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    ATCC hek 293t cells
    Hek 293t Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 5169 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hek 293 cells
    Hek 293 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12989 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC hek 293 cells
    Optogenetic activation of SthK channels by various PACs in cell lines. a Illustration of the split-PAC-K and fused-PAC-K construct design for SthK co-expression with PACs. b Photocurrents elicited by different split-PAC-K variants after 10 ms exposure to a 470 nm light pulse. Scale bars: 10 s, 0.5 nA. c Normalized integrals of photocurrents as a function of the intensity of a 10 ms light pulse. Arrows denote the EC 50 for current activation by light ( n = 3 cells, two cultures). d Combined whole-cell patch-clamp (black) and optical recording (gray) of a <t>HEK</t> 293 cell expressing fused-bPAC-K and a fluorescent cAMP sensor. bPAC was excited with two 1 ms light flashes (470 nm). The dotted line indicates the zero current level. Scale bars: 20 s, 20 pA (upper trace); 20 s, 2000 aU (lower trace). e Comparison of photocurrent amplitudes generated by bPAC or TpPAC and SthK as split or fused constructs. f Current density comparison at saturating photon exposures ( ** p
    Hek 293 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 6326 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hek293t cells
    Activation of virus entry by different human TTSPs. (A) Experiment set-up. One day before transduction, <t>HEK293T</t> target cells were transfected with the appropriate receptor and one of the TTSPs. To block the cathepsin route, E64d was added at 2 h before and during transduction. (B) SARS-2-S activating capacity of the 18 human TTSPs. At the top of the graph, the four TTSP subfamilies are indicated. (C, D) The four TTSPs that proved active in panel B were evaluated for activation of wild-type and mutant forms of SARS-2-S (panel C), or SARS-1-S, MERS-S and 229E-S (panel D). An ordinary one-way ANOVA with Dunnett’s correction was used to compare SARS-2 mutants versus WT and an unpaired two-tailed t-test was used to compare the WT and mutant forms of SARS-1 and MERS. *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001. Results are the mean ± SEM; N=3 with three independently produced stocks).
    Hek293t Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 36460 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher human embryonic kidney hek 293 cells
    Activation of virus entry by different human TTSPs. (A) Experiment set-up. One day before transduction, <t>HEK293T</t> target cells were transfected with the appropriate receptor and one of the TTSPs. To block the cathepsin route, E64d was added at 2 h before and during transduction. (B) SARS-2-S activating capacity of the 18 human TTSPs. At the top of the graph, the four TTSP subfamilies are indicated. (C, D) The four TTSPs that proved active in panel B were evaluated for activation of wild-type and mutant forms of SARS-2-S (panel C), or SARS-1-S, MERS-S and 229E-S (panel D). An ordinary one-way ANOVA with Dunnett’s correction was used to compare SARS-2 mutants versus WT and an unpaired two-tailed t-test was used to compare the WT and mutant forms of SARS-1 and MERS. *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001. Results are the mean ± SEM; N=3 with three independently produced stocks).
    Human Embryonic Kidney Hek 293 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1827 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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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    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 1112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hek 293 t 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
    Hek 293 T Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1249 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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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    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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    Thermo Fisher geneblazer cckbr nfat bla 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.
    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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    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: 92/100, based on 613 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    OriGene hek293t cells
    Biochemical characterization of interactions between CEACAM1 and TIM-3 a , hTIM-3 does not co-immunoprecipitate (co-IP) with ITGA5 despite interactions with hCEACAM1. <t>HEK293T</t> cells transfected with Flag–ITGA5 and HA–TIM-3 (ITGA5Tw) or Flag–CEACAM1 and HA–TIM-3 (CwTw). Immunoprecipitation with anti-HA antibody and immunoblotted (IB) with anti-Flag antibody are shown. Input represents anti-Flag immunoblot of lysates. b, Co-immunoprecipitation of human TIM-3 and CEACAM1 from activated primary human T cells after N -glycanase treatment of lystates followed by immunoprecipitation with anti-human TIM-3 antibodies (2E2, 2E12 or 3F9) or IgG as control and immunoblotted with anti-human CEACAM1 antibody (5F4). Protein lystates from HeLa-CEACAM1 transfectants treated with N -glycanase followed by immunoprecipitation with 5F4 and the immune complex used as positive control (pos). c , mTIM-3 interacts with mCEACAM1 in mouse T cells. Splenocytes from Ceacam1 4S Tg Ceacam1 −/− and Ceacam1-4L Tg Ceacam1 −/− mice cultured with anti-CD3 (1 μg ml −1 ) or anti-CD3 (1 μg ml −1 ) and anti-CD28 (1 μg ml −1 ) or medium for 96 h. Cell lysates immunoprecipitated with anti-mCEACAM1 antibody (cc1) or with mIgG and IB with 5D12 (anti-mTIM-3 antibody) are shown. Locations of mTIM-3 protein variants are indicated. CHO, carbohydrate. d , Immunoprecipitation and immunoblot as in a with tunicamycin treated, wild-type HA–hTIM-3 and Flag–hCEACAM1 co-transfected HEK293T cells. Arrowhead denotes core CEACAM1 protein. e , Potential hCEACAM1-interacting residues on hTIM-3 highlighted in blue. f , HEK293 T cells transiently co-transfected with Flag–hCEACAM1 and HA–hTIM-3 mutants. Immunoblotting of anti-HA were used to analyse hTIM-3 expression in HEK293T transfectants. Except for Pro50Ala mutation displaying enhanced overall protein expression, all other mutations in the IgV domain of hTIM-3 are equally detected by anti-HA antibody. g , Quantification of association of hTIM-3 mutants associated with wild-type hCEACAM1 shown in summing all experiments performed. Association between wild-type hCEACAM1 and hTIM-3 core protein are depicted as reference (set as 1, n = 3, mean ± s.e.m. shown, unpaired Student’s t -test). h , Immunoprecipitation with anti-Flag (hCEACAM1) and immunoblot with anti-HA (hTIM-3) or anti-Flag of wild-type hCEACAM1 and mutant hTIM-3 proteins are shown. i , Quantification of h as performed in g. j , HEK293T cells co-transfected with Flag–hCEACAM1 wild-type and HA– hTIM-3 mutants and immunoprecipitation/immunblot as in h revealing no effects of Cys52Ala or Cys63Ala mutations in hTIM-3 in affecting association with hCEACAM1 in contrast to Cys109Ala mutation of hTIM-3 that disrupts interactions with hCEACAM1. k , Potential hTIM-3-interacting-residues around the FG–CC′ cleft of hCEACAM1 highlighted in red. l , HEK293T cells transiently co-transfected with Flag–hCEACAM1 mutants and wild-type HA–hTIM-3. Immunoblot with anti-Flag antibody was used to analyse hCEACAM1 expression in HEK293T co-transfectants. All hCEACAM1 mutations in IgV domain equally detected. m . n–p , Analysis of Gly47Ala mutation of hCEACAM1 in hTIM-3 co-transfected HEK293T cells by immunoprecipitation with anti-HA (hTIM-3) and immunoblot with anti-Flag (hCEACAM1) to detect association ( n ), IB with anti-Flag to confirm similarity of hCEACAM1 transfection ( o ) and quantification of associated hCEACAM1 of n as shown in m. q–s , Analysis of hCEACAM1 mutants Asn42Ala and Arg43Ala association with hTIM-3 ( q ), similarity of transfections ( r ) and quantification of q as in n–p . Representative of four ( d, h ), three ( f, g, i, l–s ), two ( a–c ) and one ( j ) independent experiments. * P
    Hek293t Cells, supplied by OriGene, used in various techniques. Bioz Stars score: 92/100, based on 595 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    N/A
    GeneBLAzer EDNRA NFAT bla HEK293T cells contain the human Endothelin Type A Recpetor EDNRA stably integrated into the CellSensor NFAT bla HEK293T cell line CellSensor NFAT bla HEK293T cells Cat
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    GeneBLAzer H2 CRE bla HEK293T cells contain the human Histamine Receptor 2 H2 stably integrated into the CellSensor CRE bla HEK293T cell line CellSensor CRE bla HEK293T cells Cat no
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    N/A
    The HEK 293T cells antibody from Proteintech is a rabbit polyclonal antibody to a peptide of n a HEK 293T cells This antibody recognizes human antigen The HEK 293T cells
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    Optogenetic activation of SthK channels by various PACs in cell lines. a Illustration of the split-PAC-K and fused-PAC-K construct design for SthK co-expression with PACs. b Photocurrents elicited by different split-PAC-K variants after 10 ms exposure to a 470 nm light pulse. Scale bars: 10 s, 0.5 nA. c Normalized integrals of photocurrents as a function of the intensity of a 10 ms light pulse. Arrows denote the EC 50 for current activation by light ( n = 3 cells, two cultures). d Combined whole-cell patch-clamp (black) and optical recording (gray) of a HEK 293 cell expressing fused-bPAC-K and a fluorescent cAMP sensor. bPAC was excited with two 1 ms light flashes (470 nm). The dotted line indicates the zero current level. Scale bars: 20 s, 20 pA (upper trace); 20 s, 2000 aU (lower trace). e Comparison of photocurrent amplitudes generated by bPAC or TpPAC and SthK as split or fused constructs. f Current density comparison at saturating photon exposures ( ** p

    Journal: Nature Communications

    Article Title: Potassium channel-based optogenetic silencing

    doi: 10.1038/s41467-018-07038-8

    Figure Lengend Snippet: Optogenetic activation of SthK channels by various PACs in cell lines. a Illustration of the split-PAC-K and fused-PAC-K construct design for SthK co-expression with PACs. b Photocurrents elicited by different split-PAC-K variants after 10 ms exposure to a 470 nm light pulse. Scale bars: 10 s, 0.5 nA. c Normalized integrals of photocurrents as a function of the intensity of a 10 ms light pulse. Arrows denote the EC 50 for current activation by light ( n = 3 cells, two cultures). d Combined whole-cell patch-clamp (black) and optical recording (gray) of a HEK 293 cell expressing fused-bPAC-K and a fluorescent cAMP sensor. bPAC was excited with two 1 ms light flashes (470 nm). The dotted line indicates the zero current level. Scale bars: 20 s, 20 pA (upper trace); 20 s, 2000 aU (lower trace). e Comparison of photocurrent amplitudes generated by bPAC or TpPAC and SthK as split or fused constructs. f Current density comparison at saturating photon exposures ( ** p

    Article Snippet: Briefly, HEK 293 cells (ATCC CRL-1573) were transfected with a mix of pAD deltaF6, pAAV2/9, or pAAV 2/1, and the AAV expression vector.

    Techniques: Activation Assay, Construct, Expressing, Mass Spectrometry, Patch Clamp, Generated

    Cell viability (EC 50 ) determined by MTT assay for cells incubated with buffer (control); 10 μM phenanthriplatin (Phen); 10 μM cimetidine (Cim); 100 μM cimetidine; 10 μM phenanthriplatin + 10 μM cimetidine; and 10 μM phenanthriplatin + 100 μM cimetidine, for 10 min. (A) HEK 293 WT cells (B) HEK 293 hOCT2 cells (C) HEK293 hMATE1 and hMATE2K cells. Above the columns is the number of experiments. The asterisk ( * ) indicates a statistically significant difference compared to the other columns, # a statistically difference compared to all the other columns except than to 10 μM phenanthriplatin + 10 μM cimetidine (ANOVA).

    Journal: Frontiers in Chemistry

    Article Title: Interaction of the New Monofunctional Anticancer Agent Phenanthriplatin With Transporters for Organic Cations

    doi: 10.3389/fchem.2018.00180

    Figure Lengend Snippet: Cell viability (EC 50 ) determined by MTT assay for cells incubated with buffer (control); 10 μM phenanthriplatin (Phen); 10 μM cimetidine (Cim); 100 μM cimetidine; 10 μM phenanthriplatin + 10 μM cimetidine; and 10 μM phenanthriplatin + 100 μM cimetidine, for 10 min. (A) HEK 293 WT cells (B) HEK 293 hOCT2 cells (C) HEK293 hMATE1 and hMATE2K cells. Above the columns is the number of experiments. The asterisk ( * ) indicates a statistically significant difference compared to the other columns, # a statistically difference compared to all the other columns except than to 10 μM phenanthriplatin + 10 μM cimetidine (ANOVA).

    Article Snippet: Cell culture Experiments were performed with human embryonic kidney (HEK) 293 cells (CRL-1573; American Type Culture Collection, Rochville, MD), which stably express mOCT1, mOCT2, mOCT3 (Schlatter et al., ), hOCT1, hOCT2, hOCT3, (kind gift of Prof. H. Koepsell, University Würzburg), hMATE1 or hMATE2K (Schmidt-Lauber et al., ).

    Techniques: MTT Assay, Incubation

    Activation of virus entry by different human TTSPs. (A) Experiment set-up. One day before transduction, HEK293T target cells were transfected with the appropriate receptor and one of the TTSPs. To block the cathepsin route, E64d was added at 2 h before and during transduction. (B) SARS-2-S activating capacity of the 18 human TTSPs. At the top of the graph, the four TTSP subfamilies are indicated. (C, D) The four TTSPs that proved active in panel B were evaluated for activation of wild-type and mutant forms of SARS-2-S (panel C), or SARS-1-S, MERS-S and 229E-S (panel D). An ordinary one-way ANOVA with Dunnett’s correction was used to compare SARS-2 mutants versus WT and an unpaired two-tailed t-test was used to compare the WT and mutant forms of SARS-1 and MERS. *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001. Results are the mean ± SEM; N=3 with three independently produced stocks).

    Journal: bioRxiv

    Article Title: The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways

    doi: 10.1101/2020.11.09.374603

    Figure Lengend Snippet: Activation of virus entry by different human TTSPs. (A) Experiment set-up. One day before transduction, HEK293T target cells were transfected with the appropriate receptor and one of the TTSPs. To block the cathepsin route, E64d was added at 2 h before and during transduction. (B) SARS-2-S activating capacity of the 18 human TTSPs. At the top of the graph, the four TTSP subfamilies are indicated. (C, D) The four TTSPs that proved active in panel B were evaluated for activation of wild-type and mutant forms of SARS-2-S (panel C), or SARS-1-S, MERS-S and 229E-S (panel D). An ordinary one-way ANOVA with Dunnett’s correction was used to compare SARS-2 mutants versus WT and an unpaired two-tailed t-test was used to compare the WT and mutant forms of SARS-1 and MERS. *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001. Results are the mean ± SEM; N=3 with three independently produced stocks).

    Article Snippet: HEK293T cells (HCL4517; Thermo Fisher Scientific) and Vero E6 (ATCC CRL-1586) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS, 1 mM sodium pyruvate, 0.075% sodium bicarbonate and (only for Vero E6) 0.1 mM non-essential amino acids.

    Techniques: Activation Assay, Transduction, Transfection, Blocking Assay, Mutagenesis, Two Tailed Test, Produced

    Expression of coronavirus receptors, activating proteases and spike proteins. (A) Expression of coronavirus receptors and activating proteases, determined by qRT-PCR. The heat map shows mRNA levels relative to β-actin. Samples of human nasal (N=2) and lung tissue (N=8) were analyzed, besides the three cell lines (Calu-3, Vero E6 and HEK293T: N=2). We previously reported an expression analysis of all 18 human TTSPs in Calu-3 cells and human respiratory tissue ( 13 ). (B) Western blot analysis showing S expression and S1/S2 cleavage in HEK293T producer cells transfected with wild type (WT) or mutant forms of SARS-2-S, SARS-1-S or MERS-S.

    Journal: bioRxiv

    Article Title: The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways

    doi: 10.1101/2020.11.09.374603

    Figure Lengend Snippet: Expression of coronavirus receptors, activating proteases and spike proteins. (A) Expression of coronavirus receptors and activating proteases, determined by qRT-PCR. The heat map shows mRNA levels relative to β-actin. Samples of human nasal (N=2) and lung tissue (N=8) were analyzed, besides the three cell lines (Calu-3, Vero E6 and HEK293T: N=2). We previously reported an expression analysis of all 18 human TTSPs in Calu-3 cells and human respiratory tissue ( 13 ). (B) Western blot analysis showing S expression and S1/S2 cleavage in HEK293T producer cells transfected with wild type (WT) or mutant forms of SARS-2-S, SARS-1-S or MERS-S.

    Article Snippet: HEK293T cells (HCL4517; Thermo Fisher Scientific) and Vero E6 (ATCC CRL-1586) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS, 1 mM sodium pyruvate, 0.075% sodium bicarbonate and (only for Vero E6) 0.1 mM non-essential amino acids.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Mutagenesis

    The SARS-2-S D614G change increases stability and entry via the cathepsin route, and reduces the preference for 33°C. (A) Thermostability analysis. The pseudoparticles were incubated at the indicated temperatures for 1 h, followed by 2 h entry into HEK293T target cells and luminescence reading after 72 h. Results are expressed as transduction efficiency, relative to the condition incubated at 4°C (mean ± SEM, N=3). (B) analysis of the four CoV pseudotypes; (C) comparison of SARS-2-S D614 , the three S1/S2 loop mutants, and the SARS-2-S G614 mutant. (D) Pseudovirions carrying SARS-2-S D614 or S G614 were produced at 33°C or 37°C, then pelleted to determine their S content by western blot. The graphs show the S0 and S2 band intensities, normalized to the band of MLV-gag (mean ± SEM, N=3). (E) Cell entry efficiency (mean ± SEM, N=3) of the two pseudovirus variants in Calu-3 and Vero E6. (F) Particle entry in HEK293T cells transfected with an empty plasmid ( empty ) or one of the TTSPs. In all panels: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001, unpaired two-tailed t-test.

    Journal: bioRxiv

    Article Title: The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways

    doi: 10.1101/2020.11.09.374603

    Figure Lengend Snippet: The SARS-2-S D614G change increases stability and entry via the cathepsin route, and reduces the preference for 33°C. (A) Thermostability analysis. The pseudoparticles were incubated at the indicated temperatures for 1 h, followed by 2 h entry into HEK293T target cells and luminescence reading after 72 h. Results are expressed as transduction efficiency, relative to the condition incubated at 4°C (mean ± SEM, N=3). (B) analysis of the four CoV pseudotypes; (C) comparison of SARS-2-S D614 , the three S1/S2 loop mutants, and the SARS-2-S G614 mutant. (D) Pseudovirions carrying SARS-2-S D614 or S G614 were produced at 33°C or 37°C, then pelleted to determine their S content by western blot. The graphs show the S0 and S2 band intensities, normalized to the band of MLV-gag (mean ± SEM, N=3). (E) Cell entry efficiency (mean ± SEM, N=3) of the two pseudovirus variants in Calu-3 and Vero E6. (F) Particle entry in HEK293T cells transfected with an empty plasmid ( empty ) or one of the TTSPs. In all panels: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001, unpaired two-tailed t-test.

    Article Snippet: HEK293T cells (HCL4517; Thermo Fisher Scientific) and Vero E6 (ATCC CRL-1586) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS, 1 mM sodium pyruvate, 0.075% sodium bicarbonate and (only for Vero E6) 0.1 mM non-essential amino acids.

    Techniques: Incubation, Transduction, Mutagenesis, Produced, Western Blot, Transfection, Plasmid Preparation, Two Tailed Test

    Spike incorporation into pseudovirions is temperature-dependent. (A) Experiment set-up. S-bearing pseudoviruses were produced in HEK293T cells at either 33°C or 37°C, and the released particles were pelleted to determine S content by western blot. In parallel, they were used to transduce HEK293T target cells expressing the appropriate receptor and TMPRSS2. (B) The graphs show S content relative to that of MLV-gag (mean ± SEM; of three independently produced stocks). Representative blots show uncleaved and cleaved S protein bands. (C) Particle infectivity was measured by luminescence read-out at day 3 post transduction. A two-tailed unpaired t-test was used to compare the 33°C and 37°C results, regarding total S content or particle infectivity. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

    Journal: bioRxiv

    Article Title: The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways

    doi: 10.1101/2020.11.09.374603

    Figure Lengend Snippet: Spike incorporation into pseudovirions is temperature-dependent. (A) Experiment set-up. S-bearing pseudoviruses were produced in HEK293T cells at either 33°C or 37°C, and the released particles were pelleted to determine S content by western blot. In parallel, they were used to transduce HEK293T target cells expressing the appropriate receptor and TMPRSS2. (B) The graphs show S content relative to that of MLV-gag (mean ± SEM; of three independently produced stocks). Representative blots show uncleaved and cleaved S protein bands. (C) Particle infectivity was measured by luminescence read-out at day 3 post transduction. A two-tailed unpaired t-test was used to compare the 33°C and 37°C results, regarding total S content or particle infectivity. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

    Article Snippet: HEK293T cells (HCL4517; Thermo Fisher Scientific) and Vero E6 (ATCC CRL-1586) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS, 1 mM sodium pyruvate, 0.075% sodium bicarbonate and (only for Vero E6) 0.1 mM non-essential amino acids.

    Techniques: Produced, Western Blot, Transduction, Expressing, Infection, Two Tailed Test

    Sequence conservation in selected domains of MGRN1 and MEGF8, Related to Figure 2 (A) Sequence alignment showing conservation and secondary structure features of the cytoplasmic tail of several members of the MEGF8-ATRN family. The top three proteins are three paralogs (MEGF8, Attractin (ATRN) or Attractin-like (ATRNL1) found in humans and the rest are homologs of these proteins identified in various species. Proteins are named by their GenBank identifiers (GIs), followed by the complete names of the species from which they are derived. Predicted secondary structure elements are shown at the top: the red rectangle denotes a predicted α-helix and the multiple green arrows denote predicted β-strands. Low complexity regions between the strands are hidden and denoted as gaps with numbers. The MASRPFA sequence motif is highlighted in black. The alignment is colored based on 70% consensus with the following scheme as shown in the consensus sequence: h (hydrophobic), l (aliphatic), and a (aromatic) are shaded yellow; p (polar) are shaded blue; charged are shaded pale violet; s (small) and u (tiny) are shaded light green; b (big) is shaded dark gray. (B) ClustalW alignment of the RING domains of various MGRN1 homologs show conservation of the residues altered to generate the inactive MGRN1 Mut1 (C279A/C282A) and MGRN1 Mut2 (L307A/R308A) variants ( Gunn et al., 2013b ). Conservation at each position in the RING domain, color-coded according to the indicated scheme, is based on analysis of 200 homologs of MGRN1 using the ConSurf method ( Ashkenazy et al., 2016 ). (C) Mutations in the RING domain introduced in MGRN1 Mut1 and MGRN1 Mut2 do not abolish their interactions with MEGF8 based on a co-IP experiment after transient expression of the indicated proteins in HEK293T cells (identical to the assay depicted in Fig. 2C ). Asterisk (*) indicates endogenous MGRN1 from HEK293T cells that co-precipitated with MEGF8.

    Journal: bioRxiv

    Article Title: A ubiquitin-based mechanism for the oligogenic inheritance of heterotaxy and heart defects

    doi: 10.1101/2020.05.25.113944

    Figure Lengend Snippet: Sequence conservation in selected domains of MGRN1 and MEGF8, Related to Figure 2 (A) Sequence alignment showing conservation and secondary structure features of the cytoplasmic tail of several members of the MEGF8-ATRN family. The top three proteins are three paralogs (MEGF8, Attractin (ATRN) or Attractin-like (ATRNL1) found in humans and the rest are homologs of these proteins identified in various species. Proteins are named by their GenBank identifiers (GIs), followed by the complete names of the species from which they are derived. Predicted secondary structure elements are shown at the top: the red rectangle denotes a predicted α-helix and the multiple green arrows denote predicted β-strands. Low complexity regions between the strands are hidden and denoted as gaps with numbers. The MASRPFA sequence motif is highlighted in black. The alignment is colored based on 70% consensus with the following scheme as shown in the consensus sequence: h (hydrophobic), l (aliphatic), and a (aromatic) are shaded yellow; p (polar) are shaded blue; charged are shaded pale violet; s (small) and u (tiny) are shaded light green; b (big) is shaded dark gray. (B) ClustalW alignment of the RING domains of various MGRN1 homologs show conservation of the residues altered to generate the inactive MGRN1 Mut1 (C279A/C282A) and MGRN1 Mut2 (L307A/R308A) variants ( Gunn et al., 2013b ). Conservation at each position in the RING domain, color-coded according to the indicated scheme, is based on analysis of 200 homologs of MGRN1 using the ConSurf method ( Ashkenazy et al., 2016 ). (C) Mutations in the RING domain introduced in MGRN1 Mut1 and MGRN1 Mut2 do not abolish their interactions with MEGF8 based on a co-IP experiment after transient expression of the indicated proteins in HEK293T cells (identical to the assay depicted in Fig. 2C ). Asterisk (*) indicates endogenous MGRN1 from HEK293T cells that co-precipitated with MEGF8.

    Article Snippet: NIH/3T3 and HEK293T cell culture Flp-In-3T3 (a derivative of NIH/3T3 cells and referred to as “NIH/3T3” cells throughout the text) and HEK293T cell lines were purchased from Thermo Fisher Scientific and ATCC, respectively.

    Techniques: Sequencing, Derivative Assay, Co-Immunoprecipitation Assay, Expressing

    The MEGF8/MGRN1 complex ubiquitinates Smoothened, Related to Figure 4 (A) Ubiquitination of SMO-eGFP in the presence of wild-type HA-Ubiquitin (HA-UB) or a mutant where all the lysine residues have been mutated to arginine (UB K0 ) to prevent ubiquitin chain elongation. Ubiquitination was assessed as described in Fig. 4A after transient expression of the indicated proteins in HEK293T cells. (B) To determine if the MEGF8-MGRN1 complex can regulate the abundance of other G-protein coupled receptors (GPCRs), HEK293T cells were transiently transfected with constructs encoding MEGF8, functional or catalytically inactive MGRN1 (MGRN1 Mut1 ), and either SMO (top) or Somatostatin receptor type 3 (SSTR3) (bottom). Immunoblots indicate that co-expression of MEGF8 and MGRN1 complex reduced the abundance of SMO, but had no effect on SSTR3. (C) Ubiquitination of wild type SMO or variants carrying mutations in cytoplasmically-exposed lysine residues by the MEGF8-MGRN1 complex. The following five SMO mutants were tested: (1) SMO-K0 (all 21 cytoplasmic lysine residues were changed to arginines), (2) Smo-Ctail K0 (all 16 lysines in the C-terminal cytoplasmic tail were changed to arginines), (3) SMO-ICL K0 (all 5 lysines in the three cytoplasmic loops were changed to arginine), (4) SMO-ICL2 K0 (both lysines in the second intracellular loop were changed to arginines), and (5) SMO-ICL3 K0 (all three lysines in the third intracellular loop were changed to arginines). Cells were lysed under denaturing conditions, native SMO was purified by IP using beads covalently linked to an anti-SMO antibody, and the amount of HA-UB covalently conjugated to SMO was assessed using immunoblotting with an anti-HA antibody (bottom panel). (D) Chimeric proteins were used to identify the minimal region of MEGF8 sufficient to support SMO ubiquitination. Using the assay shown in Fig. 4A , SMO ubiquitination was assessed after transient co-expression of the following in 293T cells: SMO-eGFP, HA-UB, MGRN1 (or the inactive mutant MGRN1 Mut1 ), and the MEGF8 mutant or chimera shown above the blot. See Fig. 2A and associated text for a description of these chimeras. (E) Flow cytometry was used to measure cell surface labeled CD16 in live Megf8 -/- cells stably expressing various CD16/CD7/MEGF8 chimeras (diagrammed in Fig. S5D ). These are the same stable cell lines analyzed in Figs. 4C and 4D . 4000 cells were analyzed per condition.

    Journal: bioRxiv

    Article Title: A ubiquitin-based mechanism for the oligogenic inheritance of heterotaxy and heart defects

    doi: 10.1101/2020.05.25.113944

    Figure Lengend Snippet: The MEGF8/MGRN1 complex ubiquitinates Smoothened, Related to Figure 4 (A) Ubiquitination of SMO-eGFP in the presence of wild-type HA-Ubiquitin (HA-UB) or a mutant where all the lysine residues have been mutated to arginine (UB K0 ) to prevent ubiquitin chain elongation. Ubiquitination was assessed as described in Fig. 4A after transient expression of the indicated proteins in HEK293T cells. (B) To determine if the MEGF8-MGRN1 complex can regulate the abundance of other G-protein coupled receptors (GPCRs), HEK293T cells were transiently transfected with constructs encoding MEGF8, functional or catalytically inactive MGRN1 (MGRN1 Mut1 ), and either SMO (top) or Somatostatin receptor type 3 (SSTR3) (bottom). Immunoblots indicate that co-expression of MEGF8 and MGRN1 complex reduced the abundance of SMO, but had no effect on SSTR3. (C) Ubiquitination of wild type SMO or variants carrying mutations in cytoplasmically-exposed lysine residues by the MEGF8-MGRN1 complex. The following five SMO mutants were tested: (1) SMO-K0 (all 21 cytoplasmic lysine residues were changed to arginines), (2) Smo-Ctail K0 (all 16 lysines in the C-terminal cytoplasmic tail were changed to arginines), (3) SMO-ICL K0 (all 5 lysines in the three cytoplasmic loops were changed to arginine), (4) SMO-ICL2 K0 (both lysines in the second intracellular loop were changed to arginines), and (5) SMO-ICL3 K0 (all three lysines in the third intracellular loop were changed to arginines). Cells were lysed under denaturing conditions, native SMO was purified by IP using beads covalently linked to an anti-SMO antibody, and the amount of HA-UB covalently conjugated to SMO was assessed using immunoblotting with an anti-HA antibody (bottom panel). (D) Chimeric proteins were used to identify the minimal region of MEGF8 sufficient to support SMO ubiquitination. Using the assay shown in Fig. 4A , SMO ubiquitination was assessed after transient co-expression of the following in 293T cells: SMO-eGFP, HA-UB, MGRN1 (or the inactive mutant MGRN1 Mut1 ), and the MEGF8 mutant or chimera shown above the blot. See Fig. 2A and associated text for a description of these chimeras. (E) Flow cytometry was used to measure cell surface labeled CD16 in live Megf8 -/- cells stably expressing various CD16/CD7/MEGF8 chimeras (diagrammed in Fig. S5D ). These are the same stable cell lines analyzed in Figs. 4C and 4D . 4000 cells were analyzed per condition.

    Article Snippet: NIH/3T3 and HEK293T cell culture Flp-In-3T3 (a derivative of NIH/3T3 cells and referred to as “NIH/3T3” cells throughout the text) and HEK293T cell lines were purchased from Thermo Fisher Scientific and ATCC, respectively.

    Techniques: Mutagenesis, Expressing, Transfection, Construct, Functional Assay, Western Blot, Purification, Flow Cytometry, Labeling, Stable Transfection

    The interaction between MGRN1 and MEGF8 is required to attenuate Hedgehog signaling (A) Depictions of full length MEGF8, truncated MEGF8 (MEGF8 ΔN , MEGF8 ΔCtail , MEGF8 ΔMASRPFA ), functional MGRN1, and catalytically inactive MGRN1 (MGRN1 Mut1 and MGRN1 Mut2 ) proteins. The multiple domains in the extracellular region of MEGF8 are shown as circles and colored as in Fig. S1A. (B) Sequence logo showing the conservation in sequence entropy bits of the MASRPFA sequence (yellow shading) in the cytoplasmic tail of MEGF8 and related proteins (alignment shown in Fig. S4A ). The logo spans the region from the penultimate residue of the TM helix to the extended β-strand-like region immediately distal to the MASRPFA motif (shown by a green arrow). Deletion boundaries for the MEGF8 mutants shown in Fig. 2A are noted below the logo. (C) The interaction between MEGF8 or MEGF8 mutants (shown in Fig. 2A , all 1D4 tagged) and MGRN1 (FLAG tagged) was tested by transient co-expression in HEK293T cells, followed by immunoprecipitation (IP) of MEGF8 proteins using the appended 1D4 tag. Immunoblots of input samples show the abundance of indicated proteins in extracts and IP samples show the amount of endogenous and transfected MGRN1 (detected with an anti-MGRN1 antibody) that co-precipitated with MEGF8. Asterisk (*) indicates endogenous MGRN1 from HEK293T cells present in input and IP samples. (D and E) GLI1 abundance was measured as a metric of Hh signaling strength by immunoblotting (D) and SMO ciliary abundance by confocal fluorescence microscopy (E) in Megf8 -/- NIH/3T3 cells stably expressing 1D4-tagged MEGF8 or MEGF8 ΔC (see Fig. 2A ). (D) The interaction between each MEGF8 variant and endogenous MGRN1 was also tested by co-IP. (F and G) Total GLI, SMO and MEGF8 abundances were measured by immunoblotting (F) and SMO ciliary abundance by confocal fluorescence microscopy (G) in Mgrn1 -/- ;Rnf157 -/- NIH/3T3 cells stably expressing wild-type MGRN1 or variants carrying inactivating mutations in the RING domain (MGRN1 Mut1 and MGRN1 Mut2 , see Figs. 2A and S4B ). (D, F) Cells used for IP and immunoblotting were treated with the indicated concentrations of SHH; (E, G) cells used for ciliary SMO measurements were left untreated. (E, G) Horizontally positioned violin plots summarize the quantification of SMO fluorescence (red) at ∼50 individual cilia (green, ARL13B) per cell line from representative images of the type shown immediately to the left. (E, G) Arrowheads identify individual cilia captured in the zoomed images to the right of each panel. Statistical significance was determined by the Kruskal-Wallis test; not-significant (ns) > 0.05 and **** p -value ≤ 0.0001. Scale bars, 10 µm in merged panels and 2 µm in zoomed displays.

    Journal: bioRxiv

    Article Title: A ubiquitin-based mechanism for the oligogenic inheritance of heterotaxy and heart defects

    doi: 10.1101/2020.05.25.113944

    Figure Lengend Snippet: The interaction between MGRN1 and MEGF8 is required to attenuate Hedgehog signaling (A) Depictions of full length MEGF8, truncated MEGF8 (MEGF8 ΔN , MEGF8 ΔCtail , MEGF8 ΔMASRPFA ), functional MGRN1, and catalytically inactive MGRN1 (MGRN1 Mut1 and MGRN1 Mut2 ) proteins. The multiple domains in the extracellular region of MEGF8 are shown as circles and colored as in Fig. S1A. (B) Sequence logo showing the conservation in sequence entropy bits of the MASRPFA sequence (yellow shading) in the cytoplasmic tail of MEGF8 and related proteins (alignment shown in Fig. S4A ). The logo spans the region from the penultimate residue of the TM helix to the extended β-strand-like region immediately distal to the MASRPFA motif (shown by a green arrow). Deletion boundaries for the MEGF8 mutants shown in Fig. 2A are noted below the logo. (C) The interaction between MEGF8 or MEGF8 mutants (shown in Fig. 2A , all 1D4 tagged) and MGRN1 (FLAG tagged) was tested by transient co-expression in HEK293T cells, followed by immunoprecipitation (IP) of MEGF8 proteins using the appended 1D4 tag. Immunoblots of input samples show the abundance of indicated proteins in extracts and IP samples show the amount of endogenous and transfected MGRN1 (detected with an anti-MGRN1 antibody) that co-precipitated with MEGF8. Asterisk (*) indicates endogenous MGRN1 from HEK293T cells present in input and IP samples. (D and E) GLI1 abundance was measured as a metric of Hh signaling strength by immunoblotting (D) and SMO ciliary abundance by confocal fluorescence microscopy (E) in Megf8 -/- NIH/3T3 cells stably expressing 1D4-tagged MEGF8 or MEGF8 ΔC (see Fig. 2A ). (D) The interaction between each MEGF8 variant and endogenous MGRN1 was also tested by co-IP. (F and G) Total GLI, SMO and MEGF8 abundances were measured by immunoblotting (F) and SMO ciliary abundance by confocal fluorescence microscopy (G) in Mgrn1 -/- ;Rnf157 -/- NIH/3T3 cells stably expressing wild-type MGRN1 or variants carrying inactivating mutations in the RING domain (MGRN1 Mut1 and MGRN1 Mut2 , see Figs. 2A and S4B ). (D, F) Cells used for IP and immunoblotting were treated with the indicated concentrations of SHH; (E, G) cells used for ciliary SMO measurements were left untreated. (E, G) Horizontally positioned violin plots summarize the quantification of SMO fluorescence (red) at ∼50 individual cilia (green, ARL13B) per cell line from representative images of the type shown immediately to the left. (E, G) Arrowheads identify individual cilia captured in the zoomed images to the right of each panel. Statistical significance was determined by the Kruskal-Wallis test; not-significant (ns) > 0.05 and **** p -value ≤ 0.0001. Scale bars, 10 µm in merged panels and 2 µm in zoomed displays.

    Article Snippet: NIH/3T3 and HEK293T cell culture Flp-In-3T3 (a derivative of NIH/3T3 cells and referred to as “NIH/3T3” cells throughout the text) and HEK293T cell lines were purchased from Thermo Fisher Scientific and ATCC, respectively.

    Techniques: Functional Assay, Sequencing, Expressing, Immunoprecipitation, Western Blot, Transfection, Fluorescence, Microscopy, Stable Transfection, Variant Assay, Co-Immunoprecipitation Assay

    PCR tagging enables C-terminal tagging of the majority of human genes. (a) Search space for Cas12a-PAM sites suitable for C-terminal protein tagging. PCR cassette insertion into the genome using PAM sites located in the confined search space (blue) led to a disruption of the crRNA target sequence. This would not be the case for PAM sites in the extended search space (orange). To prevent recleavage after insertion, the homology arm of the PCR fragment (provided by the M2 tagging oligo) is designed such that a small deletion in the region after the STOP codon does lead to the disruption of the crRNA target site. (b) Fraction (in percentage) of human genes with suitable PAM sites near the STOP codon, as a function of the confined and extended search spaces (a) and different Cas12a variants as indicated. For calculation, we used the following PAM sites: hLbCas12a/hAsCas12a: TTTV; LbCas12a RR variant: TYCV, TYTV; AsCas12a, RVR variant: TATV; AsCas12a RR variant: TTTV, TYCV; enAsCas12a: TTYN, VTTV, TRTV, VTCC, HSCC, TACA, TTAC, CACC (Tier 1 and 2 PAM sites). (c) Tagging of the indicated genes in HEK293T cells. Helper plasmids with different Cas12a genes, as indicated. PCR cassettes contained crRNA genes with matching PAM site specificity. For TOMM70 , three different Cas12a variants were tested using three different crRNA sequences for AsCas12a, as indicated. Tagging efficiency was determined 3 d after transfection.

    Journal: The Journal of Cell Biology

    Article Title: CRISPR-Cas12a–assisted PCR tagging of mammalian genes

    doi: 10.1083/jcb.201910210

    Figure Lengend Snippet: PCR tagging enables C-terminal tagging of the majority of human genes. (a) Search space for Cas12a-PAM sites suitable for C-terminal protein tagging. PCR cassette insertion into the genome using PAM sites located in the confined search space (blue) led to a disruption of the crRNA target sequence. This would not be the case for PAM sites in the extended search space (orange). To prevent recleavage after insertion, the homology arm of the PCR fragment (provided by the M2 tagging oligo) is designed such that a small deletion in the region after the STOP codon does lead to the disruption of the crRNA target site. (b) Fraction (in percentage) of human genes with suitable PAM sites near the STOP codon, as a function of the confined and extended search spaces (a) and different Cas12a variants as indicated. For calculation, we used the following PAM sites: hLbCas12a/hAsCas12a: TTTV; LbCas12a RR variant: TYCV, TYTV; AsCas12a, RVR variant: TATV; AsCas12a RR variant: TTTV, TYCV; enAsCas12a: TTYN, VTTV, TRTV, VTCC, HSCC, TACA, TTAC, CACC (Tier 1 and 2 PAM sites). (c) Tagging of the indicated genes in HEK293T cells. Helper plasmids with different Cas12a genes, as indicated. PCR cassettes contained crRNA genes with matching PAM site specificity. For TOMM70 , three different Cas12a variants were tested using three different crRNA sequences for AsCas12a, as indicated. Tagging efficiency was determined 3 d after transfection.

    Article Snippet: Chemical transfection Transfection of HEK293T, HeLa, and U2OS cells was performed using Lipofectamine 2000 (Invitrogen) according to protocol of the manufacturer and using a 24-well format.

    Techniques: Polymerase Chain Reaction, Sequencing, Variant Assay, Transfection

    Tagging efficiency as a function of different parameters. (a) Length of homology arms. M1 and M2 tagging oligos containing the indicated sequence lengths of homology arm (5′-HA and 3′-HA, respectively) to the destination locus were used for PCR tagging of the HNRNPA1 locus in HEK293T cells. Tagging efficiency was estimated 3 d after transfection as described before. Data from three replicates are shown. Error bars indicate SD. (b) PCR cassettes containing various types of ends to direct the choice of DNA repair pathway: homology arms (90-bp and 55-bp homology, for HR; A), blunt ended arms without homology to the target locus (blunt; B), HgaI cut (D), and uncut ends (C). Cutting with the type IIS restriction enzyme HgaI results in 5-nt 3′ overhangs that are complementary to the overhangs generated by the crRNA directed Cas12a-cleavage of the destination locus. Tagging efficiency was estimated 3 d later as described in panel a using HEK293T cells. Data from three replicates are shown. Error bars indicate SD. (c) Use of modified and/or purified oligos. M1/M2 tagging oligos with the indicated number of phosphorothioate bonds and/or biotin as indicated were used for generation of PCR cassettes. All oligos were cartridge purified except for the ones denoted with PAGE, which were size selected using PAGE. Tagging efficiency was estimated 3 d after transfection as described before using HEK293T cells. Data from three replicates are shown. Error bars indicate SD.

    Journal: The Journal of Cell Biology

    Article Title: CRISPR-Cas12a–assisted PCR tagging of mammalian genes

    doi: 10.1083/jcb.201910210

    Figure Lengend Snippet: Tagging efficiency as a function of different parameters. (a) Length of homology arms. M1 and M2 tagging oligos containing the indicated sequence lengths of homology arm (5′-HA and 3′-HA, respectively) to the destination locus were used for PCR tagging of the HNRNPA1 locus in HEK293T cells. Tagging efficiency was estimated 3 d after transfection as described before. Data from three replicates are shown. Error bars indicate SD. (b) PCR cassettes containing various types of ends to direct the choice of DNA repair pathway: homology arms (90-bp and 55-bp homology, for HR; A), blunt ended arms without homology to the target locus (blunt; B), HgaI cut (D), and uncut ends (C). Cutting with the type IIS restriction enzyme HgaI results in 5-nt 3′ overhangs that are complementary to the overhangs generated by the crRNA directed Cas12a-cleavage of the destination locus. Tagging efficiency was estimated 3 d later as described in panel a using HEK293T cells. Data from three replicates are shown. Error bars indicate SD. (c) Use of modified and/or purified oligos. M1/M2 tagging oligos with the indicated number of phosphorothioate bonds and/or biotin as indicated were used for generation of PCR cassettes. All oligos were cartridge purified except for the ones denoted with PAGE, which were size selected using PAGE. Tagging efficiency was estimated 3 d after transfection as described before using HEK293T cells. Data from three replicates are shown. Error bars indicate SD.

    Article Snippet: Chemical transfection Transfection of HEK293T, HeLa, and U2OS cells was performed using Lipofectamine 2000 (Invitrogen) according to protocol of the manufacturer and using a 24-well format.

    Techniques: Sequencing, Polymerase Chain Reaction, Transfection, Generated, Modification, Purification, Polyacrylamide Gel Electrophoresis

    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

    Inhibition of JEV translation and replication by ZAP. (A and C) A549-EGFP, ZAP-L and ZAP-S cells absorbed with JEV (MOI = 10) on ice for 2 h were washed and then incubated at 37°C. (A) At the indicated time points, proteins were harvested for western blot with the indicated antibodies. (B) Relative NS3 protein levels normalized with actin from two independent experiments were quantified by ImageJ software. (C) RNA was collected for viral RNA determination by using RT-qPCR. Relative JEV RNA level was normalized by that of GAPDH. (D) Illustration of SP6 promoter-driven RdRP-dead JEV replicon (E) Western blot of 293T/17 cells expressing EGFP, ZAP-L-V5 and ZAP-S-V5 with the indicated antibodies. (F) 293T/17-EGFP and -ZAP-S cell were cotransfected with 5′-capped RdRP-dead JEV replicon RNA and control firefly luciferase RNA for 1 and 2 h. At the indicated time points, cells were collected and separated into two portions for the measurements of luciferase activity and RNA level, respectively. The relative luciferase activity and RNA level of Renilla luciferase reporter normalized with transfection control firefly luciferase are shown as the percentage to that of EGFP at 1 h post transfection. (G) 5′-capped RdRP-dead JEV replicon RNA and control firefly luciferase RNA cotransfected 293T/17-EGFP and -ZAP-S cells were harvested at 3 and 9 h post-transfection to determine the replicon RNA level. The relative replicon RNA level normalized with that of firefly luciferase is shown. Representative data are shown as mean ± SD from 3 independent experiments and analyzed by two-tailed Student’s t test. * P ≤0.05; ** P ≤0.01; *** P ≤0.001.

    Journal: PLoS Pathogens

    Article Title: Inhibition of Japanese encephalitis virus infection by the host zinc-finger antiviral protein

    doi: 10.1371/journal.ppat.1007166

    Figure Lengend Snippet: Inhibition of JEV translation and replication by ZAP. (A and C) A549-EGFP, ZAP-L and ZAP-S cells absorbed with JEV (MOI = 10) on ice for 2 h were washed and then incubated at 37°C. (A) At the indicated time points, proteins were harvested for western blot with the indicated antibodies. (B) Relative NS3 protein levels normalized with actin from two independent experiments were quantified by ImageJ software. (C) RNA was collected for viral RNA determination by using RT-qPCR. Relative JEV RNA level was normalized by that of GAPDH. (D) Illustration of SP6 promoter-driven RdRP-dead JEV replicon (E) Western blot of 293T/17 cells expressing EGFP, ZAP-L-V5 and ZAP-S-V5 with the indicated antibodies. (F) 293T/17-EGFP and -ZAP-S cell were cotransfected with 5′-capped RdRP-dead JEV replicon RNA and control firefly luciferase RNA for 1 and 2 h. At the indicated time points, cells were collected and separated into two portions for the measurements of luciferase activity and RNA level, respectively. The relative luciferase activity and RNA level of Renilla luciferase reporter normalized with transfection control firefly luciferase are shown as the percentage to that of EGFP at 1 h post transfection. (G) 5′-capped RdRP-dead JEV replicon RNA and control firefly luciferase RNA cotransfected 293T/17-EGFP and -ZAP-S cells were harvested at 3 and 9 h post-transfection to determine the replicon RNA level. The relative replicon RNA level normalized with that of firefly luciferase is shown. Representative data are shown as mean ± SD from 3 independent experiments and analyzed by two-tailed Student’s t test. * P ≤0.05; ** P ≤0.01; *** P ≤0.001.

    Article Snippet: Human embryonic kidney 293T/17 cells (ATCC, CRL-11268) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS and 2 mM L-glutamine.

    Techniques: Inhibition, Incubation, Western Blot, Software, Quantitative RT-PCR, Expressing, Luciferase, Activity Assay, Transfection, Two Tailed Test

    Mapping of ZAP-responsive element (ZRE) in JEV 3′-UTR. (A) The CLIP-seq diagram of ZAP-S binding RNA mapped to JEV 3′-UTR. Read coverage, the reads of each position normalized to the total number of reads mapping to the viral genome. The JEV 3′-UTR is divided into domain I, II and III as indicated. The positions of CG dinucleotide are shown by dots. (B) Biotin-labeled JEV 3′-UTR RNA probes (full-length and individual domain I, II, III, and I+II) incubated with A549-ZAP-S cell extracts were pulled down with streptavidin beads and determined for ZAP-S-V5 protein by western blot (left panel). The intensity of ZAP-S was quantified by ImageJ software. Mean ± SD was calculated from 3 independent experiments and analyzed by two-tailed Student’s t test (right panel). (C) 5′-capped full-length and deleted Fluc/5′+3′-UTR RNA (left panel) and control Rluc RNA were cotransfected into 293T/17-EGFP and -ZAP-S cells. At 18 h post-transfection, cell lysates were collected to perform dual-luciferase assay (right panel). Representative data from three independent experiments shown as mean ± SD (n = 3) were analyzed by two-tailed Student’s t test. ** P ≤0.01; *** P ≤0.001; NS , not significant.

    Journal: PLoS Pathogens

    Article Title: Inhibition of Japanese encephalitis virus infection by the host zinc-finger antiviral protein

    doi: 10.1371/journal.ppat.1007166

    Figure Lengend Snippet: Mapping of ZAP-responsive element (ZRE) in JEV 3′-UTR. (A) The CLIP-seq diagram of ZAP-S binding RNA mapped to JEV 3′-UTR. Read coverage, the reads of each position normalized to the total number of reads mapping to the viral genome. The JEV 3′-UTR is divided into domain I, II and III as indicated. The positions of CG dinucleotide are shown by dots. (B) Biotin-labeled JEV 3′-UTR RNA probes (full-length and individual domain I, II, III, and I+II) incubated with A549-ZAP-S cell extracts were pulled down with streptavidin beads and determined for ZAP-S-V5 protein by western blot (left panel). The intensity of ZAP-S was quantified by ImageJ software. Mean ± SD was calculated from 3 independent experiments and analyzed by two-tailed Student’s t test (right panel). (C) 5′-capped full-length and deleted Fluc/5′+3′-UTR RNA (left panel) and control Rluc RNA were cotransfected into 293T/17-EGFP and -ZAP-S cells. At 18 h post-transfection, cell lysates were collected to perform dual-luciferase assay (right panel). Representative data from three independent experiments shown as mean ± SD (n = 3) were analyzed by two-tailed Student’s t test. ** P ≤0.01; *** P ≤0.001; NS , not significant.

    Article Snippet: Human embryonic kidney 293T/17 cells (ATCC, CRL-11268) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS and 2 mM L-glutamine.

    Techniques: Cross-linking Immunoprecipitation, Binding Assay, Labeling, Incubation, Western Blot, Software, Two Tailed Test, Transfection, Luciferase

    ZAP mainly targets JEV 3′-UTR. (A) Map of ZAP-S binding sites in full-length JEV genome by CLIP-seq of RNA isolated from JEV-infected ZAP-S overexpressing A549 cells. Read coverage, the reads of each position normalized to the total number of reads mapping to the viral genome. (B) Schematic diagram of the reporter RNAs (left panel). 293T/17-EGFP and -ZAP-S cells were cotransfected with 5′-capped firefly luciferase (Fluc) flanked by JEV 5′-UTR 197 and/or 3′-UTR RNA and control Renilla luciferase (Rluc) RNA for 18 h. Relative luciferase activity was measured by dual-luciferase reporter assay (right panel). Representative data from two independent experiments are mean ± SD (n = 3) and analyzed by two-tailed Student’s t test. *** P ≤0.001; NS , not significant. (C) Biotin-labeled JEV 3′-UTR RNA (at the indicated amounts) was incubated with ZAP-S or ZAP-S-del4ZF overexpressing A549 cell extracts and then pulled down by using streptavidin beads. The co-precipitated ZAP-S-V5 (WT and del4ZFs) was assayed by western blot.

    Journal: PLoS Pathogens

    Article Title: Inhibition of Japanese encephalitis virus infection by the host zinc-finger antiviral protein

    doi: 10.1371/journal.ppat.1007166

    Figure Lengend Snippet: ZAP mainly targets JEV 3′-UTR. (A) Map of ZAP-S binding sites in full-length JEV genome by CLIP-seq of RNA isolated from JEV-infected ZAP-S overexpressing A549 cells. Read coverage, the reads of each position normalized to the total number of reads mapping to the viral genome. (B) Schematic diagram of the reporter RNAs (left panel). 293T/17-EGFP and -ZAP-S cells were cotransfected with 5′-capped firefly luciferase (Fluc) flanked by JEV 5′-UTR 197 and/or 3′-UTR RNA and control Renilla luciferase (Rluc) RNA for 18 h. Relative luciferase activity was measured by dual-luciferase reporter assay (right panel). Representative data from two independent experiments are mean ± SD (n = 3) and analyzed by two-tailed Student’s t test. *** P ≤0.001; NS , not significant. (C) Biotin-labeled JEV 3′-UTR RNA (at the indicated amounts) was incubated with ZAP-S or ZAP-S-del4ZF overexpressing A549 cell extracts and then pulled down by using streptavidin beads. The co-precipitated ZAP-S-V5 (WT and del4ZFs) was assayed by western blot.

    Article Snippet: Human embryonic kidney 293T/17 cells (ATCC, CRL-11268) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS and 2 mM L-glutamine.

    Techniques: Binding Assay, Cross-linking Immunoprecipitation, Isolation, Infection, Luciferase, Activity Assay, Reporter Assay, Two Tailed Test, Labeling, Incubation, Western Blot

    Zinc-finger motifs of ZAP are required for JEV RNA binding and its antiviral activity. (A) Schematic representation of human ZAP isoforms (ZAP-L, 902 a.a. and ZAP-S, 699 a.a.). The four tandem CCCH-type zinc-finger (ZF) motifs within the N-terminus of ZAP are shown as solid black boxes. Deletion of the four ZFs (a.a. 73–193) are indicated with a dashed line. (B) 293T/17 cells infected with JEV (MOI = 5) for 16 h were transfected with plasmids expressing EGFP, WT or ZF-deleted ZAP-L-V5 and ZAP-S-V5 for additional 24 h. The viral RNA bound with V5-tagged proteins was pulled down by anti-V5 agarose affinity gel and amplified by RT-PCR with JEV 3′-UTR specific primers (middle panel). RT-PCR of input viral RNA in JEV-infected cells (lower panel). Western blot analysis of the immunoprecipitated ZAP-L and ZAP-S (WT and del4ZFs) (upper panel). (C-E) The indicated cells were infected with JEV (MOI = 5) for 10 h. Cell lysates, total RNA, and culture supernatants were determined for the indicated proteins by western blot (C), viral RNA by RT-PCR (D), and viral titer by plaque assay (E). Representative data from three independent experiments shown as mean ± SD (n = 3) were analyzed by two-tailed Student’s t test. *** P ≤0.001.

    Journal: PLoS Pathogens

    Article Title: Inhibition of Japanese encephalitis virus infection by the host zinc-finger antiviral protein

    doi: 10.1371/journal.ppat.1007166

    Figure Lengend Snippet: Zinc-finger motifs of ZAP are required for JEV RNA binding and its antiviral activity. (A) Schematic representation of human ZAP isoforms (ZAP-L, 902 a.a. and ZAP-S, 699 a.a.). The four tandem CCCH-type zinc-finger (ZF) motifs within the N-terminus of ZAP are shown as solid black boxes. Deletion of the four ZFs (a.a. 73–193) are indicated with a dashed line. (B) 293T/17 cells infected with JEV (MOI = 5) for 16 h were transfected with plasmids expressing EGFP, WT or ZF-deleted ZAP-L-V5 and ZAP-S-V5 for additional 24 h. The viral RNA bound with V5-tagged proteins was pulled down by anti-V5 agarose affinity gel and amplified by RT-PCR with JEV 3′-UTR specific primers (middle panel). RT-PCR of input viral RNA in JEV-infected cells (lower panel). Western blot analysis of the immunoprecipitated ZAP-L and ZAP-S (WT and del4ZFs) (upper panel). (C-E) The indicated cells were infected with JEV (MOI = 5) for 10 h. Cell lysates, total RNA, and culture supernatants were determined for the indicated proteins by western blot (C), viral RNA by RT-PCR (D), and viral titer by plaque assay (E). Representative data from three independent experiments shown as mean ± SD (n = 3) were analyzed by two-tailed Student’s t test. *** P ≤0.001.

    Article Snippet: Human embryonic kidney 293T/17 cells (ATCC, CRL-11268) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS and 2 mM L-glutamine.

    Techniques: RNA Binding Assay, Activity Assay, Infection, Transfection, Expressing, Amplification, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunoprecipitation, Plaque Assay, Two Tailed Test

    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

    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

    Biochemical characterization of interactions between CEACAM1 and TIM-3 a , hTIM-3 does not co-immunoprecipitate (co-IP) with ITGA5 despite interactions with hCEACAM1. HEK293T cells transfected with Flag–ITGA5 and HA–TIM-3 (ITGA5Tw) or Flag–CEACAM1 and HA–TIM-3 (CwTw). Immunoprecipitation with anti-HA antibody and immunoblotted (IB) with anti-Flag antibody are shown. Input represents anti-Flag immunoblot of lysates. b, Co-immunoprecipitation of human TIM-3 and CEACAM1 from activated primary human T cells after N -glycanase treatment of lystates followed by immunoprecipitation with anti-human TIM-3 antibodies (2E2, 2E12 or 3F9) or IgG as control and immunoblotted with anti-human CEACAM1 antibody (5F4). Protein lystates from HeLa-CEACAM1 transfectants treated with N -glycanase followed by immunoprecipitation with 5F4 and the immune complex used as positive control (pos). c , mTIM-3 interacts with mCEACAM1 in mouse T cells. Splenocytes from Ceacam1 4S Tg Ceacam1 −/− and Ceacam1-4L Tg Ceacam1 −/− mice cultured with anti-CD3 (1 μg ml −1 ) or anti-CD3 (1 μg ml −1 ) and anti-CD28 (1 μg ml −1 ) or medium for 96 h. Cell lysates immunoprecipitated with anti-mCEACAM1 antibody (cc1) or with mIgG and IB with 5D12 (anti-mTIM-3 antibody) are shown. Locations of mTIM-3 protein variants are indicated. CHO, carbohydrate. d , Immunoprecipitation and immunoblot as in a with tunicamycin treated, wild-type HA–hTIM-3 and Flag–hCEACAM1 co-transfected HEK293T cells. Arrowhead denotes core CEACAM1 protein. e , Potential hCEACAM1-interacting residues on hTIM-3 highlighted in blue. f , HEK293 T cells transiently co-transfected with Flag–hCEACAM1 and HA–hTIM-3 mutants. Immunoblotting of anti-HA were used to analyse hTIM-3 expression in HEK293T transfectants. Except for Pro50Ala mutation displaying enhanced overall protein expression, all other mutations in the IgV domain of hTIM-3 are equally detected by anti-HA antibody. g , Quantification of association of hTIM-3 mutants associated with wild-type hCEACAM1 shown in summing all experiments performed. Association between wild-type hCEACAM1 and hTIM-3 core protein are depicted as reference (set as 1, n = 3, mean ± s.e.m. shown, unpaired Student’s t -test). h , Immunoprecipitation with anti-Flag (hCEACAM1) and immunoblot with anti-HA (hTIM-3) or anti-Flag of wild-type hCEACAM1 and mutant hTIM-3 proteins are shown. i , Quantification of h as performed in g. j , HEK293T cells co-transfected with Flag–hCEACAM1 wild-type and HA– hTIM-3 mutants and immunoprecipitation/immunblot as in h revealing no effects of Cys52Ala or Cys63Ala mutations in hTIM-3 in affecting association with hCEACAM1 in contrast to Cys109Ala mutation of hTIM-3 that disrupts interactions with hCEACAM1. k , Potential hTIM-3-interacting-residues around the FG–CC′ cleft of hCEACAM1 highlighted in red. l , HEK293T cells transiently co-transfected with Flag–hCEACAM1 mutants and wild-type HA–hTIM-3. Immunoblot with anti-Flag antibody was used to analyse hCEACAM1 expression in HEK293T co-transfectants. All hCEACAM1 mutations in IgV domain equally detected. m . n–p , Analysis of Gly47Ala mutation of hCEACAM1 in hTIM-3 co-transfected HEK293T cells by immunoprecipitation with anti-HA (hTIM-3) and immunoblot with anti-Flag (hCEACAM1) to detect association ( n ), IB with anti-Flag to confirm similarity of hCEACAM1 transfection ( o ) and quantification of associated hCEACAM1 of n as shown in m. q–s , Analysis of hCEACAM1 mutants Asn42Ala and Arg43Ala association with hTIM-3 ( q ), similarity of transfections ( r ) and quantification of q as in n–p . Representative of four ( d, h ), three ( f, g, i, l–s ), two ( a–c ) and one ( j ) independent experiments. * P

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: Biochemical characterization of interactions between CEACAM1 and TIM-3 a , hTIM-3 does not co-immunoprecipitate (co-IP) with ITGA5 despite interactions with hCEACAM1. HEK293T cells transfected with Flag–ITGA5 and HA–TIM-3 (ITGA5Tw) or Flag–CEACAM1 and HA–TIM-3 (CwTw). Immunoprecipitation with anti-HA antibody and immunoblotted (IB) with anti-Flag antibody are shown. Input represents anti-Flag immunoblot of lysates. b, Co-immunoprecipitation of human TIM-3 and CEACAM1 from activated primary human T cells after N -glycanase treatment of lystates followed by immunoprecipitation with anti-human TIM-3 antibodies (2E2, 2E12 or 3F9) or IgG as control and immunoblotted with anti-human CEACAM1 antibody (5F4). Protein lystates from HeLa-CEACAM1 transfectants treated with N -glycanase followed by immunoprecipitation with 5F4 and the immune complex used as positive control (pos). c , mTIM-3 interacts with mCEACAM1 in mouse T cells. Splenocytes from Ceacam1 4S Tg Ceacam1 −/− and Ceacam1-4L Tg Ceacam1 −/− mice cultured with anti-CD3 (1 μg ml −1 ) or anti-CD3 (1 μg ml −1 ) and anti-CD28 (1 μg ml −1 ) or medium for 96 h. Cell lysates immunoprecipitated with anti-mCEACAM1 antibody (cc1) or with mIgG and IB with 5D12 (anti-mTIM-3 antibody) are shown. Locations of mTIM-3 protein variants are indicated. CHO, carbohydrate. d , Immunoprecipitation and immunoblot as in a with tunicamycin treated, wild-type HA–hTIM-3 and Flag–hCEACAM1 co-transfected HEK293T cells. Arrowhead denotes core CEACAM1 protein. e , Potential hCEACAM1-interacting residues on hTIM-3 highlighted in blue. f , HEK293 T cells transiently co-transfected with Flag–hCEACAM1 and HA–hTIM-3 mutants. Immunoblotting of anti-HA were used to analyse hTIM-3 expression in HEK293T transfectants. Except for Pro50Ala mutation displaying enhanced overall protein expression, all other mutations in the IgV domain of hTIM-3 are equally detected by anti-HA antibody. g , Quantification of association of hTIM-3 mutants associated with wild-type hCEACAM1 shown in summing all experiments performed. Association between wild-type hCEACAM1 and hTIM-3 core protein are depicted as reference (set as 1, n = 3, mean ± s.e.m. shown, unpaired Student’s t -test). h , Immunoprecipitation with anti-Flag (hCEACAM1) and immunoblot with anti-HA (hTIM-3) or anti-Flag of wild-type hCEACAM1 and mutant hTIM-3 proteins are shown. i , Quantification of h as performed in g. j , HEK293T cells co-transfected with Flag–hCEACAM1 wild-type and HA– hTIM-3 mutants and immunoprecipitation/immunblot as in h revealing no effects of Cys52Ala or Cys63Ala mutations in hTIM-3 in affecting association with hCEACAM1 in contrast to Cys109Ala mutation of hTIM-3 that disrupts interactions with hCEACAM1. k , Potential hTIM-3-interacting-residues around the FG–CC′ cleft of hCEACAM1 highlighted in red. l , HEK293T cells transiently co-transfected with Flag–hCEACAM1 mutants and wild-type HA–hTIM-3. Immunoblot with anti-Flag antibody was used to analyse hCEACAM1 expression in HEK293T co-transfectants. All hCEACAM1 mutations in IgV domain equally detected. m . n–p , Analysis of Gly47Ala mutation of hCEACAM1 in hTIM-3 co-transfected HEK293T cells by immunoprecipitation with anti-HA (hTIM-3) and immunoblot with anti-Flag (hCEACAM1) to detect association ( n ), IB with anti-Flag to confirm similarity of hCEACAM1 transfection ( o ) and quantification of associated hCEACAM1 of n as shown in m. q–s , Analysis of hCEACAM1 mutants Asn42Ala and Arg43Ala association with hTIM-3 ( q ), similarity of transfections ( r ) and quantification of q as in n–p . Representative of four ( d, h ), three ( f, g, i, l–s ), two ( a–c ) and one ( j ) independent experiments. * P

    Article Snippet: HEK293T cells transfected with the 1,200 ng of Flag-tagged human CEACAM1 wild-type or mutant vectors or 1,200 ng of Flag-tagged ITGA5 ( ; Origene) and 1,200 ng of HA-tagged human TIM-3 wild-type or mutant vectors or 1,200 ng of vector controls when mono-transfections were performed and cells transfected for 48 h. In some experiments, 6 h after transfection, transfected cells were treated with 2 µg ml−1 or 10 µg ml−1 tunicamyin provided in DMSO for the last 24 h of transfection.

    Techniques: Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Positive Control, Mouse Assay, Cell Culture, Expressing, Mutagenesis

    CEACAM1 is essential for TIM-3 mediated T cell tolerance a , Schematic diagram of OVA antigen-specific tolerance induction model. b , Schematic diagram of OVA immunization. c , Tracking in vivo antigen-specific T-cell responses of CFSE-labelled OT-II transgenic Rag2 −/− T cells in total lymphocyte gate of mesenteric lymph nodes, peripheral lymph node or spleen of wild-type or Ceacam1 −/− recipients after gating on CFSE-positive cells and staining for CEACAM1 in PBS and OVA 323–339 immunized mice. Hyper-responsiveness of OT-II transgenic Rag2 −/− T cells in Ceacam1 −/− mice was not due to decreased regulatory T-cell induction (data not shown) or increased initial parking on the basis of cell numbers shown. d , TIM-3 expression on CEACAM1-positive and -negative CFSE + cells as in c. e , Schematic diagram of SEB-induced T-cell tolerance model. f , mCEACAM1 and mTIM-3 expression on CD4 + Vβ8 + T cells after SEB tolerance induction. g , hCEACAM1 and hTIM-3 expression on activated primary human T cells defined by staining with indicated antibodies. h , CEACAM1 expression on TIM-3-silenced primary human T cells after re-activation by flow cytometry. Relative TIM-3, CEACAM1 or CD4 expression on T cells expressing control shRNA ( lacZ control, red) or three independent shRNAs directed at TIM3 (overlay, blue). shRNA target sequences shown. i – l , CEACAM1 and TIM-3 expression and functional consequences on T cells in HIV infection. CD4 + IFN-γ + T cells are decreased among CEACAM1 + TIM-3 + CD4 + T cells in HIV infection in response to Gag peptides ( i ). Although proportions of CEACAM1 + TIM-3 + CD8 + T cells are similar in HIV-infected and -uninfected subjects ( j ), CEACAM1 + TIM-3 + CD8 + T cells express little IFN-γ after stimulation with HIV Gag peptides or SEB relative to TIM-3 + CEACAM1 − CD8 + T cells ( k, l ). C, hCEACAM1; T, hTIM-3 ( n = 4 per group, mean ± s.e.m.). m – o , In situ proximity ligation analysis (PLA) of CEACAM1 and TIM-3. m , HEK293T cells transiently co-transfected with Flag–hCEACAM1 or HA–hTIM-3. Cells stained with DAPI (left), anti-tubulin (middle), anti-HA (rabbit) and anti-Flag (mouse) (middle right) or merged (right). Several examples of a positive PLA signal (middle right and right panels: red fluorescent dots) indicative of a maximum distance of 30–40 nm between hCEACAM1 and hTIM-3. n , Negative control, co-expression of Flag–PLK1 (protein kinase I) and HA–TIM-3 failed to generate fluorescent dots (that is, PLA negative). Cells stained with DAPI, anti-tubulin, anti-HA/anti-Flag or merged as in m. o , Negative control, co-expression of HA–ADAP (adhesion and degranulation promoting adaptor protein) failed to show a signal (that is, PLA negative) with staining as in m. p, q , CEACAM1 and TIM-3 colocalization at immunological synapse of primary human CD4 and CD8 T cells. Confocal microscopy of hTIM-3 + hCEACAM1 + primary CD4 + and CD8 + T cells forming conjugates with SEB-loaded B cells. DIC, differential interference contrast. Blue denotes B cell; red denotes CD3; purple denotes CEACAM1; green denotes TIM-3. White indicates colocalization between CEACAM1 and TIM-3 ( p ). Average Pearson correlation coefficients for CD4 + and CD8 + T cells were 0.543 and 0.566, respectively, representing strong co-localization ( q ). Data are mean ± s.e.m. and representative of five ( f, g ), four ( p, q ), three ( c, d, m–o ) and two ( h ) independent experiments. * P

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: CEACAM1 is essential for TIM-3 mediated T cell tolerance a , Schematic diagram of OVA antigen-specific tolerance induction model. b , Schematic diagram of OVA immunization. c , Tracking in vivo antigen-specific T-cell responses of CFSE-labelled OT-II transgenic Rag2 −/− T cells in total lymphocyte gate of mesenteric lymph nodes, peripheral lymph node or spleen of wild-type or Ceacam1 −/− recipients after gating on CFSE-positive cells and staining for CEACAM1 in PBS and OVA 323–339 immunized mice. Hyper-responsiveness of OT-II transgenic Rag2 −/− T cells in Ceacam1 −/− mice was not due to decreased regulatory T-cell induction (data not shown) or increased initial parking on the basis of cell numbers shown. d , TIM-3 expression on CEACAM1-positive and -negative CFSE + cells as in c. e , Schematic diagram of SEB-induced T-cell tolerance model. f , mCEACAM1 and mTIM-3 expression on CD4 + Vβ8 + T cells after SEB tolerance induction. g , hCEACAM1 and hTIM-3 expression on activated primary human T cells defined by staining with indicated antibodies. h , CEACAM1 expression on TIM-3-silenced primary human T cells after re-activation by flow cytometry. Relative TIM-3, CEACAM1 or CD4 expression on T cells expressing control shRNA ( lacZ control, red) or three independent shRNAs directed at TIM3 (overlay, blue). shRNA target sequences shown. i – l , CEACAM1 and TIM-3 expression and functional consequences on T cells in HIV infection. CD4 + IFN-γ + T cells are decreased among CEACAM1 + TIM-3 + CD4 + T cells in HIV infection in response to Gag peptides ( i ). Although proportions of CEACAM1 + TIM-3 + CD8 + T cells are similar in HIV-infected and -uninfected subjects ( j ), CEACAM1 + TIM-3 + CD8 + T cells express little IFN-γ after stimulation with HIV Gag peptides or SEB relative to TIM-3 + CEACAM1 − CD8 + T cells ( k, l ). C, hCEACAM1; T, hTIM-3 ( n = 4 per group, mean ± s.e.m.). m – o , In situ proximity ligation analysis (PLA) of CEACAM1 and TIM-3. m , HEK293T cells transiently co-transfected with Flag–hCEACAM1 or HA–hTIM-3. Cells stained with DAPI (left), anti-tubulin (middle), anti-HA (rabbit) and anti-Flag (mouse) (middle right) or merged (right). Several examples of a positive PLA signal (middle right and right panels: red fluorescent dots) indicative of a maximum distance of 30–40 nm between hCEACAM1 and hTIM-3. n , Negative control, co-expression of Flag–PLK1 (protein kinase I) and HA–TIM-3 failed to generate fluorescent dots (that is, PLA negative). Cells stained with DAPI, anti-tubulin, anti-HA/anti-Flag or merged as in m. o , Negative control, co-expression of HA–ADAP (adhesion and degranulation promoting adaptor protein) failed to show a signal (that is, PLA negative) with staining as in m. p, q , CEACAM1 and TIM-3 colocalization at immunological synapse of primary human CD4 and CD8 T cells. Confocal microscopy of hTIM-3 + hCEACAM1 + primary CD4 + and CD8 + T cells forming conjugates with SEB-loaded B cells. DIC, differential interference contrast. Blue denotes B cell; red denotes CD3; purple denotes CEACAM1; green denotes TIM-3. White indicates colocalization between CEACAM1 and TIM-3 ( p ). Average Pearson correlation coefficients for CD4 + and CD8 + T cells were 0.543 and 0.566, respectively, representing strong co-localization ( q ). Data are mean ± s.e.m. and representative of five ( f, g ), four ( p, q ), three ( c, d, m–o ) and two ( h ) independent experiments. * P

    Article Snippet: HEK293T cells transfected with the 1,200 ng of Flag-tagged human CEACAM1 wild-type or mutant vectors or 1,200 ng of Flag-tagged ITGA5 ( ; Origene) and 1,200 ng of HA-tagged human TIM-3 wild-type or mutant vectors or 1,200 ng of vector controls when mono-transfections were performed and cells transfected for 48 h. In some experiments, 6 h after transfection, transfected cells were treated with 2 µg ml−1 or 10 µg ml−1 tunicamyin provided in DMSO for the last 24 h of transfection.

    Techniques: In Vivo, Transgenic Assay, Staining, Mouse Assay, Expressing, Activation Assay, Flow Cytometry, Cytometry, shRNA, Functional Assay, Infection, In Situ, Ligation, Proximity Ligation Assay, Transfection, Negative Control, Negative Staining, Confocal Microscopy

    CEACAM1 determines TIM-3 expression and function a , HEK293T cells transiently co-transfected with Flag–hCEACAM1 and wild-type or mutants of HA–hTIM-3. Flow cytometry detecting HA–hTIM-3 (detected with anti-HA) and Flag–hCEACAM1 (detected with 5F4) proteins at cell surface (top), Golgi apparatus (middle) or endoplasmic reticulum (bottom) using monensin and brefeldin A, respectively. b , Cellular distribution of wild-type or mutant hTIM-3 when co-expressed with wild-type hCEACAM1. Total counts of hTIM-3 at surface, Golgi apparatus and endoplasmic reticulum summed up to 100%. Depicted as percentage of hTIM-3. c , HEK293T cells transiently co-transfected with wild-type HA–hTIM-3 (detected with 2E2) and wild-type or mutant Flag–hCEACAM1 (detected with anti-Flag). Flow cytometry analyses as in a. d , Cellular distribution of c , as in b . Depicted as percentage of hTIM-3. e , Immunoblot for wild-type or Thr101Ile variant of hTIM-3 showing maturation status in presence of wild-type or mutated (Gln44Leu) hCEACAM1. f , Normal association of Thr101Ile variant of hTIM-3 with hCEACAM1. g , Analysis of CD4 + Vβ8 + T cells after SEB tolerance induction from experimental mice of indicated genotypes. h , Galectin-9 induction of apoptosis. Annexin V + propidium iodide staining of T H 1 cells polarized from Tim3 Tg or Tim3 Tg Ceacam1 −/− mice after treatment with galectin-9 (2 μg ml −1 ) for 8 h. Note decreased apoptosis in Tim3 Tg Ceacam1 −/− T cells. i , Schematic diagram of protocol used for protein pull-down using in-column IgV domain of GST–hTIM-3 incubated with hCEACAM1 protein derived from transfected HEK293T cells as in . j , GST or GST–hTIM-3 staining of hCEACAM1-4L–transfected Jurkat T cells. k , Wild-type CD4 + T cells stimulated with anti-CD3 and/or anti-CD28 in the presence or absence of mCEACAM1 NFc, or IgG1-Fc as control, and cells analysed for secretion of IFN-γ and IL-2. l, m , Characterization of tolerance in SEB model. Tim3 Tg (l) and Tim3 Tg Ceacam1 −/− ( m . Lymph node cells collected after SEB treatment and re-stimulated with soluble anti-CD3 at indicated doses and IL-2 measured by ELISA after 72 h. Note tolerance in Tim3 Tg but not Tim3 Tg Ceacam1 −/− mice. n = 3 per group. n , Anti-mTIM-3 blockade with 2C12 antibody of mCEACAM1 NFc or control IgG-Fc staining of CD4 + T cells from indicated genotypes expressed as levels relative to Ceacam1 −/− mice. o, p , Analysis of mTIM-3 cytoplasmic tail function in transmitting mCEACAM1-induced signals. Activated mouse CD4 + T cells from wild-type ( o ) or Ceacam1 −/− ( p ) mice were retrovirally transduced, sorted and stimulated with anti-CD3 with either human IgG-Fc (IgG, control) or mCEACAM1 N-terminal domain as NFc and TNF-α secretion assessed by ELISA after 72 h. Note ability of CEACAM1 N-terminal domain to transduce a signal associated with inhibition of TNF-α secretion in wild-type but not Ceacam1 −/− T cells. n = 3 per group. Data are mean ± s.e.m. and represent three ( f, g, k–p ) and two ( a–e, h, j ) independent experiments. * P

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: CEACAM1 determines TIM-3 expression and function a , HEK293T cells transiently co-transfected with Flag–hCEACAM1 and wild-type or mutants of HA–hTIM-3. Flow cytometry detecting HA–hTIM-3 (detected with anti-HA) and Flag–hCEACAM1 (detected with 5F4) proteins at cell surface (top), Golgi apparatus (middle) or endoplasmic reticulum (bottom) using monensin and brefeldin A, respectively. b , Cellular distribution of wild-type or mutant hTIM-3 when co-expressed with wild-type hCEACAM1. Total counts of hTIM-3 at surface, Golgi apparatus and endoplasmic reticulum summed up to 100%. Depicted as percentage of hTIM-3. c , HEK293T cells transiently co-transfected with wild-type HA–hTIM-3 (detected with 2E2) and wild-type or mutant Flag–hCEACAM1 (detected with anti-Flag). Flow cytometry analyses as in a. d , Cellular distribution of c , as in b . Depicted as percentage of hTIM-3. e , Immunoblot for wild-type or Thr101Ile variant of hTIM-3 showing maturation status in presence of wild-type or mutated (Gln44Leu) hCEACAM1. f , Normal association of Thr101Ile variant of hTIM-3 with hCEACAM1. g , Analysis of CD4 + Vβ8 + T cells after SEB tolerance induction from experimental mice of indicated genotypes. h , Galectin-9 induction of apoptosis. Annexin V + propidium iodide staining of T H 1 cells polarized from Tim3 Tg or Tim3 Tg Ceacam1 −/− mice after treatment with galectin-9 (2 μg ml −1 ) for 8 h. Note decreased apoptosis in Tim3 Tg Ceacam1 −/− T cells. i , Schematic diagram of protocol used for protein pull-down using in-column IgV domain of GST–hTIM-3 incubated with hCEACAM1 protein derived from transfected HEK293T cells as in . j , GST or GST–hTIM-3 staining of hCEACAM1-4L–transfected Jurkat T cells. k , Wild-type CD4 + T cells stimulated with anti-CD3 and/or anti-CD28 in the presence or absence of mCEACAM1 NFc, or IgG1-Fc as control, and cells analysed for secretion of IFN-γ and IL-2. l, m , Characterization of tolerance in SEB model. Tim3 Tg (l) and Tim3 Tg Ceacam1 −/− ( m . Lymph node cells collected after SEB treatment and re-stimulated with soluble anti-CD3 at indicated doses and IL-2 measured by ELISA after 72 h. Note tolerance in Tim3 Tg but not Tim3 Tg Ceacam1 −/− mice. n = 3 per group. n , Anti-mTIM-3 blockade with 2C12 antibody of mCEACAM1 NFc or control IgG-Fc staining of CD4 + T cells from indicated genotypes expressed as levels relative to Ceacam1 −/− mice. o, p , Analysis of mTIM-3 cytoplasmic tail function in transmitting mCEACAM1-induced signals. Activated mouse CD4 + T cells from wild-type ( o ) or Ceacam1 −/− ( p ) mice were retrovirally transduced, sorted and stimulated with anti-CD3 with either human IgG-Fc (IgG, control) or mCEACAM1 N-terminal domain as NFc and TNF-α secretion assessed by ELISA after 72 h. Note ability of CEACAM1 N-terminal domain to transduce a signal associated with inhibition of TNF-α secretion in wild-type but not Ceacam1 −/− T cells. n = 3 per group. Data are mean ± s.e.m. and represent three ( f, g, k–p ) and two ( a–e, h, j ) independent experiments. * P

    Article Snippet: HEK293T cells transfected with the 1,200 ng of Flag-tagged human CEACAM1 wild-type or mutant vectors or 1,200 ng of Flag-tagged ITGA5 ( ; Origene) and 1,200 ng of HA-tagged human TIM-3 wild-type or mutant vectors or 1,200 ng of vector controls when mono-transfections were performed and cells transfected for 48 h. In some experiments, 6 h after transfection, transfected cells were treated with 2 µg ml−1 or 10 µg ml−1 tunicamyin provided in DMSO for the last 24 h of transfection.

    Techniques: Expressing, Transfection, Flow Cytometry, Cytometry, Mutagenesis, Variant Assay, Mouse Assay, Staining, Incubation, Derivative Assay, Enzyme-linked Immunosorbent Assay, Transduction, Inhibition

    CEACAM1 and TIM-3 heterodimerize and serve as heterophilic ligands a, b , Co-immunoprecipitation (IP) and immunoblot (IB) of wild-type hCEACAM1 and hTIM-3 in co-transfected HEK293T cells, c, d , Co-immunoprecipitation and immunoblot of wild-type hCEACAM1 and hTIM-3 mutants ( c ) or wild-type hTIM-3 and hCEACAM1 mutants ( d ) as in a and b. e , Human CEACAM1 (IgV)-TIM-3 (IgV) heterodimer structure, f, g , 2 F o — F c maps contoured at 0.9σ showing electron densities, h, i , Autoradiogram of anti-haemagglutinin (HA) (hTIM-3) immunoprecipitate from metabolic-labelled ( h ) and pulse-chase metabolic-labelled ( i ) co-transfected HEK293T cells. CHO, carbohydrate; core T, non-glycosylated hTIM-3; Cw, wild-type hCEACAM1; EndoH, endoglycosidaseH; H2-MA, HA-tagged influenza virus A M2 protein; T, hTIM-3 (Thr101Ile); Tw, wild-type hTIM-3. hTIM-3 isoforms noted. j , Quantification of densities in i ( n = 3 per group). k , Immunoblot for mTIM-3 from PBS-treated (−) or SEB-treated (+) CD4 + T cells. Labelling as in h and i. 1 , mTIM-3 expression after SEB tolerance induction, m , Column-bound glutathione S -transferase (GST)-hTIM-3 IgV-domain pull-down of hCEACAM1 detected by immunoblot. GST 2 , GST-hTIM-3 dimer. Ft, flow through, n , Suppression of mouse CD4 + T-cell proliferation by mCEACAM1 N-terminal domain-Fc fusion protein (NFc). o , Immunoprecipitation of mTIM-3 and immunoblot for BAT3 or mTIM-3 from lysates of CD4 + T cells. p, q , Proliferation of CD4 + T cells from wild-type ( p ) and CeaCAM1 −/− ( q ) mice transduced with wild-type mTIM-3 (Tw), mTIM-3 Δ252–281 (Tmut) or vector exposed to anti-CD3 and either NFc or IgG1-Fc (IgG1). Data are mean ± s.e.m. and represent five ( a, b ), four ( c, d ), three ( h-j, l, n, p, q ) and two ( k, m, o ) independent experiments. NS, not significant; * P

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: CEACAM1 and TIM-3 heterodimerize and serve as heterophilic ligands a, b , Co-immunoprecipitation (IP) and immunoblot (IB) of wild-type hCEACAM1 and hTIM-3 in co-transfected HEK293T cells, c, d , Co-immunoprecipitation and immunoblot of wild-type hCEACAM1 and hTIM-3 mutants ( c ) or wild-type hTIM-3 and hCEACAM1 mutants ( d ) as in a and b. e , Human CEACAM1 (IgV)-TIM-3 (IgV) heterodimer structure, f, g , 2 F o — F c maps contoured at 0.9σ showing electron densities, h, i , Autoradiogram of anti-haemagglutinin (HA) (hTIM-3) immunoprecipitate from metabolic-labelled ( h ) and pulse-chase metabolic-labelled ( i ) co-transfected HEK293T cells. CHO, carbohydrate; core T, non-glycosylated hTIM-3; Cw, wild-type hCEACAM1; EndoH, endoglycosidaseH; H2-MA, HA-tagged influenza virus A M2 protein; T, hTIM-3 (Thr101Ile); Tw, wild-type hTIM-3. hTIM-3 isoforms noted. j , Quantification of densities in i ( n = 3 per group). k , Immunoblot for mTIM-3 from PBS-treated (−) or SEB-treated (+) CD4 + T cells. Labelling as in h and i. 1 , mTIM-3 expression after SEB tolerance induction, m , Column-bound glutathione S -transferase (GST)-hTIM-3 IgV-domain pull-down of hCEACAM1 detected by immunoblot. GST 2 , GST-hTIM-3 dimer. Ft, flow through, n , Suppression of mouse CD4 + T-cell proliferation by mCEACAM1 N-terminal domain-Fc fusion protein (NFc). o , Immunoprecipitation of mTIM-3 and immunoblot for BAT3 or mTIM-3 from lysates of CD4 + T cells. p, q , Proliferation of CD4 + T cells from wild-type ( p ) and CeaCAM1 −/− ( q ) mice transduced with wild-type mTIM-3 (Tw), mTIM-3 Δ252–281 (Tmut) or vector exposed to anti-CD3 and either NFc or IgG1-Fc (IgG1). Data are mean ± s.e.m. and represent five ( a, b ), four ( c, d ), three ( h-j, l, n, p, q ) and two ( k, m, o ) independent experiments. NS, not significant; * P

    Article Snippet: HEK293T cells transfected with the 1,200 ng of Flag-tagged human CEACAM1 wild-type or mutant vectors or 1,200 ng of Flag-tagged ITGA5 ( ; Origene) and 1,200 ng of HA-tagged human TIM-3 wild-type or mutant vectors or 1,200 ng of vector controls when mono-transfections were performed and cells transfected for 48 h. In some experiments, 6 h after transfection, transfected cells were treated with 2 µg ml−1 or 10 µg ml−1 tunicamyin provided in DMSO for the last 24 h of transfection.

    Techniques: Immunoprecipitation, Transfection, Pulse Chase, Expressing, Flow Cytometry, Mouse Assay, Transduction, Plasmid Preparation

    TIM-3 and CEACAM1 are co-expressed on T cells during induction of tolerance a, b , Tolerance induction in indicated mice. Median c.p.m., counts per minute. c, d , Responses of CFSE-labelled transgenic OT-II Rag2 −/− T cells in mesenteric lymph nodes (MLN), peripheral lymph node (LN) or spleen of wild-type (WT) or Ceacam1 −/− recipients to PBS ( n = 3 per group) or OVA ( n = 5 per group) for proliferation ( c ) and CEACAM1 or TIM-3 ( d ) expression. ND, not detectable. e , hCEACAM1 and hTIM-3 expression in co-transfected HEK293T cells. Percentage and mean fluorescence intensity (MFI) of hTIM-3 indicated. BFA, brefeldin A; ER, endoplasmic reticulum. f , hCEACAM1 and hTIM-3 expression on activated primary CD4 + human T cells. g, h , CEACAM1 + TIM-3 + CD4 + T cells ( g ) and intracellular cytokine staining for IFN-γ in CD4 + T cells after SEB stimulation ( h ) in HIV infection. C, CEACAM1; T, TIM-3 ( n = 4 per group). i , In situ proximity ligation assay of hCEACAM1 and hTIM-3 co-transfected HEK293T as in e . DAPI, 4′,6-diamidino-2-phenylindole. All data are mean ± s.e.m. and represent five ( e, f ), three ( c, d, i ) and two ( a, b ) independent experiments. * P

    Journal: Nature

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    doi: 10.1038/nature13848

    Figure Lengend Snippet: TIM-3 and CEACAM1 are co-expressed on T cells during induction of tolerance a, b , Tolerance induction in indicated mice. Median c.p.m., counts per minute. c, d , Responses of CFSE-labelled transgenic OT-II Rag2 −/− T cells in mesenteric lymph nodes (MLN), peripheral lymph node (LN) or spleen of wild-type (WT) or Ceacam1 −/− recipients to PBS ( n = 3 per group) or OVA ( n = 5 per group) for proliferation ( c ) and CEACAM1 or TIM-3 ( d ) expression. ND, not detectable. e , hCEACAM1 and hTIM-3 expression in co-transfected HEK293T cells. Percentage and mean fluorescence intensity (MFI) of hTIM-3 indicated. BFA, brefeldin A; ER, endoplasmic reticulum. f , hCEACAM1 and hTIM-3 expression on activated primary CD4 + human T cells. g, h , CEACAM1 + TIM-3 + CD4 + T cells ( g ) and intracellular cytokine staining for IFN-γ in CD4 + T cells after SEB stimulation ( h ) in HIV infection. C, CEACAM1; T, TIM-3 ( n = 4 per group). i , In situ proximity ligation assay of hCEACAM1 and hTIM-3 co-transfected HEK293T as in e . DAPI, 4′,6-diamidino-2-phenylindole. All data are mean ± s.e.m. and represent five ( e, f ), three ( c, d, i ) and two ( a, b ) independent experiments. * P

    Article Snippet: HEK293T cells transfected with the 1,200 ng of Flag-tagged human CEACAM1 wild-type or mutant vectors or 1,200 ng of Flag-tagged ITGA5 ( ; Origene) and 1,200 ng of HA-tagged human TIM-3 wild-type or mutant vectors or 1,200 ng of vector controls when mono-transfections were performed and cells transfected for 48 h. In some experiments, 6 h after transfection, transfected cells were treated with 2 µg ml−1 or 10 µg ml−1 tunicamyin provided in DMSO for the last 24 h of transfection.

    Techniques: Mouse Assay, Transgenic Assay, Expressing, Transfection, Fluorescence, Staining, Infection, In Situ, Proximity Ligation Assay