1 dimerization from HEK293T cells pre-treated with Bardoxolone methyl followed by CBR-470-1 treatment for 4 hours ( n =3). f, 1 H-NMR of CR-MGx peptide (isolated product of MGx incubated with Ac-NH-VVCGGGRGG-C(O)NH 2 peptide). 1 H NMR (500MHz, d6-DMSO) δ 12.17 (s, 1H), 12.02 (s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 8.32-8.29 (m, 2H), 8.23 (t, J = 5.6 Hz, 1H), 8.14 (t, J = 5.9 Hz, 1H), 8.05 (t, J = 5.9 Hz, 1H), 8.01 (t, J = 5.9 Hz, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.26 (s, 1H), 7.09 (s, 1H), 4.33-4.28 (m, 1H), 4.25-4.16 (m, 3H), 3.83 (dd, J = 6.9 Hz, J = 16.2 Hz, 1H), 3.79-3.67 (m, 6H), 3.63 (d, J = 5.7 Hz, 2H), 3.54 (dd, J = 4.9 Hz, J = 16.2 Hz, 1H), 3.18-3.13 (m, 2H), 3.04 (dd, J = 4.9 Hz, J = 13.9 Hz, 1H), 2.88 (dd, J = 8.6 Hz, J = 13.6 Hz, 1H), 2.04 (s, 3H), 1.96 (sep, J = 6.8 Hz, 2H), 1.87 (s, 3H), 1.80-1.75 (m, 1H), 1.56-1.47 (m, 3H), .87-.82 (m, 12H). g, 1 H-NMR of CR peptide (Ac-NH-VVCGGGRGG-C(O)NH 2 ). 1 H NMR (500MHz, d6-DMSO) δ 8.27-8.24 (m, 2H), 8.18 (t, J = 5.7 Hz, 1H), 8.13-8.08 (m, 3H), 8.04 (t, J = 5.7 Hz, 1H), 7.91 (d, J = 8.8 Hz), 7.86 (d, J = 8.8 Hz, 1H), 7.43 (t, J = 5.4 Hz, 1H), 7.28 (s, 1H), 7.10 (s, 1H), 4.39 (dt, J = 5.6 Hz, J = 7.4 Hz, 1H), 4.28 (dt, J = 5.7 Hz, J = 7.2 Hz, 1H), 4.21-4.13 (m, 2H), 3.82-3.70 (m, 8H), 3.64 (d, J = 5.8, 2H), 3.08 (dt, J = 6.5 Hz, J = 6.5 Hz, 2H), 2.80-2.67 (m, 2H), 2.43 (t, J = 8.6 Hz, 1H), 1.94 (sep, J = 6.8 Hz, 2H), 1.85 (s, 3H), 1.75-1.68 (m, 1H), 1.54-1.42 (m, 3H), .85-.81 (m, 12H) h, 1 H- 1 H TOCSY of CR-MGx peptide. i, Peak assignment for CR-MGx peptide TOCSY spectrum. Data are mean ± SEM of biologically independent samples.

    Article Snippet: IMR32, HLF, SH-SY5Y, HeLa, and HEK293T cells were propagated in DMEM (Corning) supplemented with 10% fetal bovine serum (FBS, Corning) and Anti-anti (Gibco).

    Techniques: Nuclear Magnetic Resonance, Labeling, Cell Culture, Expressing, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Modification, Western Blot, Isolation, Incubation

    Modulation of PGK1 induces HMW-KEAP1. a, Anti-pgK (phosphoglyceryl-lysine) and anti-GAPDH Western blots analysis of CBR-470-1 or DMSO-treated IMR32 cells at early (30 min) and late (24 hr) time points ( n =6). b, Anti-FLAG (left) and anti-pgK (right) Western blot analysis of affinity purified FLAG-KEAP1 from HEK293T cells treated with DMSO or CBR-470-1 for 30 min. Duplicate samples were run under non-reducing (left) and reducing (DTT, right) conditions (n=6). c, Densitometry quantification of total endogenous KEAP1 levels (combined bands at ∼70 and 140 kDa) in IMR32 cells treated with DMSO or CBR-470-1 for the indicated times ( n =6). d , Western blot detection of FLAG-KEAP1 in HEK293T cells comparing no-reducing reagent to DTT (left), and stability of CBR-470-1-dependent HMW-KEAP1 to the presence of DTT (12.5 mM final concentration, middle) and beta-mercaptoethanol (5% v/v final concentration, right) during sample preparation. treated with DMSO or CBR-470-1 for 8 hours ( n =8). e, Time-dependent CBR-470-1 treatment of HEK293T cells expressing FLAG-KEAP1. Time-dependent assays were run with 20 μM CBR-470-1 with Western blot analysis at the indicated time-points ( n =8). f, g, Western blot detection ( f ) and quantification ( g ) of endogenous KEAP1 and β-actin in IMR32 cells treated with DMSO or CBR-470-1 for the indicated times ( n =6). Arrows indicate monomeric (∼70 kDa) and HMW-KEAP1 (∼140 kDa) bands. h, i, Western blot ( h ) detection and quantification ( i ) of FLAG-KEAP1 in HEK293T cells exposed to increasing doses of CBR-470-1 ( n =3). j, Kinetic qRT-PCR measurement of NQO1 mRNA levels from IMR32 cells treated with tBHQ (10 μM) or CBR-470-1 (10 μM) for the indicated times ( n =3). k, Quantification of HMW-KEAP1 formation upon treatment with CBR-470-1 or the direct KEAP1 alkylator TBHQ, in the presence or absence of reduced glutathione (GSH) or N -acetylcysteine (NAC) ( n =3). All measurements taken after 8 hour of treatment in FLAG-KEAP1 expressing HEK293T cells. l, Transient shRNA knockdown of PGK1 induced HMW-KEAP1 formation, which was blocked by co-treatment of cells by GSH ( n =3). m, Anti-FLAG Western blot analysis of FLAG-KEAP1 monomer and HMW-KEAP1 fraction with dose-dependent incubation of distilled MGx in lysate from HEK-293T cells expressing FLAG-KEAP1 ( n =4). n, SDS-PAGE gel (silver stain) and anti-FLAG Western blot analysis of purified KEAP1 treated with the MGx under the indicated reducing conditions for 2 hr at 37°C ( n =3). Purified protein reactions were quenched in 4x SDS loading buffer containing βME and processed for gel analysis as in (d). Data shown represent mean ± SEM of biologically independent samples.

    Journal: Nature

    Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling

    doi: 10.1038/s41586-018-0622-0

    Figure Lengend Snippet: Modulation of PGK1 induces HMW-KEAP1. a, Anti-pgK (phosphoglyceryl-lysine) and anti-GAPDH Western blots analysis of CBR-470-1 or DMSO-treated IMR32 cells at early (30 min) and late (24 hr) time points ( n =6). b, Anti-FLAG (left) and anti-pgK (right) Western blot analysis of affinity purified FLAG-KEAP1 from HEK293T cells treated with DMSO or CBR-470-1 for 30 min. Duplicate samples were run under non-reducing (left) and reducing (DTT, right) conditions (n=6). c, Densitometry quantification of total endogenous KEAP1 levels (combined bands at ∼70 and 140 kDa) in IMR32 cells treated with DMSO or CBR-470-1 for the indicated times ( n =6). d , Western blot detection of FLAG-KEAP1 in HEK293T cells comparing no-reducing reagent to DTT (left), and stability of CBR-470-1-dependent HMW-KEAP1 to the presence of DTT (12.5 mM final concentration, middle) and beta-mercaptoethanol (5% v/v final concentration, right) during sample preparation. treated with DMSO or CBR-470-1 for 8 hours ( n =8). e, Time-dependent CBR-470-1 treatment of HEK293T cells expressing FLAG-KEAP1. Time-dependent assays were run with 20 μM CBR-470-1 with Western blot analysis at the indicated time-points ( n =8). f, g, Western blot detection ( f ) and quantification ( g ) of endogenous KEAP1 and β-actin in IMR32 cells treated with DMSO or CBR-470-1 for the indicated times ( n =6). Arrows indicate monomeric (∼70 kDa) and HMW-KEAP1 (∼140 kDa) bands. h, i, Western blot ( h ) detection and quantification ( i ) of FLAG-KEAP1 in HEK293T cells exposed to increasing doses of CBR-470-1 ( n =3). j, Kinetic qRT-PCR measurement of NQO1 mRNA levels from IMR32 cells treated with tBHQ (10 μM) or CBR-470-1 (10 μM) for the indicated times ( n =3). k, Quantification of HMW-KEAP1 formation upon treatment with CBR-470-1 or the direct KEAP1 alkylator TBHQ, in the presence or absence of reduced glutathione (GSH) or N -acetylcysteine (NAC) ( n =3). All measurements taken after 8 hour of treatment in FLAG-KEAP1 expressing HEK293T cells. l, Transient shRNA knockdown of PGK1 induced HMW-KEAP1 formation, which was blocked by co-treatment of cells by GSH ( n =3). m, Anti-FLAG Western blot analysis of FLAG-KEAP1 monomer and HMW-KEAP1 fraction with dose-dependent incubation of distilled MGx in lysate from HEK-293T cells expressing FLAG-KEAP1 ( n =4). n, SDS-PAGE gel (silver stain) and anti-FLAG Western blot analysis of purified KEAP1 treated with the MGx under the indicated reducing conditions for 2 hr at 37°C ( n =3). Purified protein reactions were quenched in 4x SDS loading buffer containing βME and processed for gel analysis as in (d). Data shown represent mean ± SEM of biologically independent samples.

    Article Snippet: IMR32, HLF, SH-SY5Y, HeLa, and HEK293T cells were propagated in DMEM (Corning) supplemented with 10% fetal bovine serum (FBS, Corning) and Anti-anti (Gibco).

    Techniques: Western Blot, Affinity Purification, Concentration Assay, Sample Prep, Expressing, Quantitative RT-PCR, shRNA, Incubation, SDS Page, Silver Staining, Purification

    A photoactivatable affinity probe-based approach identifies PGK1 as the relevant cellular target of CBR-470-1. a, Structure of CBR-470-PAP. b, Relative ARE-LUC luminance values from IMR32 cells transfected with pTI-ARE-LUC and then treated with the indicated doses of CBR-470-PAP for 24 hours ( n =3). c, Silver staining and anti-biotin Western blots of ammonium sulfate fractionated lysates from UV-irradiated IMR32 cells treated with 5 μM for 1 hour with or without CBR-470-1 competition (250 μM)( n =3). Shown on the right are initial proteomic target results from gel-band digestion and LC-MS/MS analysis. d, Anti-biotin Western blots from in vitro crosslinking assays with recombinant PGK1 and EBP1 in the presence of the indicated doses of CBR-470-PAP ( n =2). e, Anti-biotin Western blot analyses from an in vitro crosslinking assay with recombinant PGK1 in the presence of CBR-470-PAP (1 μM) and indicated concentration of soluble CBR-470-1 competitor ( n =2). f , Anti-biotin Western blot analyses of cells treated with 5 μM CBR-470-PAP after transduction with anti-PGK1 and anti-EBP1 shRNA for 48 hours. Depletion of PGK1 protein selectively reduces CBR-470-PAP-dependent labeling ( n =2). g, Dye-based thermal denaturation assay with recombinant PGK1 in the presence CBR-470-1 (20 μM) or vehicle alone ( n =3). Calculated T m values are listed. h, i , Dose-dependent thermal stability assay of recombinant PGK1 and GAPDH in the presence of increasing doses of CBR-470-1 near the T m of both proteins (57°C) ( h ) ( n =5) or room temperature ( i ) ( n =3). Western blot of sample supernatants after centrifugation (13,000 rpm) detected total PGK1 and GAPDH protein, which were plotted in Prism (below). j , ARE-LUC reporter activity in HEK293T cells with transient shRNA knockdown of ENO1 ( n =3). Data shown represent mean ± SEM of biologically independent samples.

    Journal: Nature

    Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling

    doi: 10.1038/s41586-018-0622-0

    Figure Lengend Snippet: A photoactivatable affinity probe-based approach identifies PGK1 as the relevant cellular target of CBR-470-1. a, Structure of CBR-470-PAP. b, Relative ARE-LUC luminance values from IMR32 cells transfected with pTI-ARE-LUC and then treated with the indicated doses of CBR-470-PAP for 24 hours ( n =3). c, Silver staining and anti-biotin Western blots of ammonium sulfate fractionated lysates from UV-irradiated IMR32 cells treated with 5 μM for 1 hour with or without CBR-470-1 competition (250 μM)( n =3). Shown on the right are initial proteomic target results from gel-band digestion and LC-MS/MS analysis. d, Anti-biotin Western blots from in vitro crosslinking assays with recombinant PGK1 and EBP1 in the presence of the indicated doses of CBR-470-PAP ( n =2). e, Anti-biotin Western blot analyses from an in vitro crosslinking assay with recombinant PGK1 in the presence of CBR-470-PAP (1 μM) and indicated concentration of soluble CBR-470-1 competitor ( n =2). f , Anti-biotin Western blot analyses of cells treated with 5 μM CBR-470-PAP after transduction with anti-PGK1 and anti-EBP1 shRNA for 48 hours. Depletion of PGK1 protein selectively reduces CBR-470-PAP-dependent labeling ( n =2). g, Dye-based thermal denaturation assay with recombinant PGK1 in the presence CBR-470-1 (20 μM) or vehicle alone ( n =3). Calculated T m values are listed. h, i , Dose-dependent thermal stability assay of recombinant PGK1 and GAPDH in the presence of increasing doses of CBR-470-1 near the T m of both proteins (57°C) ( h ) ( n =5) or room temperature ( i ) ( n =3). Western blot of sample supernatants after centrifugation (13,000 rpm) detected total PGK1 and GAPDH protein, which were plotted in Prism (below). j , ARE-LUC reporter activity in HEK293T cells with transient shRNA knockdown of ENO1 ( n =3). Data shown represent mean ± SEM of biologically independent samples.

    Article Snippet: IMR32, HLF, SH-SY5Y, HeLa, and HEK293T cells were propagated in DMEM (Corning) supplemented with 10% fetal bovine serum (FBS, Corning) and Anti-anti (Gibco).

    Techniques: Transfection, Silver Staining, Western Blot, Irradiation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, In Vitro, Recombinant, Concentration Assay, Transduction, shRNA, Labeling, Thermal Denaturation Assay, Stability Assay, Centrifugation, Activity Assay

    Methylglyoxal modifies KEAP1 to form a covalent, high molecular weight dimer and activate NRF2 signaling. a, Time-course, anti-FLAG Western blot analysis of whole cell lysates from HEK293T cells expressing FLAG-KEAP1 treated with DMSO or CBR-470-1. b, Western blot monitoring of FLAG-KEAP1 migration in HEK293T lysates after incubation with central glycolytic metabolites in vitro (1 and 5 mM, left and right for each metabolite). c, FLAG-KEAP1 (red) and β-actin (green) from HEK293T cells treated with MGx (5 mM) for 8 hr. d, Relative NQO1 and HMOX1 mRNA levels in IMR32 cells treated with MGx (1 mM) or water control ( n =3). e, LC-MS/MS quantitation of cellular MGx levels in IMR32 cells treated with CBR-470-1 relative to DMSO ( n =4). f, ARE-LUC reporter activity in HEK293T cells with transient shRNA knockdown of GLO1 ( n =8). Univariate two-sided t-test ( d, f ); data are mean ± SEM of biologically independent samples.

    Journal: Nature

    Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling

    doi: 10.1038/s41586-018-0622-0

    Figure Lengend Snippet: Methylglyoxal modifies KEAP1 to form a covalent, high molecular weight dimer and activate NRF2 signaling. a, Time-course, anti-FLAG Western blot analysis of whole cell lysates from HEK293T cells expressing FLAG-KEAP1 treated with DMSO or CBR-470-1. b, Western blot monitoring of FLAG-KEAP1 migration in HEK293T lysates after incubation with central glycolytic metabolites in vitro (1 and 5 mM, left and right for each metabolite). c, FLAG-KEAP1 (red) and β-actin (green) from HEK293T cells treated with MGx (5 mM) for 8 hr. d, Relative NQO1 and HMOX1 mRNA levels in IMR32 cells treated with MGx (1 mM) or water control ( n =3). e, LC-MS/MS quantitation of cellular MGx levels in IMR32 cells treated with CBR-470-1 relative to DMSO ( n =4). f, ARE-LUC reporter activity in HEK293T cells with transient shRNA knockdown of GLO1 ( n =8). Univariate two-sided t-test ( d, f ); data are mean ± SEM of biologically independent samples.

    Article Snippet: IMR32, HLF, SH-SY5Y, HeLa, and HEK293T cells were propagated in DMEM (Corning) supplemented with 10% fetal bovine serum (FBS, Corning) and Anti-anti (Gibco).

    Techniques: Molecular Weight, Western Blot, Expressing, Migration, Incubation, In Vitro, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Quantitation Assay, Activity Assay, shRNA

    A high throughput screen identifies a non-covalent NRF2 activator chemical series which activate a robust NRF2 transcriptional program in multiple cell types. a, Plate-based Z-scores of ARE-LUC luminance measurements of all test compounds from a 30k compound screen in IMR32 cells. b, Structure of screening hit CBR-470-0. c, Relative ARE-LUC luminance measurements from IMR32 cells treated for 24 hours with a concentration response of CBR-470-0 and reported NRF2 activators TBHQ and AI-1 ( n =3 biologically independent samples, mean and s.e.m.). d, LC-MS quantification of CBR-470-1 (50μM) incubated in the presence or absence of GSH (1mM) in PBS for 1 hour (left) and 48 hours (right). Relative ion intensities within each time point were compared with representative chromatograms shown ( n =2). e, Relative ARE-LUC luminance values from IMR32 cells transfected with wild type (wt) or mutant (mt, two core nucleotides necessary for NRF2 binding were changed from GC to AT) ARE-LUC reporter constructs and treated with the indicated doses of CBR-470-1 for 24 hours ( n =3, mean and s.e.m.). f, Relative abundance of NRF2-dependent transcripts as determined by qRT-PCR from IMR32 cells treated for 24 hours with 5 μM CBR-470-1 ( n =3). g, Western blot analyses of total NRF2 protein content or NRF2-controlled genes ( NQO1, HMOX1 ) from IMR32 cells treated for 24 hours with 5 μM CBR-470-1 ( n =5). h, Western blot analyses of total NRF2 protein content from the indicated cell types treated for 4 hours with 5 μM CBR-470-1 ( n =3). i, Relative expression levels of NQO1 and HMOX1 from the indicated cell types treated for 24 hours with 5 μM CBR-470-1 ( n =3, mean and s.d.). j, Relative ARE-LUC luminescence values from HEK293T cells transfected with the indicated shRNA constructs and pTI-ARE-LUC and then treated with TBHQ (10 μM) or CBR-470-1 (5 μM) for 24 hours ( n =3). k, Relative viability measurements of SH-SY5Y cells treated with either CBR-470-1 (5 μM) or TBHQ (10 μM) for 48 hours and then challenged with the indicated doses of tert-Butyl hydroperoxide (TBHP) for 8 hours ( n =4). Data are mean and s.d. of biologically independent samples ( P *

    Journal: Nature

    Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling

    doi: 10.1038/s41586-018-0622-0

    Figure Lengend Snippet: A high throughput screen identifies a non-covalent NRF2 activator chemical series which activate a robust NRF2 transcriptional program in multiple cell types. a, Plate-based Z-scores of ARE-LUC luminance measurements of all test compounds from a 30k compound screen in IMR32 cells. b, Structure of screening hit CBR-470-0. c, Relative ARE-LUC luminance measurements from IMR32 cells treated for 24 hours with a concentration response of CBR-470-0 and reported NRF2 activators TBHQ and AI-1 ( n =3 biologically independent samples, mean and s.e.m.). d, LC-MS quantification of CBR-470-1 (50μM) incubated in the presence or absence of GSH (1mM) in PBS for 1 hour (left) and 48 hours (right). Relative ion intensities within each time point were compared with representative chromatograms shown ( n =2). e, Relative ARE-LUC luminance values from IMR32 cells transfected with wild type (wt) or mutant (mt, two core nucleotides necessary for NRF2 binding were changed from GC to AT) ARE-LUC reporter constructs and treated with the indicated doses of CBR-470-1 for 24 hours ( n =3, mean and s.e.m.). f, Relative abundance of NRF2-dependent transcripts as determined by qRT-PCR from IMR32 cells treated for 24 hours with 5 μM CBR-470-1 ( n =3). g, Western blot analyses of total NRF2 protein content or NRF2-controlled genes ( NQO1, HMOX1 ) from IMR32 cells treated for 24 hours with 5 μM CBR-470-1 ( n =5). h, Western blot analyses of total NRF2 protein content from the indicated cell types treated for 4 hours with 5 μM CBR-470-1 ( n =3). i, Relative expression levels of NQO1 and HMOX1 from the indicated cell types treated for 24 hours with 5 μM CBR-470-1 ( n =3, mean and s.d.). j, Relative ARE-LUC luminescence values from HEK293T cells transfected with the indicated shRNA constructs and pTI-ARE-LUC and then treated with TBHQ (10 μM) or CBR-470-1 (5 μM) for 24 hours ( n =3). k, Relative viability measurements of SH-SY5Y cells treated with either CBR-470-1 (5 μM) or TBHQ (10 μM) for 48 hours and then challenged with the indicated doses of tert-Butyl hydroperoxide (TBHP) for 8 hours ( n =4). Data are mean and s.d. of biologically independent samples ( P *

    Article Snippet: IMR32, HLF, SH-SY5Y, HeLa, and HEK293T cells were propagated in DMEM (Corning) supplemented with 10% fetal bovine serum (FBS, Corning) and Anti-anti (Gibco).

    Techniques: High Throughput Screening Assay, Concentration Assay, Liquid Chromatography with Mass Spectroscopy, Incubation, Transfection, Mutagenesis, Binding Assay, Construct, Quantitative RT-PCR, Western Blot, Expressing, shRNA

    Methylglyoxal forms a novel posttranslational modification between proximal cysteine and arginine residues in KEAP1. a, Quantified HMW-KEAP1 formation of wild-type or mutant FLAG-KEAP1 from HEK293T cells treated with DMSO or CBR-470-1 for 8 hr ( n =23 for WT; n =16 for R15A; n =13 for C151S; n =7 for K39R, R135A; n =4 for R6A, R50A, all other C-to-S mutations, and R15/135A C151S triple-mutant; n =3 for R15/135A, and all K-to-M mutations). b, Schematic of the model peptide screen for intramolecular modifications formed by MGx and nucleophilic residues. c, Total ion- (TIC) and extracted ion chromatograms (EIC) from MGx- and mock-treated peptide, with a new peak in the former condition marked with an asterisk. EICs are specific to the indicated m/ z . ( n =3 independent biological replicates). d, 1 H-NMR spectra of the unmodified (top) and MICA-modified (bottom) model peptide, with pertinent protons highlighted in each. Notable changes in the MICA-modified spectrum include the appearance of a singlet at 2.04 p.p.m. (allyl methyl in MICA), loss of the thiol proton at 2.43 p.p.m., and changes in chemical shift and splitting pattern of the cysteine beta protons and the arginine delta and epsilon protons. Full spectra and additional multidimensional NMR spectra can be found in Extended Data Fig. 7 . e, EIC from LC-MS/MS analyses of gel-isolated and digested HMW-KEAP1 (CBR-470-1 and MGx-induced) and monomeric KEAP1 for the C151-R135 crosslinked peptide. Slight retention time variation was observed on commercial columns ( n= 3 independent biological replicates). f, PRM chromatograms for the parent and six parent-to-daughter transitions in representative targeted proteomic runs from HMW-KEAP1 and monomeric digests ( n =6). g, Schematic depicting the direct communication between glucose metabolism and KEAP1-NRF2 signaling mediated by MGx modification of KEAP1 and subsequent activation of the NRF2 transcriptional program. Univariate two-sided t-test ( a ); data are mean ± SEM of biologically independent samples.

    Journal: Nature

    Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling

    doi: 10.1038/s41586-018-0622-0

    Figure Lengend Snippet: Methylglyoxal forms a novel posttranslational modification between proximal cysteine and arginine residues in KEAP1. a, Quantified HMW-KEAP1 formation of wild-type or mutant FLAG-KEAP1 from HEK293T cells treated with DMSO or CBR-470-1 for 8 hr ( n =23 for WT; n =16 for R15A; n =13 for C151S; n =7 for K39R, R135A; n =4 for R6A, R50A, all other C-to-S mutations, and R15/135A C151S triple-mutant; n =3 for R15/135A, and all K-to-M mutations). b, Schematic of the model peptide screen for intramolecular modifications formed by MGx and nucleophilic residues. c, Total ion- (TIC) and extracted ion chromatograms (EIC) from MGx- and mock-treated peptide, with a new peak in the former condition marked with an asterisk. EICs are specific to the indicated m/ z . ( n =3 independent biological replicates). d, 1 H-NMR spectra of the unmodified (top) and MICA-modified (bottom) model peptide, with pertinent protons highlighted in each. Notable changes in the MICA-modified spectrum include the appearance of a singlet at 2.04 p.p.m. (allyl methyl in MICA), loss of the thiol proton at 2.43 p.p.m., and changes in chemical shift and splitting pattern of the cysteine beta protons and the arginine delta and epsilon protons. Full spectra and additional multidimensional NMR spectra can be found in Extended Data Fig. 7 . e, EIC from LC-MS/MS analyses of gel-isolated and digested HMW-KEAP1 (CBR-470-1 and MGx-induced) and monomeric KEAP1 for the C151-R135 crosslinked peptide. Slight retention time variation was observed on commercial columns ( n= 3 independent biological replicates). f, PRM chromatograms for the parent and six parent-to-daughter transitions in representative targeted proteomic runs from HMW-KEAP1 and monomeric digests ( n =6). g, Schematic depicting the direct communication between glucose metabolism and KEAP1-NRF2 signaling mediated by MGx modification of KEAP1 and subsequent activation of the NRF2 transcriptional program. Univariate two-sided t-test ( a ); data are mean ± SEM of biologically independent samples.

    Article Snippet: IMR32, HLF, SH-SY5Y, HeLa, and HEK293T cells were propagated in DMEM (Corning) supplemented with 10% fetal bovine serum (FBS, Corning) and Anti-anti (Gibco).

    Techniques: Modification, Mutagenesis, Nuclear Magnetic Resonance, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Isolation, Activation Assay

    MGx and glyoxylase activity regulates NRF2 activation. CBR-470-1 causes elevated MGx levels in cells. a, Schematic depicting chemical derivatization and trapping of cellular MGx for analysis by targeted metabolomics using two unique fragment ions. b, c, Daughter ion fragments ( b ) and resulting MS/MS quantification of MGx levels ( c ) in IMR32 cells treated with CBR-470-1, relative to DMSO ( n =4). d, Quantitative LC-MS/MS measurement of cellular MGx levels in IMR32 cells treated for 2 hours with CBR-470-1 or co-treated for 2 hours with CBR-470-1 and NAC (2 mM) relative to DMSO ( n =4). e, Relative ARE-LUC luminance values from IMR32 cells transfected with pTI-ARE-LUC and co-treated with the indicated doses of CBR-470-1 and GSH ( n =3). f, Relative levels of transcripts NQO1 and HMOX1 from IMR32 cells co-treated with CBR-470-1 (10 μM) and the indicated concentrations of GSH for 24 hours ( n =3). g, Fractional ARE-LUC values from HEK293T cells transiently co-transfected with pTI-ARE-LUC and the indicated shRNAs and then treated for 24 hours with the indicated doses of CBR-470-1 ( n =3). h, ARE-LUC reporter activity in HEK293T cells treated with CBR-470-1 alone (black) and with a cell-permeable small molecule GLO1 inhibitor (red) ( n =3). Univariate two-sided t-test (Extended Data Fig 7d, h) ; data are mean ± SEM of biologically independent samples.

    Journal: Nature

    Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling

    doi: 10.1038/s41586-018-0622-0

    Figure Lengend Snippet: MGx and glyoxylase activity regulates NRF2 activation. CBR-470-1 causes elevated MGx levels in cells. a, Schematic depicting chemical derivatization and trapping of cellular MGx for analysis by targeted metabolomics using two unique fragment ions. b, c, Daughter ion fragments ( b ) and resulting MS/MS quantification of MGx levels ( c ) in IMR32 cells treated with CBR-470-1, relative to DMSO ( n =4). d, Quantitative LC-MS/MS measurement of cellular MGx levels in IMR32 cells treated for 2 hours with CBR-470-1 or co-treated for 2 hours with CBR-470-1 and NAC (2 mM) relative to DMSO ( n =4). e, Relative ARE-LUC luminance values from IMR32 cells transfected with pTI-ARE-LUC and co-treated with the indicated doses of CBR-470-1 and GSH ( n =3). f, Relative levels of transcripts NQO1 and HMOX1 from IMR32 cells co-treated with CBR-470-1 (10 μM) and the indicated concentrations of GSH for 24 hours ( n =3). g, Fractional ARE-LUC values from HEK293T cells transiently co-transfected with pTI-ARE-LUC and the indicated shRNAs and then treated for 24 hours with the indicated doses of CBR-470-1 ( n =3). h, ARE-LUC reporter activity in HEK293T cells treated with CBR-470-1 alone (black) and with a cell-permeable small molecule GLO1 inhibitor (red) ( n =3). Univariate two-sided t-test (Extended Data Fig 7d, h) ; data are mean ± SEM of biologically independent samples.

    Article Snippet: IMR32, HLF, SH-SY5Y, HeLa, and HEK293T cells were propagated in DMEM (Corning) supplemented with 10% fetal bovine serum (FBS, Corning) and Anti-anti (Gibco).

    Techniques: Activity Assay, Activation Assay, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Transfection

    CBR-470-1-dependent inhibition of glycolysis activates NRF2 signaling. a, Anti-biotin Western blot analysis of IMR32 cells treated with CBR-470-PAP (10 μM) for one hour and exposed to UV light to induce photocrosslinking (representative shown from n = 4 biological replicates). b, Transient transfection of shRNA constructs targeting PGK1 in HEK293T cells activates the ARE-LUC reporter. PGK1 and β-actin protein levels shown from representative experiments ( n =4 biological replicates). c, Viral shRNA knockdown of PGK1 induces NQO1 mRNA levels in IMR32 cells. PGK1 and Tubulin protein levels are shown from representative experiments ( n =3). d,e, CBR-470-1 activation of ARE-LUC reporter in HEK293T cells with transient knockdown ( d ) or overexpression ( e ) of PGK1 demonstrates opposing effects on compound potency. PGK1, Actin and Tubulin protein levels are shown from representative experiments ( n =3). f, Heat map depiction of relative metabolite levels in IMR32 cells treated for 30 min with CBR-470-1 (left) or viral shRNA knockdown of PGK1 (right) relative to DMSO and scramble shRNA controls, respectively. BPG refers to both 2,3-BPG and 1,3-BPG, whereas 1,3-BPG specifically refers to the 1,3-isomer. g, ARE-LUC reporter activity in IMR32 cells co-treated with CBR-470-1 (5 μM) and 2DG for 24 hr. ( n =12). Statistical analyses are univariate two-sided t-tests ( b, c, g ). Data are mean and s.d. of biologically independent samples.

    Journal: Nature

    Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling

    doi: 10.1038/s41586-018-0622-0

    Figure Lengend Snippet: CBR-470-1-dependent inhibition of glycolysis activates NRF2 signaling. a, Anti-biotin Western blot analysis of IMR32 cells treated with CBR-470-PAP (10 μM) for one hour and exposed to UV light to induce photocrosslinking (representative shown from n = 4 biological replicates). b, Transient transfection of shRNA constructs targeting PGK1 in HEK293T cells activates the ARE-LUC reporter. PGK1 and β-actin protein levels shown from representative experiments ( n =4 biological replicates). c, Viral shRNA knockdown of PGK1 induces NQO1 mRNA levels in IMR32 cells. PGK1 and Tubulin protein levels are shown from representative experiments ( n =3). d,e, CBR-470-1 activation of ARE-LUC reporter in HEK293T cells with transient knockdown ( d ) or overexpression ( e ) of PGK1 demonstrates opposing effects on compound potency. PGK1, Actin and Tubulin protein levels are shown from representative experiments ( n =3). f, Heat map depiction of relative metabolite levels in IMR32 cells treated for 30 min with CBR-470-1 (left) or viral shRNA knockdown of PGK1 (right) relative to DMSO and scramble shRNA controls, respectively. BPG refers to both 2,3-BPG and 1,3-BPG, whereas 1,3-BPG specifically refers to the 1,3-isomer. g, ARE-LUC reporter activity in IMR32 cells co-treated with CBR-470-1 (5 μM) and 2DG for 24 hr. ( n =12). Statistical analyses are univariate two-sided t-tests ( b, c, g ). Data are mean and s.d. of biologically independent samples.

    Article Snippet: IMR32, HLF, SH-SY5Y, HeLa, and HEK293T cells were propagated in DMEM (Corning) supplemented with 10% fetal bovine serum (FBS, Corning) and Anti-anti (Gibco).

    Techniques: Inhibition, Western Blot, Transfection, shRNA, Construct, Activation Assay, Over Expression, Activity Assay

    Effect of nucleotide substitution on RNA replication and translational capacity mediated by CA16 5′UTR. ( A ) Construction of reporter plasmid to determine translational activity mediated by CA16 5′UTR. ( B ) Effect of base 104 of 5′UTR on RNA replication in cells. WT or M2 vector expressing luciferase were transfected into HEK293T cells, and cells were harvested at 12 h, 24 h, 36 h and 48 h after transfection. RNA was extracted from a portion of each cell sample and analyzed by RT-qPCR with primers specific for GAPDH RNA or CA16 5′UTR RNA. GAPDH was used as a control. The RNA level obtained from transfection with WT 5′UTR was normalized to 100%. ( C ) Effect of base 104 of 5′UTR on translational activity in cells. Luciferase activiy was detected in a portion of each cell sample.and the viral replication rate was expressed as a fold increase in luciferase activity. * P

    Journal: Scientific Reports

    Article Title: Identification of a nucleotide in 5′ untranslated region contributing to virus replication and virulence of Coxsackievirus A16

    doi: 10.1038/srep20839

    Figure Lengend Snippet: Effect of nucleotide substitution on RNA replication and translational capacity mediated by CA16 5′UTR. ( A ) Construction of reporter plasmid to determine translational activity mediated by CA16 5′UTR. ( B ) Effect of base 104 of 5′UTR on RNA replication in cells. WT or M2 vector expressing luciferase were transfected into HEK293T cells, and cells were harvested at 12 h, 24 h, 36 h and 48 h after transfection. RNA was extracted from a portion of each cell sample and analyzed by RT-qPCR with primers specific for GAPDH RNA or CA16 5′UTR RNA. GAPDH was used as a control. The RNA level obtained from transfection with WT 5′UTR was normalized to 100%. ( C ) Effect of base 104 of 5′UTR on translational activity in cells. Luciferase activiy was detected in a portion of each cell sample.and the viral replication rate was expressed as a fold increase in luciferase activity. * P

    Article Snippet: A 70% confluent monolayer of HEK293T cells in a T25 flask (BD Biosciences, Franklin Lakes, NJ, USA) was transfected with pcDNA3.1-T7 DNA Pol (1 μg) and CA16 infectious clone (2 μg) or mutants digested by the restriction enzyme Sac I (Takara) using 9 μl Lipofectamine 2000 reagent (Invitrogen) and then incubated at 37 °C in 5 ml of DMEM (10% FCS).

    Techniques: Plasmid Preparation, Activity Assay, Expressing, Luciferase, Transfection, Quantitative RT-PCR

    Interactions of WT or M2 mutant 5′UTR of CA16 with cellular proteins hnRNP K, hnRNP A1 and PCBP1. ( A ) Structural prediction of CA16 5′UTR using Mfold software. The nucleotide change C104T is located at the linker between loop I and II. ( B ) Expression of cellular proteins after transfection with 5′UTR of CA16. VR1012, hnRNP K-HA, hnRNP A1-HA or PCBP1-HA was co-transfected with WT 5′UTR or M2 mutant expression vector into HEK293T cells. Cell lysate were prepared at 48 h after transfection. Part of each cell lysate was dissolved in 1 × loading buffer for immunoblotting analysis. ( C ) Immunoprecipitation assay. Most of each cell lysates was incubated with anti-HA agarose beads at 4 °C for 3 h. Following washing and dissociation, part of each bead pellet was re-suspended in 1 × loading buffer for immunoblotting analysis, and another part was used for RNA extraction. ( D ) RNA levels of CA16 5′UTR in cell lysates. RNA was extracted from a portion of each set of transfected HEK293T cells and then analyzed by RT-qPCR using primers specific for GAPDH RNA or CA16 5′UTR RNA. GAPDH was used as a control. The RNA level in the presence of VR1012 and WT 5′UTR was normalized to 100%. ( E ) Interaction of various cellular proteins with CA16 5′UTR. RNA extract from immunoprecipitation was subjected to RT-qPCR analysis with primers specific for CA16 5′UTR RNA. The RNA level in the presence of PCBP1, hnRNP K or hnRNP A1 and WT 5′UTR was normalized to 100%. The RNA level in the presence of PCBP1 and WT 5′UTR was normalized to 100% in the VR1012 negative control group. Errors bars represent the SD from triplicate wells within one experiment. Results represent at least three independent experiments. * P

    Journal: Scientific Reports

    Article Title: Identification of a nucleotide in 5′ untranslated region contributing to virus replication and virulence of Coxsackievirus A16

    doi: 10.1038/srep20839

    Figure Lengend Snippet: Interactions of WT or M2 mutant 5′UTR of CA16 with cellular proteins hnRNP K, hnRNP A1 and PCBP1. ( A ) Structural prediction of CA16 5′UTR using Mfold software. The nucleotide change C104T is located at the linker between loop I and II. ( B ) Expression of cellular proteins after transfection with 5′UTR of CA16. VR1012, hnRNP K-HA, hnRNP A1-HA or PCBP1-HA was co-transfected with WT 5′UTR or M2 mutant expression vector into HEK293T cells. Cell lysate were prepared at 48 h after transfection. Part of each cell lysate was dissolved in 1 × loading buffer for immunoblotting analysis. ( C ) Immunoprecipitation assay. Most of each cell lysates was incubated with anti-HA agarose beads at 4 °C for 3 h. Following washing and dissociation, part of each bead pellet was re-suspended in 1 × loading buffer for immunoblotting analysis, and another part was used for RNA extraction. ( D ) RNA levels of CA16 5′UTR in cell lysates. RNA was extracted from a portion of each set of transfected HEK293T cells and then analyzed by RT-qPCR using primers specific for GAPDH RNA or CA16 5′UTR RNA. GAPDH was used as a control. The RNA level in the presence of VR1012 and WT 5′UTR was normalized to 100%. ( E ) Interaction of various cellular proteins with CA16 5′UTR. RNA extract from immunoprecipitation was subjected to RT-qPCR analysis with primers specific for CA16 5′UTR RNA. The RNA level in the presence of PCBP1, hnRNP K or hnRNP A1 and WT 5′UTR was normalized to 100%. The RNA level in the presence of PCBP1 and WT 5′UTR was normalized to 100% in the VR1012 negative control group. Errors bars represent the SD from triplicate wells within one experiment. Results represent at least three independent experiments. * P

    Article Snippet: A 70% confluent monolayer of HEK293T cells in a T25 flask (BD Biosciences, Franklin Lakes, NJ, USA) was transfected with pcDNA3.1-T7 DNA Pol (1 μg) and CA16 infectious clone (2 μg) or mutants digested by the restriction enzyme Sac I (Takara) using 9 μl Lipofectamine 2000 reagent (Invitrogen) and then incubated at 37 °C in 5 ml of DMEM (10% FCS).

    Techniques: Mutagenesis, Software, Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Incubation, RNA Extraction, Quantitative RT-PCR, Negative Control

    Pathological analysis of WT or M2 infected neonatal mice. One-day-old ICR mice were intracerebrally inoculated with medium or WT or M2 virus from transfected HEK293T cells at 10 4 CCID 50 ml −1 . No histological change was observed in the brain of the non-infected medium control ( A ) or WT ( B ) or M2 ( C ) infected mice. No histological change was observed in the lung ( D,F ) or hind limb muscle ( G,I ) or spinal muscle ( J,L ) of the non-infected medium control or M2 infected mice. Mice infected with WT viruses exhibited severe alveolar shrinkage ( E ) in the lung tissue. Mice infected with WT viruses (grades 4 to 5) exhibited signs of severe necrosis, including muscle bundle fracture, dissolution of muscle fiber cells, nuclei shrinkage and swelling (H and K). A to L, magnification 400× .

    Journal: Scientific Reports

    Article Title: Identification of a nucleotide in 5′ untranslated region contributing to virus replication and virulence of Coxsackievirus A16

    doi: 10.1038/srep20839

    Figure Lengend Snippet: Pathological analysis of WT or M2 infected neonatal mice. One-day-old ICR mice were intracerebrally inoculated with medium or WT or M2 virus from transfected HEK293T cells at 10 4 CCID 50 ml −1 . No histological change was observed in the brain of the non-infected medium control ( A ) or WT ( B ) or M2 ( C ) infected mice. No histological change was observed in the lung ( D,F ) or hind limb muscle ( G,I ) or spinal muscle ( J,L ) of the non-infected medium control or M2 infected mice. Mice infected with WT viruses exhibited severe alveolar shrinkage ( E ) in the lung tissue. Mice infected with WT viruses (grades 4 to 5) exhibited signs of severe necrosis, including muscle bundle fracture, dissolution of muscle fiber cells, nuclei shrinkage and swelling (H and K). A to L, magnification 400× .

    Article Snippet: A 70% confluent monolayer of HEK293T cells in a T25 flask (BD Biosciences, Franklin Lakes, NJ, USA) was transfected with pcDNA3.1-T7 DNA Pol (1 μg) and CA16 infectious clone (2 μg) or mutants digested by the restriction enzyme Sac I (Takara) using 9 μl Lipofectamine 2000 reagent (Invitrogen) and then incubated at 37 °C in 5 ml of DMEM (10% FCS).

    Techniques: Infection, Mouse Assay, Transfection

    Correlation of 5′UTR nucleotides with viral virulence of CA16 in neonatal mice. ( A,C ) Viruses from HEK293T cells transfected with WT or various mutant infectious clones (10 μL) were intracerebrally inoculated into neonatal mice at 10 3.0 CCID 50 ml −1 ( A ) or 10 4.0 CCID 50 ml −1 ( C ), and survival rates and clinical scores were monitored and recorded daily after infection. Mock-infected mice were given culture medium from cells transfected with negative empty vector. ( B,D ) Viral load variations in various tissues of mice inoculated with WT or M2 viruses at 10 3.0 CCID 50 ml −1 ( B ) or 10 4.0 CCID 50 ml −1 ( D ). Virus loads were assessed by RT-qPCR with primers specific for the GAPDH or for CA16 VP1 RNA in samples of the brain, lung, spine skeletal muscle, hind-limb muscle and blood from infected mice. GAPDH was used as a control. The results represent the mean virus loads [log 10 copies (mg tissue) −1 or log 10 copies (ml blood) −1 ] ± SD (three mice per group, repeated three times). * P

    Journal: Scientific Reports

    Article Title: Identification of a nucleotide in 5′ untranslated region contributing to virus replication and virulence of Coxsackievirus A16

    doi: 10.1038/srep20839

    Figure Lengend Snippet: Correlation of 5′UTR nucleotides with viral virulence of CA16 in neonatal mice. ( A,C ) Viruses from HEK293T cells transfected with WT or various mutant infectious clones (10 μL) were intracerebrally inoculated into neonatal mice at 10 3.0 CCID 50 ml −1 ( A ) or 10 4.0 CCID 50 ml −1 ( C ), and survival rates and clinical scores were monitored and recorded daily after infection. Mock-infected mice were given culture medium from cells transfected with negative empty vector. ( B,D ) Viral load variations in various tissues of mice inoculated with WT or M2 viruses at 10 3.0 CCID 50 ml −1 ( B ) or 10 4.0 CCID 50 ml −1 ( D ). Virus loads were assessed by RT-qPCR with primers specific for the GAPDH or for CA16 VP1 RNA in samples of the brain, lung, spine skeletal muscle, hind-limb muscle and blood from infected mice. GAPDH was used as a control. The results represent the mean virus loads [log 10 copies (mg tissue) −1 or log 10 copies (ml blood) −1 ] ± SD (three mice per group, repeated three times). * P

    Article Snippet: A 70% confluent monolayer of HEK293T cells in a T25 flask (BD Biosciences, Franklin Lakes, NJ, USA) was transfected with pcDNA3.1-T7 DNA Pol (1 μg) and CA16 infectious clone (2 μg) or mutants digested by the restriction enzyme Sac I (Takara) using 9 μl Lipofectamine 2000 reagent (Invitrogen) and then incubated at 37 °C in 5 ml of DMEM (10% FCS).

    Techniques: Mouse Assay, Transfection, Mutagenesis, Clone Assay, Infection, Plasmid Preparation, Quantitative RT-PCR

    Effect of WT or M2 mutant 5′UTR of CA16 on viral RNA synthesis and viral translational capacity in transfected HEK293T cells. HEK293T cells were transfected with same amount of the WT or M2 infectious clone or empty vector, and were harvested at 12 h, 24 h, 36 h and 48 h after transfection. ( A ) RNA was extracted from a portion of each sample, treated with Dnase to degrade the transfected plasmid. DNA and then analyzed by RT-qPCR with primers specific for GAPDH or CA16 VP1 RNA. GAPDH was used as a control. The RNA level obtained by transfection with WT 5′UTR was normalized to 100%. ( B ) A portion of each cell sample was used to detect the VP1 protein by Western blot. ( C ) A portion of each cell sample was used to detect the viral titer. ( D ) Viral loads in supernatant of transfected HEK293T cells at 48h after transfection were detected by RT-qPCR with primers specific for CA16 VP1 RNA. ( E ) The VP1 protein of the WT or M2 virus in the supernatant was detected by Western blot using VP1 antibody. * P

    Journal: Scientific Reports

    Article Title: Identification of a nucleotide in 5′ untranslated region contributing to virus replication and virulence of Coxsackievirus A16

    doi: 10.1038/srep20839

    Figure Lengend Snippet: Effect of WT or M2 mutant 5′UTR of CA16 on viral RNA synthesis and viral translational capacity in transfected HEK293T cells. HEK293T cells were transfected with same amount of the WT or M2 infectious clone or empty vector, and were harvested at 12 h, 24 h, 36 h and 48 h after transfection. ( A ) RNA was extracted from a portion of each sample, treated with Dnase to degrade the transfected plasmid. DNA and then analyzed by RT-qPCR with primers specific for GAPDH or CA16 VP1 RNA. GAPDH was used as a control. The RNA level obtained by transfection with WT 5′UTR was normalized to 100%. ( B ) A portion of each cell sample was used to detect the VP1 protein by Western blot. ( C ) A portion of each cell sample was used to detect the viral titer. ( D ) Viral loads in supernatant of transfected HEK293T cells at 48h after transfection were detected by RT-qPCR with primers specific for CA16 VP1 RNA. ( E ) The VP1 protein of the WT or M2 virus in the supernatant was detected by Western blot using VP1 antibody. * P

    Article Snippet: A 70% confluent monolayer of HEK293T cells in a T25 flask (BD Biosciences, Franklin Lakes, NJ, USA) was transfected with pcDNA3.1-T7 DNA Pol (1 μg) and CA16 infectious clone (2 μg) or mutants digested by the restriction enzyme Sac I (Takara) using 9 μl Lipofectamine 2000 reagent (Invitrogen) and then incubated at 37 °C in 5 ml of DMEM (10% FCS).

    Techniques: Mutagenesis, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot

    ALKBH5 demethylates m 6 A but not m 6 A m in mRNA in HEK293T cells a , ALKBH5 expression does not decrease m 6 A m in HEK293T cells. The relative abundance of modified adenosines in mRNA caps of HEK293T cells expressing GST vector (Ctrl) or ALKBH5 with an N-terminal GST tag (GST–ALKBH5) was determined by 2D TLC. When determining the ratio of m 6 A m to A m , we did not observe a significant decrease of m 6 A m in ALKBH5-overexpressing cells, indicating that ALKBH5 does not convert m 6 A m to A m in vivo (representative images show n; n = 3 biologic al replic ates; me an ± s.e.m.). b , ALKBH5 knockdown does not increase m 6 A m in HEK293T cells. The relative abundance of modified adenosines in mRNA caps of HEK293T cells transfected with scrambled siRNA (siCtrl) or siRNA directed against ALKBH5 (siALKBH5) was determined by 2D TLC. When determining the ratio of m 6 A m to A m , we did not observe a significant increase of m 6 A m in ALKBH5-expressing cells, indicating that ALKBH5 does not convert m 6 A m to A m in vivo (repres entative images shown; n = 3 biological replicates; mean ± s.e.m.). c , ALKBH5 knockdown increases m 6 A in HEK293T cells. The relative abundance of m 6 A versus (A + C + U) in mRNA of HEK293T cells transfected with scrambled siRNA (siCtrl) or siRNA directed against ALKBH5 (siALKBH5) was determined by 2D TLC. We observed an approximately 30% increase of m 6 A upon ALKBH5 knockdown, indicating that ALKBH5 readily influences the levels of m 6 A in vivo (representative images shown; n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t -test, * P ≤ 0.05). d , ALKBH5 expression decreases m 6 A in HEK293T cells. The relative abundance of m 6 A versus (A + C + U) in mRNA of HEK293T cells expressing GST vector (Ctrl) or ALKBH5 with an N-terminal GST tag (GST-ALKBH5) was determined by 2D TLC. We observed a significant decrease of m 6 A upon ALKBH5 expression, indicating that SLKBH5 readily influences levels of m 6 A in vivo (representative images shown; n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t -test, ** P ≤ 0.01).

    Journal: Nature

    Article Title: Reversible methylation of m6Am in the 5′ cap controls mRNA stability

    doi: 10.1038/nature21022

    Figure Lengend Snippet: ALKBH5 demethylates m 6 A but not m 6 A m in mRNA in HEK293T cells a , ALKBH5 expression does not decrease m 6 A m in HEK293T cells. The relative abundance of modified adenosines in mRNA caps of HEK293T cells expressing GST vector (Ctrl) or ALKBH5 with an N-terminal GST tag (GST–ALKBH5) was determined by 2D TLC. When determining the ratio of m 6 A m to A m , we did not observe a significant decrease of m 6 A m in ALKBH5-overexpressing cells, indicating that ALKBH5 does not convert m 6 A m to A m in vivo (representative images show n; n = 3 biologic al replic ates; me an ± s.e.m.). b , ALKBH5 knockdown does not increase m 6 A m in HEK293T cells. The relative abundance of modified adenosines in mRNA caps of HEK293T cells transfected with scrambled siRNA (siCtrl) or siRNA directed against ALKBH5 (siALKBH5) was determined by 2D TLC. When determining the ratio of m 6 A m to A m , we did not observe a significant increase of m 6 A m in ALKBH5-expressing cells, indicating that ALKBH5 does not convert m 6 A m to A m in vivo (repres entative images shown; n = 3 biological replicates; mean ± s.e.m.). c , ALKBH5 knockdown increases m 6 A in HEK293T cells. The relative abundance of m 6 A versus (A + C + U) in mRNA of HEK293T cells transfected with scrambled siRNA (siCtrl) or siRNA directed against ALKBH5 (siALKBH5) was determined by 2D TLC. We observed an approximately 30% increase of m 6 A upon ALKBH5 knockdown, indicating that ALKBH5 readily influences the levels of m 6 A in vivo (representative images shown; n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t -test, * P ≤ 0.05). d , ALKBH5 expression decreases m 6 A in HEK293T cells. The relative abundance of m 6 A versus (A + C + U) in mRNA of HEK293T cells expressing GST vector (Ctrl) or ALKBH5 with an N-terminal GST tag (GST-ALKBH5) was determined by 2D TLC. We observed a significant decrease of m 6 A upon ALKBH5 expression, indicating that SLKBH5 readily influences levels of m 6 A in vivo (representative images shown; n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t -test, ** P ≤ 0.01).

    Article Snippet: FTO and ALKBH5 expression experiments were carried out in HEK293T cells using LipoD293 transfection reagent (Signagen) with Flag-tagged full length human wild-type FTO, human wild-type FTO containing a Flag tag and two nuclear export signals (NES) at the N terminus, GST-tagged ALKBH5 lacking 66 N-terminal amino acids, or respective control vectors.

    Techniques: Expressing, Modification, Plasmid Preparation, Thin Layer Chromatography, In Vivo, Transfection

    m 6 A m mRNAs show increased translation efficiency a , mRNA translation efficiency is associated with the modification state of the first encoded nucleotide in HEK293 cells. Cumulative distribution plot of the translation efficiency for mRNAs that start with m 6 A m , A m , C m , G m and U m . The translation efficiency of mRNAs starting with an m 6 A m is significantly higher compared to mRNAs starting wit h A m , C m , G m or U m ( n = 3,024 (m 6 A m ); 921 (A m ); 1,788 (C m ); 1,351 (G m ); 2,008 (U m ; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; grey dots represent outliers; one-way ANOVA with Tukey's post hoc test, * P ≤2.3 × 10 −2 versus m 6 A m ). b , Correlation of translation efficiency replicates derived HEK293T cells. The Pearson correlation coefficient (r) is shown. c , Distribution of reads between the coding sequence (CDS) and UTRs. High coverage in the CDS compared to UTRs verifies ribosome-derived footprints. d , Total number of ribosome footprints near the start and stop codon of transcripts. e , Three-nucleotide periodicity demonstrates ribosome-derived footprints. f , Position of ribosome footprints relative to the reading frame.

    Journal: Nature

    Article Title: Reversible methylation of m6Am in the 5′ cap controls mRNA stability

    doi: 10.1038/nature21022

    Figure Lengend Snippet: m 6 A m mRNAs show increased translation efficiency a , mRNA translation efficiency is associated with the modification state of the first encoded nucleotide in HEK293 cells. Cumulative distribution plot of the translation efficiency for mRNAs that start with m 6 A m , A m , C m , G m and U m . The translation efficiency of mRNAs starting with an m 6 A m is significantly higher compared to mRNAs starting wit h A m , C m , G m or U m ( n = 3,024 (m 6 A m ); 921 (A m ); 1,788 (C m ); 1,351 (G m ); 2,008 (U m ; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; grey dots represent outliers; one-way ANOVA with Tukey's post hoc test, * P ≤2.3 × 10 −2 versus m 6 A m ). b , Correlation of translation efficiency replicates derived HEK293T cells. The Pearson correlation coefficient (r) is shown. c , Distribution of reads between the coding sequence (CDS) and UTRs. High coverage in the CDS compared to UTRs verifies ribosome-derived footprints. d , Total number of ribosome footprints near the start and stop codon of transcripts. e , Three-nucleotide periodicity demonstrates ribosome-derived footprints. f , Position of ribosome footprints relative to the reading frame.

    Article Snippet: FTO and ALKBH5 expression experiments were carried out in HEK293T cells using LipoD293 transfection reagent (Signagen) with Flag-tagged full length human wild-type FTO, human wild-type FTO containing a Flag tag and two nuclear export signals (NES) at the N terminus, GST-tagged ALKBH5 lacking 66 N-terminal amino acids, or respective control vectors.

    Techniques: Modification, Derivative Assay, Sequencing

    Newly mapped m 6 A m clusters overlap with transcription start sites (TSS) and the YYANW initiator core motif and mark mRNAs for increased half-life a , b , To confirm that that the residues identified as m 6 A m in miCLIP reflect transcription initiation sites, we searched for known TSS and transcription initiation sequences around each m 6 A m -containing region. Notably, owing to the calling algorithm, these regions do not contain any 5′ UTR m 6 A. To identify genome-wide positions of the TSS we used published CAGE-seq datasets (see Methods). Shown is the nucleotide distance of the called m 6 A m from TSS ( a ) and YYANW ( b ). These results demonstrate that TSS and the YYANW core initiator sequence are highly clustered at m 6 A m -containing regions (5′-most nucleotide is at position 0 on the x -axis). This suggests that the called m 6 A m -containing regions reflect true TSS. c , Related to . Correlation of half-life replicates derived from Flag-transfected (Ctrl, left scatter plot) or Flag-NES-FTO-transfected (NES-FTO, right scatter plot) HEK293T cells. The Pearson correlation coefficient (r) is shown for each comparison and indicates high correlation between replicates. d , mRNA stability is determined by the modification state of the first encoded nucleotide in HeLa cells. Cumulative distribution plot of the half-life for mRNAs that start with m 6 A m , A m , C m , G m and U m . The half-life of mRNAs starting with an m 6 A m is approximately 2.5 h longer compared to mRNAs starting with A m , C m , G m or U m . Notably, for this analysis we used m 6 A m . This allowed us to determine if the stabilizing effect of m 6 A m on mRNA half-lives is conserved across different cell types. Indeed, the increase in m 6 A m (n = 2,401 (m 6 A m ); 645 (A m ); 1,310 (C m ); 988 (G m ); 1,533 (U m ); data represents the average from two independent data sets; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; grey dots represent outliers; one-way ANOVA with Tukey's post hoc test, *** P

    Journal: Nature

    Article Title: Reversible methylation of m6Am in the 5′ cap controls mRNA stability

    doi: 10.1038/nature21022

    Figure Lengend Snippet: Newly mapped m 6 A m clusters overlap with transcription start sites (TSS) and the YYANW initiator core motif and mark mRNAs for increased half-life a , b , To confirm that that the residues identified as m 6 A m in miCLIP reflect transcription initiation sites, we searched for known TSS and transcription initiation sequences around each m 6 A m -containing region. Notably, owing to the calling algorithm, these regions do not contain any 5′ UTR m 6 A. To identify genome-wide positions of the TSS we used published CAGE-seq datasets (see Methods). Shown is the nucleotide distance of the called m 6 A m from TSS ( a ) and YYANW ( b ). These results demonstrate that TSS and the YYANW core initiator sequence are highly clustered at m 6 A m -containing regions (5′-most nucleotide is at position 0 on the x -axis). This suggests that the called m 6 A m -containing regions reflect true TSS. c , Related to . Correlation of half-life replicates derived from Flag-transfected (Ctrl, left scatter plot) or Flag-NES-FTO-transfected (NES-FTO, right scatter plot) HEK293T cells. The Pearson correlation coefficient (r) is shown for each comparison and indicates high correlation between replicates. d , mRNA stability is determined by the modification state of the first encoded nucleotide in HeLa cells. Cumulative distribution plot of the half-life for mRNAs that start with m 6 A m , A m , C m , G m and U m . The half-life of mRNAs starting with an m 6 A m is approximately 2.5 h longer compared to mRNAs starting with A m , C m , G m or U m . Notably, for this analysis we used m 6 A m . This allowed us to determine if the stabilizing effect of m 6 A m on mRNA half-lives is conserved across different cell types. Indeed, the increase in m 6 A m (n = 2,401 (m 6 A m ); 645 (A m ); 1,310 (C m ); 988 (G m ); 1,533 (U m ); data represents the average from two independent data sets; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; grey dots represent outliers; one-way ANOVA with Tukey's post hoc test, *** P

    Article Snippet: FTO and ALKBH5 expression experiments were carried out in HEK293T cells using LipoD293 transfection reagent (Signagen) with Flag-tagged full length human wild-type FTO, human wild-type FTO containing a Flag tag and two nuclear export signals (NES) at the N terminus, GST-tagged ALKBH5 lacking 66 N-terminal amino acids, or respective control vectors.

    Techniques: Genome Wide, Sequencing, Derivative Assay, Transfection, Modification

    Expression changes of m 6 A m , m 6 A and A m mRNAs upon NES-FTO expression and FTO or ALKBH5 deficiency a , m 6 A m mRNAs exhibit increased half-life compared to A m mRNAs in vivo. HEK293T cells were electroporated with in vitro -synthesized mRNAsstarting with either of two extended caps: m 7 Gppp A m or m 7 Gpppm 6 A m .We then isolated cellular poly(A) RNA and determined the in vivo half-life of the electroporated A m - and m 6 A m -containing mRNA by qRT-PCR.In control siRNA-treated HEK293T cells (siCtrl), the m 6 A m mRNAshowed a trend towards increased half-life compared to the A m mRNA(unpaired Student's t -test, P = 0.08). Notably, when we performed the sameexperiment in FTO siRNA-treated cells (siFTO) to prevent demethylationof m 6 A m , the m 6 A m mRNA half-life was significantly increased ( n = 3biological replicates; mean ± s.e.m.; unpaired Student's t -test, P ≤ 0.05). b , NES-FTO expression preferentially affects the half-life of m 6 A m mRNAscompared to m 6 A mRNAs. Changes in half-life of mRNAs containingeither m 6 A m or m 6 A in HEK293T cells transfected with either Flag vector(Ctrl) or FTO with an N-terminal nuclear export signal (NES-FTO)were determined by RNA-seq. m 6 A m mRNAs are generally long-lived (see ) and show reduced half-lives after NES–FTO expression. We asked if FTO could elicit a similar effect on mRNAs containing m 6 A. For this experiment, we used a set of mRNAs with annotated m 6 , excluding those which also contain an annotated m 6 A m . NES-FTO expression reduced the half-life of m 6 A m mRNAs but did not have any substantial effect on the half-life of m 6 A mRNAs. These data support the idea that FTO preferentially targets m 6 A m compared to m 6 A ( n = 2,049 (m 6 A m ); 2,495 (m 6 A); data represent the average from two independent datasets; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; grey dots represent outliers; one-way ANOVA with Tukey's post hoc test, *** P ≤ 2.2 × 10 −16 versus m 6 A). c , NES-FTO expression preferentially affects the half-life of m 6 A m mRNAs compared to A m mRNAs. Changes in half-life of A m mRNAs ( FUCA1, PCK1, SCFD2 ) and m 6 A m mRNAs (PCNA, PSMD3, MAGOHB) in HEK293T cells transfected with either Flag vector (Ctrl) or FTO with an N-terminal nuclear export signal (NES-FTO) were determined by BrU pulse-chase analysis and subsequent qRT-PCR. m 6 A m mRNAs show a significant reduction in half-life after NES-FTO expression whereas the half-life of A m and also demonstrate the stabilization effect of m 6 A m using a different method to measure mRNA half-life (that is, BrU pulse-chase labelling) other than transcriptional inhibition ( n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t -test, * P ≤ 0.05, ** P ≤ 0.01). d , The expression of mRNAs containing either m 6 A m or A m upon Fto knockout was determined by RNA-seq. FTO depletion ( Fto −/− ) results in increased abundance of mRNAs with an annotated m 6 A m residue in liver tissue derived from Fto -knockout mice. Fold change was measured relative to the RNA levels measured in the same tissue obtained from wild-type littermates ( n = 2,048 (m 6 A m ); 1,025 (A m ); 2,081 (C m ); 1,742 (G m ); 1,242 (U m ); data represent the average from two independent data sets; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; grey dots represent outliers; one-way ANOVA with Tukey's post hoc test, *** P ≤ 7.5 × 10 −6 m 6 A m versus A m and U m ). e , Knockdown of ALKBH5 does not increase the levels of m 6 A m mRNAs. The expression of mRNAs containing either m 6 A m or A m upon ALKBH5 knockdown in HEK293T cells was determined by RNA-seq. In contrast to knockdown or knockout of FTO, m 6 A m mRNAs are slightly less abundant than A m mRNAs in ALKBH5 -knockdown cells. This suggests that ALKBH5 does not target m 6 A m -containing mRNAs in vivo ( n = 3,111 (m 6 A m ); 1,928 (A m ); 4,382 (C m ); 3,110 (G m ); 3,998 (U m ); data represent the average from two independent datasets; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; grey dots represent outliers; one-way ANOVA with Tukey's post hoc test, ** P ≤ 1.2 × 10 −3 m 6 A m versus A m and U m ). Fig. 3a

    Journal: Nature

    Article Title: Reversible methylation of m6Am in the 5′ cap controls mRNA stability

    doi: 10.1038/nature21022

    Figure Lengend Snippet: Expression changes of m 6 A m , m 6 A and A m mRNAs upon NES-FTO expression and FTO or ALKBH5 deficiency a , m 6 A m mRNAs exhibit increased half-life compared to A m mRNAs in vivo. HEK293T cells were electroporated with in vitro -synthesized mRNAsstarting with either of two extended caps: m 7 Gppp A m or m 7 Gpppm 6 A m .We then isolated cellular poly(A) RNA and determined the in vivo half-life of the electroporated A m - and m 6 A m -containing mRNA by qRT-PCR.In control siRNA-treated HEK293T cells (siCtrl), the m 6 A m mRNAshowed a trend towards increased half-life compared to the A m mRNA(unpaired Student's t -test, P = 0.08). Notably, when we performed the sameexperiment in FTO siRNA-treated cells (siFTO) to prevent demethylationof m 6 A m , the m 6 A m mRNA half-life was significantly increased ( n = 3biological replicates; mean ± s.e.m.; unpaired Student's t -test, P ≤ 0.05). b , NES-FTO expression preferentially affects the half-life of m 6 A m mRNAscompared to m 6 A mRNAs. Changes in half-life of mRNAs containingeither m 6 A m or m 6 A in HEK293T cells transfected with either Flag vector(Ctrl) or FTO with an N-terminal nuclear export signal (NES-FTO)were determined by RNA-seq. m 6 A m mRNAs are generally long-lived (see ) and show reduced half-lives after NES–FTO expression. We asked if FTO could elicit a similar effect on mRNAs containing m 6 A. For this experiment, we used a set of mRNAs with annotated m 6 , excluding those which also contain an annotated m 6 A m . NES-FTO expression reduced the half-life of m 6 A m mRNAs but did not have any substantial effect on the half-life of m 6 A mRNAs. These data support the idea that FTO preferentially targets m 6 A m compared to m 6 A ( n = 2,049 (m 6 A m ); 2,495 (m 6 A); data represent the average from two independent datasets; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; grey dots represent outliers; one-way ANOVA with Tukey's post hoc test, *** P ≤ 2.2 × 10 −16 versus m 6 A). c , NES-FTO expression preferentially affects the half-life of m 6 A m mRNAs compared to A m mRNAs. Changes in half-life of A m mRNAs ( FUCA1, PCK1, SCFD2 ) and m 6 A m mRNAs (PCNA, PSMD3, MAGOHB) in HEK293T cells transfected with either Flag vector (Ctrl) or FTO with an N-terminal nuclear export signal (NES-FTO) were determined by BrU pulse-chase analysis and subsequent qRT-PCR. m 6 A m mRNAs show a significant reduction in half-life after NES-FTO expression whereas the half-life of A m and also demonstrate the stabilization effect of m 6 A m using a different method to measure mRNA half-life (that is, BrU pulse-chase labelling) other than transcriptional inhibition ( n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t -test, * P ≤ 0.05, ** P ≤ 0.01). d , The expression of mRNAs containing either m 6 A m or A m upon Fto knockout was determined by RNA-seq. FTO depletion ( Fto −/− ) results in increased abundance of mRNAs with an annotated m 6 A m residue in liver tissue derived from Fto -knockout mice. Fold change was measured relative to the RNA levels measured in the same tissue obtained from wild-type littermates ( n = 2,048 (m 6 A m ); 1,025 (A m ); 2,081 (C m ); 1,742 (G m ); 1,242 (U m ); data represent the average from two independent data sets; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; grey dots represent outliers; one-way ANOVA with Tukey's post hoc test, *** P ≤ 7.5 × 10 −6 m 6 A m versus A m and U m ). e , Knockdown of ALKBH5 does not increase the levels of m 6 A m mRNAs. The expression of mRNAs containing either m 6 A m or A m upon ALKBH5 knockdown in HEK293T cells was determined by RNA-seq. In contrast to knockdown or knockout of FTO, m 6 A m mRNAs are slightly less abundant than A m mRNAs in ALKBH5 -knockdown cells. This suggests that ALKBH5 does not target m 6 A m -containing mRNAs in vivo ( n = 3,111 (m 6 A m ); 1,928 (A m ); 4,382 (C m ); 3,110 (G m ); 3,998 (U m ); data represent the average from two independent datasets; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; grey dots represent outliers; one-way ANOVA with Tukey's post hoc test, ** P ≤ 1.2 × 10 −3 m 6 A m versus A m and U m ). Fig. 3a

    Article Snippet: FTO and ALKBH5 expression experiments were carried out in HEK293T cells using LipoD293 transfection reagent (Signagen) with Flag-tagged full length human wild-type FTO, human wild-type FTO containing a Flag tag and two nuclear export signals (NES) at the N terminus, GST-tagged ALKBH5 lacking 66 N-terminal amino acids, or respective control vectors.

    Techniques: Expressing, In Vivo, In Vitro, Synthesized, Isolation, Quantitative RT-PCR, Transfection, Plasmid Preparation, RNA Sequencing Assay, Pulse Chase, Inhibition, Knock-Out, Derivative Assay, Mouse Assay

    m 6 A m mRNAs are resistant to DCP2-mediated decapping and microRNA-mediated gene silencing a , DCP2 decapping products are m 7 GDP. Here we confirm the identity of the putative m 7 GDP decapping product in the decapping assay by treatment with nucleoside-diphosphate kinase (NDPK). The shift to the m 7 GTP position confirms that the released product is m 7 GDP. A cap-labelled RNA with a guanosine as the first nucleotide was used as a positive control (lanes 3, 6, 9; the red ‘p’ denotes the position of the 32 P). b , Michaelis-Menten curves of 10 nM DCP2 reacting with m 7 Gpppm 6 A m (blue) or m 7 GpppA m (orange) for 30 min at 37 °C. DCP2 shows higher decapping activity towards m 7 GpppA m than to m 7 Gpppm 6 A m (the dashed lines indicate the K m on the × axis; n = 3 biological replicates; mean ± s.e.m.). c , DCP2 depletion preferentially stabilizes A m mRNAs compared to m 6 A m mRNAs. Changes in half-life of A m mRNAs ( FUCA1, PCK1, SCFD2 ) and m 6 A m mRNAs (PCNA, PSMD3, MAGOHB) in HEK293T cells transfected with either Flag vector (Ctrl) or DCP2 -knockout cells ( DCP2 −/− ) were determined by BrU pulse-chase analysis and subsequent qRT-PCR. A m mRNAs show a significant increase in half-life after DCP2 depletion whereas the half-life of m 6 A m mRNAs was not significantly increased. These data are related to the whole-transcriptome expression analysis presented in and indicate that, in addition to the observed abundance changes of non-m 6 A m mRNAs versus m 6 A m mRNAs, DCP2 also selectively affects the half-life of specifically examined mRNAs ( n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t -test, * P ≤ 0.05, ** P ≤ 0.01). d , we found that m 6 A m mRNAs show less upregulation upon DICER knockdown than mRNAs beginning with other nucleotides. We wanted to further examine this concept using additional independent datasets of gene expression following depletion of proteins required for microRNA-mediated mRNA degradation, such as members of the Argonaute protein family. Measurement of mRNA expression in AGO2 revealed more pronounced upregulation of non-m 6 A m mRNAs compared to those that have m 6 A m (n = 2,080 (m 6 A m ); 596 (A m ); 1,085 (C m ); 805 (G m ); 1,274 (U m ); data represent the average from two independent datasets; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; grey dots represent outliers; one-way ANOVA with Tukey's post hoc test, *** P ≤ 1 × 10 −4 m 6 A m versus A m , C m and U m ). e , but here we only look at the expression changes of mRNAs that contain TargetScan-predicted microRNA-binding sites. Applying this filter criteria, we also observed that DICER resulted in more pronounced upregulation of non-m 6 A m miRNA target mRNAs compared to those that have m 6 A m (n = 1,208 (m 6 A m ); 359 (A m ); 607 (C m ); 467 (G m ); 713 (U m ); data represent the average from two independent datasets; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; grey dots represent outliers; one-way ANOVA with Tukey's post hoc test, *** P ≤ 9.6 × 10 −4 versus m 6 A m N m , where N m = A m , C m , G m or U m ). f we show that m 6 A m mRNAs exhibit less upregulation upon DICER knockdown than mRNAs beginning with other nucleotides. We wanted to examine this concept further using additional filtering criteria. Thus, we asked if m 6 A m mRNA resistance to DICER depletion is dependent on the number of microRNA-binding sites. Therefore, we divided mRNAs into five groups: mRNAs that do not contain a predicted microRNA-binding site (0) and mRNAs that belong to specific quartiles that we assigned depending on the number of microRNA-binding sites (low (1) to high (4)). Notably, we did not observe any expression difference between m 6 A m mRNAs and non-m 6 A m mRNAs that do not carry predicted microRNA-binding sites. However, there was a clear increase in mRNA expression for mRNAs that contain microRNA-binding sites, and this increase was dependent on the number of microRNA-binding sites. Notably, for each quartile, m 6 A m mRNAs were significantly less upregulated than N m mRNAs ( n = 91 versus 89 (m 6 A m versus N m ; 1), 252 versus 339 (m 6 A m versus N m ; 1), 311 versus 454 (m 6 A m versus N m ; 2), 247 versus 541 (m 6 A m versus N m ; 3), 229 versus 512 (m 6 A m versus N m ; 4); data represent the average from two independent datasets; number of microRNA-binding sites in each quartile: 1 = 1–3; 2 = 4-6; 3 = 7–12; 4 = 13-54; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; one-way ANOVA with Tukey's post hoc test, * P ≤ 0.05, *** P ≤ 0.001, n.s., not significant). g we show that m 6 A m mRNAs are largely resistant to expression changes upon global inhibition of the microRNA machinery. We next asked whether introduction of a single microRNA also leads to differential responses of m 6 A m mRNAs compared to non-m 6 A m . For this analysis, we used m 6 A m and non-target mRNAs in the HeLa cell dataset. Indeed, miR-155 target mRNAs were significantly more suppressed in miR-155-transfected HeLa cells. This confirms that miR-155 target mRNA degradation can be detected in this dataset ( n = 1,131 (target); 7,700 (non-target; data represent the average from two independent datasets; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; grey dots represent outliers; one-way ANOVA with Tukey's post hoc test, ** P ≤ 2.2 × 10 −16 ). h , m 6 A m mRNAs show resistance to miR-155-mediated mRNA degradation. We tested if the identity of the first nucleotide affects the response of miR-155 target mRNAs to miR-155-mediated mRNA degradation. We observed that miR-155 target mRNAs that start with m 6 A m show no significant suppression upon miR-155 transfection compared to non-target mRNAs that start with m 6 A m . However, expression of miR-155 target mRNAs that start with A m , C m , G m or U m was significantly suppressed compared to non-target mRNAs that start with A m , C m , G m or U m . These data suggest that the presence m 6 A m can reduce the silencing efficiency of a single microRNA in vivo (n = 1,714 versus 232 (m 6 A m , non-target versus target); 953 versus 158 (A m , non-target versus target); 1,848 versus 281 (C m , non-target versus target); 1,394 versus 182 (G m ); 1,809 versus 278 (U m , non-target versus target); each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; one-way ANOVA with Tukey's post hoc test, * P ≤ 0.05 non-target versus miR-155 target, ** P ≤ 0.01 non-target versus miR-155 target, *** P ≤ 0.001 non-target versus miR-155 target). Fig. 4c

    Journal: Nature

    Article Title: Reversible methylation of m6Am in the 5′ cap controls mRNA stability

    doi: 10.1038/nature21022

    Figure Lengend Snippet: m 6 A m mRNAs are resistant to DCP2-mediated decapping and microRNA-mediated gene silencing a , DCP2 decapping products are m 7 GDP. Here we confirm the identity of the putative m 7 GDP decapping product in the decapping assay by treatment with nucleoside-diphosphate kinase (NDPK). The shift to the m 7 GTP position confirms that the released product is m 7 GDP. A cap-labelled RNA with a guanosine as the first nucleotide was used as a positive control (lanes 3, 6, 9; the red ‘p’ denotes the position of the 32 P). b , Michaelis-Menten curves of 10 nM DCP2 reacting with m 7 Gpppm 6 A m (blue) or m 7 GpppA m (orange) for 30 min at 37 °C. DCP2 shows higher decapping activity towards m 7 GpppA m than to m 7 Gpppm 6 A m (the dashed lines indicate the K m on the × axis; n = 3 biological replicates; mean ± s.e.m.). c , DCP2 depletion preferentially stabilizes A m mRNAs compared to m 6 A m mRNAs. Changes in half-life of A m mRNAs ( FUCA1, PCK1, SCFD2 ) and m 6 A m mRNAs (PCNA, PSMD3, MAGOHB) in HEK293T cells transfected with either Flag vector (Ctrl) or DCP2 -knockout cells ( DCP2 −/− ) were determined by BrU pulse-chase analysis and subsequent qRT-PCR. A m mRNAs show a significant increase in half-life after DCP2 depletion whereas the half-life of m 6 A m mRNAs was not significantly increased. These data are related to the whole-transcriptome expression analysis presented in and indicate that, in addition to the observed abundance changes of non-m 6 A m mRNAs versus m 6 A m mRNAs, DCP2 also selectively affects the half-life of specifically examined mRNAs ( n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t -test, * P ≤ 0.05, ** P ≤ 0.01). d , we found that m 6 A m mRNAs show less upregulation upon DICER knockdown than mRNAs beginning with other nucleotides. We wanted to further examine this concept using additional independent datasets of gene expression following depletion of proteins required for microRNA-mediated mRNA degradation, such as members of the Argonaute protein family. Measurement of mRNA expression in AGO2 revealed more pronounced upregulation of non-m 6 A m mRNAs compared to those that have m 6 A m (n = 2,080 (m 6 A m ); 596 (A m ); 1,085 (C m ); 805 (G m ); 1,274 (U m ); data represent the average from two independent datasets; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; grey dots represent outliers; one-way ANOVA with Tukey's post hoc test, *** P ≤ 1 × 10 −4 m 6 A m versus A m , C m and U m ). e , but here we only look at the expression changes of mRNAs that contain TargetScan-predicted microRNA-binding sites. Applying this filter criteria, we also observed that DICER resulted in more pronounced upregulation of non-m 6 A m miRNA target mRNAs compared to those that have m 6 A m (n = 1,208 (m 6 A m ); 359 (A m ); 607 (C m ); 467 (G m ); 713 (U m ); data represent the average from two independent datasets; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; grey dots represent outliers; one-way ANOVA with Tukey's post hoc test, *** P ≤ 9.6 × 10 −4 versus m 6 A m N m , where N m = A m , C m , G m or U m ). f we show that m 6 A m mRNAs exhibit less upregulation upon DICER knockdown than mRNAs beginning with other nucleotides. We wanted to examine this concept further using additional filtering criteria. Thus, we asked if m 6 A m mRNA resistance to DICER depletion is dependent on the number of microRNA-binding sites. Therefore, we divided mRNAs into five groups: mRNAs that do not contain a predicted microRNA-binding site (0) and mRNAs that belong to specific quartiles that we assigned depending on the number of microRNA-binding sites (low (1) to high (4)). Notably, we did not observe any expression difference between m 6 A m mRNAs and non-m 6 A m mRNAs that do not carry predicted microRNA-binding sites. However, there was a clear increase in mRNA expression for mRNAs that contain microRNA-binding sites, and this increase was dependent on the number of microRNA-binding sites. Notably, for each quartile, m 6 A m mRNAs were significantly less upregulated than N m mRNAs ( n = 91 versus 89 (m 6 A m versus N m ; 1), 252 versus 339 (m 6 A m versus N m ; 1), 311 versus 454 (m 6 A m versus N m ; 2), 247 versus 541 (m 6 A m versus N m ; 3), 229 versus 512 (m 6 A m versus N m ; 4); data represent the average from two independent datasets; number of microRNA-binding sites in each quartile: 1 = 1–3; 2 = 4-6; 3 = 7–12; 4 = 13-54; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; one-way ANOVA with Tukey's post hoc test, * P ≤ 0.05, *** P ≤ 0.001, n.s., not significant). g we show that m 6 A m mRNAs are largely resistant to expression changes upon global inhibition of the microRNA machinery. We next asked whether introduction of a single microRNA also leads to differential responses of m 6 A m mRNAs compared to non-m 6 A m . For this analysis, we used m 6 A m and non-target mRNAs in the HeLa cell dataset. Indeed, miR-155 target mRNAs were significantly more suppressed in miR-155-transfected HeLa cells. This confirms that miR-155 target mRNA degradation can be detected in this dataset ( n = 1,131 (target); 7,700 (non-target; data represent the average from two independent datasets; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; grey dots represent outliers; one-way ANOVA with Tukey's post hoc test, ** P ≤ 2.2 × 10 −16 ). h , m 6 A m mRNAs show resistance to miR-155-mediated mRNA degradation. We tested if the identity of the first nucleotide affects the response of miR-155 target mRNAs to miR-155-mediated mRNA degradation. We observed that miR-155 target mRNAs that start with m 6 A m show no significant suppression upon miR-155 transfection compared to non-target mRNAs that start with m 6 A m . However, expression of miR-155 target mRNAs that start with A m , C m , G m or U m was significantly suppressed compared to non-target mRNAs that start with A m , C m , G m or U m . These data suggest that the presence m 6 A m can reduce the silencing efficiency of a single microRNA in vivo (n = 1,714 versus 232 (m 6 A m , non-target versus target); 953 versus 158 (A m , non-target versus target); 1,848 versus 281 (C m , non-target versus target); 1,394 versus 182 (G m ); 1,809 versus 278 (U m , non-target versus target); each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; one-way ANOVA with Tukey's post hoc test, * P ≤ 0.05 non-target versus miR-155 target, ** P ≤ 0.01 non-target versus miR-155 target, *** P ≤ 0.001 non-target versus miR-155 target). Fig. 4c

    Article Snippet: FTO and ALKBH5 expression experiments were carried out in HEK293T cells using LipoD293 transfection reagent (Signagen) with Flag-tagged full length human wild-type FTO, human wild-type FTO containing a Flag tag and two nuclear export signals (NES) at the N terminus, GST-tagged ALKBH5 lacking 66 N-terminal amino acids, or respective control vectors.

    Techniques: Positive Control, Activity Assay, Transfection, Plasmid Preparation, Knock-Out, Pulse Chase, Quantitative RT-PCR, Expressing, Binding Assay, Inhibition, In Vivo

    6 A m is the preferred substrate for FTO in vivo a , Modifications of the extended mRNA cap. The first nucleotide adjacent to the m 7 G and the 5′-to-5′-triphosphate (ppp) linker is subjected to 2′- O -methylation (orange) on the ribose, forming cap1. Cap1 can be further 2′- O -methylated at the second nucleotide to form cap2 (not depicted here). If cap1 contains a 2′- O -methyladenosine (A m ), it can be further converted to cap1m by N 6 -methylation (blue), which results in N 6 ,2′- O -dimethyladenosine (m 6 A m ). b , Relative abundance of m 6 A in mRNA treated with recombinant FTO. Internal m 6 . The relative abundance of m 6 A versus (A + C + U) in 400 ng mRNA that was either left untreated (—FTO) or incubated for 1 h with 1 μM bacterially expressed recombinant human FTO (+FTO) was determined by 2D TLC. We did not observe any decrease of m 6 A in FTO-treated mRNA, indicating that FTO does not efficiently demethylate m 6 A in its physiological context in mRNA in vitro (representative images shown; n = 3 biological replicates; mean ± s.e.m.). c , FTO with a nuclear export signal is localized in the cytoplasm. Immunofluorescence staining of DDDDK/Flag tag in HEK293T cells transfected with Flag-tagged wild type FTO (Flag-FTO) or Flag-tagged FTO with an N-terminal nuclear export signal (NES-FTO). FTO is primarily nuclear while NES-FTO is readily detected in the cytosol. DAPI was used to stain nuclei (representative images shown). d , Western blot analyses were performed to verify successful knockdown, overexpression and knockout. Top left, cell extracts from HEK293T cells with FTO . An antibody directed against β-actin was used as a loading control. The lower band represents endogenous FTO, whereas the upper band represents exogenous NES-FTO, which showed approximately tenfold overexpression. Top left, cell extracts from ALKBH5- . β-Actin was used as loading control. Top right, western blot analysis of three different HEK293T clonal lines with CRISPR-mediated knockout of DCP2 that were used for RNA-seq analysis. GAPDH was used as a loading control. e , FTO expression decreases m 6 A m in HEK293T cells. The relative abundance of modified adenosines in mRNA caps of HEK293T expressing Flag vector (Ctrl) or Flag-tagged FTO with an N-terminal nuclear export signal (Flag-NES-FTO) was determined by 2D TLC. When determining the ratio of m 6 A m to A m , we observed a significant decrease of m 6 A m in Flag-NES-FTO-overexpressing cells, indicating that FTO can convert cytoplasmic m 6 A m to A m in vivo. Notably, the ratios of m 6 A m :A m that we observed upon FTO expression (both with and without the NES) may under-represent the true effect of FTO: A m mRNAs are generally less stable than m 6 A m ). Thus the A m mRNAs generated by FTO-mediated demethylation of m 6 A m may not efficiently accumulate in cells compared to m 6 A m mRNAs (representative images shown; n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t -test, * P ≤ 0.01). f , FTO expression does not affect m 6 A in HEK293T cells. The relative abundance of m 6 A versus (A + C + U) in mRNA of HEK293T expressing empty vector (Ctrl) or FTO with an N-terminal nuclear export signal (NES-FTO) was determined by 2D TLC. We did not observe any decrease of m 6 A upon NES-FTO expression, indicating that FTO does not readily influence levels of m 6 A in HEK293T cells at this level of expression. Notably, under these same expression conditions, m 6 A m ) (representative images shown; n = 3 biological replicates; mean ± s.e.m.). Control experiments measuring m 6 A and m 6 A m levels following ALKBH5 . g , FTO deficiency increases m 6 A m in vivo. Relative abundance of modified adenosines in mRNA caps of embryonic day (E) 14 wild-type (WT) littermate controls and Fto knockout (Fto –/ – ) mouse embryos (representative images shown; n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t -test, ** P ≤ 0.01). h , FTO knockdown does not affect m 6 A in HEK293T cells. The relative abundance of m 6 A versus (A + C + U) in mRNA of HEK293T cells transfected with scrambled siRNA (siCtrl) or siRNA directed against FTO (siFTO) was determined by 2D TLC. We did not observe any increase of m 6 A upon FTO knockdown, indicating that FTO does not readily influence levels of m 6 A in vivo (representative images shown; n = 3 biological replicates; mean ± s.e.m.). i , Relative abundance of m 6 A in Fto -knockout mouse embryos. The relative abundance of m 6 A versus (A + C + U) in mRNA of embryonic day 14 wild-type littermate controls and Fto -knockout ( Fto −/− ) mouse embryos was determined by 2D TLC. We did not observe any increase of m 6 A in Fto -deficient embryos, indicating that FTO does not influence the levels of m 6 A in this embryonic stage (representative images shown; n = 3 biological replicates; mean ± s.e.m.). Fig. 3d

    Journal: Nature

    Article Title: Reversible methylation of m6Am in the 5′ cap controls mRNA stability

    doi: 10.1038/nature21022

    Figure Lengend Snippet: 6 A m is the preferred substrate for FTO in vivo a , Modifications of the extended mRNA cap. The first nucleotide adjacent to the m 7 G and the 5′-to-5′-triphosphate (ppp) linker is subjected to 2′- O -methylation (orange) on the ribose, forming cap1. Cap1 can be further 2′- O -methylated at the second nucleotide to form cap2 (not depicted here). If cap1 contains a 2′- O -methyladenosine (A m ), it can be further converted to cap1m by N 6 -methylation (blue), which results in N 6 ,2′- O -dimethyladenosine (m 6 A m ). b , Relative abundance of m 6 A in mRNA treated with recombinant FTO. Internal m 6 . The relative abundance of m 6 A versus (A + C + U) in 400 ng mRNA that was either left untreated (—FTO) or incubated for 1 h with 1 μM bacterially expressed recombinant human FTO (+FTO) was determined by 2D TLC. We did not observe any decrease of m 6 A in FTO-treated mRNA, indicating that FTO does not efficiently demethylate m 6 A in its physiological context in mRNA in vitro (representative images shown; n = 3 biological replicates; mean ± s.e.m.). c , FTO with a nuclear export signal is localized in the cytoplasm. Immunofluorescence staining of DDDDK/Flag tag in HEK293T cells transfected with Flag-tagged wild type FTO (Flag-FTO) or Flag-tagged FTO with an N-terminal nuclear export signal (NES-FTO). FTO is primarily nuclear while NES-FTO is readily detected in the cytosol. DAPI was used to stain nuclei (representative images shown). d , Western blot analyses were performed to verify successful knockdown, overexpression and knockout. Top left, cell extracts from HEK293T cells with FTO . An antibody directed against β-actin was used as a loading control. The lower band represents endogenous FTO, whereas the upper band represents exogenous NES-FTO, which showed approximately tenfold overexpression. Top left, cell extracts from ALKBH5- . β-Actin was used as loading control. Top right, western blot analysis of three different HEK293T clonal lines with CRISPR-mediated knockout of DCP2 that were used for RNA-seq analysis. GAPDH was used as a loading control. e , FTO expression decreases m 6 A m in HEK293T cells. The relative abundance of modified adenosines in mRNA caps of HEK293T expressing Flag vector (Ctrl) or Flag-tagged FTO with an N-terminal nuclear export signal (Flag-NES-FTO) was determined by 2D TLC. When determining the ratio of m 6 A m to A m , we observed a significant decrease of m 6 A m in Flag-NES-FTO-overexpressing cells, indicating that FTO can convert cytoplasmic m 6 A m to A m in vivo. Notably, the ratios of m 6 A m :A m that we observed upon FTO expression (both with and without the NES) may under-represent the true effect of FTO: A m mRNAs are generally less stable than m 6 A m ). Thus the A m mRNAs generated by FTO-mediated demethylation of m 6 A m may not efficiently accumulate in cells compared to m 6 A m mRNAs (representative images shown; n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t -test, * P ≤ 0.01). f , FTO expression does not affect m 6 A in HEK293T cells. The relative abundance of m 6 A versus (A + C + U) in mRNA of HEK293T expressing empty vector (Ctrl) or FTO with an N-terminal nuclear export signal (NES-FTO) was determined by 2D TLC. We did not observe any decrease of m 6 A upon NES-FTO expression, indicating that FTO does not readily influence levels of m 6 A in HEK293T cells at this level of expression. Notably, under these same expression conditions, m 6 A m ) (representative images shown; n = 3 biological replicates; mean ± s.e.m.). Control experiments measuring m 6 A and m 6 A m levels following ALKBH5 . g , FTO deficiency increases m 6 A m in vivo. Relative abundance of modified adenosines in mRNA caps of embryonic day (E) 14 wild-type (WT) littermate controls and Fto knockout (Fto –/ – ) mouse embryos (representative images shown; n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t -test, ** P ≤ 0.01). h , FTO knockdown does not affect m 6 A in HEK293T cells. The relative abundance of m 6 A versus (A + C + U) in mRNA of HEK293T cells transfected with scrambled siRNA (siCtrl) or siRNA directed against FTO (siFTO) was determined by 2D TLC. We did not observe any increase of m 6 A upon FTO knockdown, indicating that FTO does not readily influence levels of m 6 A in vivo (representative images shown; n = 3 biological replicates; mean ± s.e.m.). i , Relative abundance of m 6 A in Fto -knockout mouse embryos. The relative abundance of m 6 A versus (A + C + U) in mRNA of embryonic day 14 wild-type littermate controls and Fto -knockout ( Fto −/− ) mouse embryos was determined by 2D TLC. We did not observe any increase of m 6 A in Fto -deficient embryos, indicating that FTO does not influence the levels of m 6 A in this embryonic stage (representative images shown; n = 3 biological replicates; mean ± s.e.m.). Fig. 3d

    Article Snippet: FTO and ALKBH5 expression experiments were carried out in HEK293T cells using LipoD293 transfection reagent (Signagen) with Flag-tagged full length human wild-type FTO, human wild-type FTO containing a Flag tag and two nuclear export signals (NES) at the N terminus, GST-tagged ALKBH5 lacking 66 N-terminal amino acids, or respective control vectors.

    Techniques: In Vivo, Methylation, Recombinant, Incubation, Thin Layer Chromatography, In Vitro, Immunofluorescence, Staining, FLAG-tag, Transfection, Western Blot, Over Expression, Knock-Out, CRISPR, RNA Sequencing Assay, Expressing, Modification, Plasmid Preparation, Generated