EMBO/SourceData huggingface dataset:
figf2a, RAW 264.7macrophages infected for 4 or 8 h were analysed by TEM. Shown are spacious vacuoles (SLAPs) containing multiple bacteria. The arrow indicates septum of dividing bacteria. Scale bars, 0.5 µm.b, The arrow indicates a LAMP1+ SLAP colocalizing with green fluorescent protein (GFP)-LC3 in cells infected for 4 h.The percentage of GFP-LC3+ (c)d, The arrowhead indicates a LAMP1+ SLAP devoid of cathepsin D in cells infected for 4 h. Scale bars, 5 µm.The percentage of GFP-LC3+ (c) or cathepsin D+ (e)f, GFP-LC3-transfected macrophages were infected with wild-type or D actA bacteria. Where indicated, chloramphenicol (CM) was added to the media at 3 h post infection. The number of bacteria per SLAP was quantified. Brackets indicate significant differences, and corresponding P values are shown.g, Macrophages were infected with wild-type bacteria for 7 h, pulsed with BrdU for 1 h, and stained for LAMP1 (red), bacteria (blue) and BrdU (green). Magnified images and the arrow indicate SLAPs containing actively replicating bacteria (BrdU+). h, The percentage of BrdU+ bacteria in SLAPs, compared to bacteria and PFA-killed bacteria, was quantified as in c.
figf3a, GFP-LC3-transfected macrophages were infected for 4 h, and the percentage of infected cells exhibiting SLAPs was quantified. Mean ± s.e.m. for three independent experiments. P values for strains with significant differences from wild-type levels are shown.b, The arrow indicates a GFP-LC3+ SLAP with internal LLO expression in cells infected for 4 h. Scale bar, 5 µm.c, Arrowheads indicate GFP-LC3+ SLAPs devoid of Lysotracker Red in cells infected for 4 h.e, The arrow indicates a LAMP1+ SLAP colocalizing with v-ATPase staining in cells infected for 4 h. Scale bar, 5 µm. f, The percentage of v-ATPase+ SLAPs was quantified as in Fig. 2c. Mean ± s.e.m. for three independent experiments. P values for conditions significantly different from PFA-killed bacteria are shown.
figf4a, Arrows indicate LAMP1- wild-type bacteria with actin 'comet tails' in cells infected for 6 h. The boxed region is magnified in the bottom left panels. The merged image for this region is shown at the bottom right. Scale bar, 5 µm.b, Macrophages were infected with IPTG-induced iLLO bacteria. Arrowheads indicate actin- iLLO bacteria within LAMP1+ vacuoles.c, Macrophages were infected with wild-type (triangles) or IPTG-induced iLLO (circles) bacteria, and the percentage of LAMP1+ bacteria was quantified. Mean ± s.e.m. for three independent experiments.d, Macrophages were infected as in c with or without IPTG induction. Intracellular bacterial replication was determined using a gentamicin-protection assay. Shown is fold replication compared to 2 h post infection. Clear triangles, wild type; filled triangles, wild type + IPTG; clear circles, iLLO; filled circles, iLLO + IPTG. Mean ± s.e.m. or range for three (wild type, wild type + IPTG, iLLO + IPTG) or two (iLLO-IPTG) independent experiments, respectively.e, Wild-type or Atg5-/- MEFs were infected with iLLO bacteria, and intracellular bacterial replication was determined as in d. Mean ± s.e.m. for three independent experiments. Clear circles, wild-type MEFs; filled circles, wild-type MEFs + IPTG; clear squares, Atg5-/- MEFs; filled squares, Atg5-/- MEFs + IPTG.f, Wild-type (circles) or Atg5-/- (squares) MEFs were infected as in e with IPTG induction. The percentage of LAMP1+ bacteria was quantified as in c. Mean ± s.e.m. for three independent experiments. g
Figure 1(A) Isolation of FTCD from rat liver extract. The p47(1-170) fragment, which does not contain the p97-binding site, was immobilized on beads and incubated with rat liver extract. Bound proteins were fractionated by SDS-PAGE, and stained with CBB.(B) FTCD binds directly to p47. GST-tagged p47 (0.54 μg) or GST-tagged p37 (0.45 μg) was incubated with His-tagged FTCD (0.80 μg), and then isolated on glutathione beads. The blots were probed with antibodies to FTCD and GST.(C) FTCD and p47 form a complex in cells. HepG2 cells were solubilized and incubated with anti-p47 antibodies. The immunoprecipitates were fractionated by SDS-PAGE, followed by Western blotting with antibodies to p47 and FTCD.(D) FTCD, p47 and p97 form a complex in cells. HepG2 cells were solubilized and incubated with anti-FTCD antibodies. The immunoprecipitates were fractionated by SDS-PAGE, followed by Western blotting with antibodies to FTCD, VCIP135, p97, p47 and syntaxin5.(E) p47 binds to the CD domain of FTCD. GST-tagged p47 (0.50 μg) was incubated with either His-tagged FTCD(1-326) (FT, 0.88 μg) or His-tagged FTCD(327-541) (CD, 0.63 μg). The blots were probed with antibodies to His-tag and GST.(F) p47(E86K, E88K) does not bind to FTCD. GST-tagged p47wt/mutant (0.50 μg) was incubated with the indicated His-tagged proteins (p97, 1.0 μg; FTCD, 0.75 μg). The blots were probed with antibodies to p97, FTCD and GST.(G) FTCD(R382A), which lacks binding ability to polyglutamates, does not bind to p47. GST-tagged p47 (0.35 μg) was incubated with His-tagged FTCDwt/mutant (0.53 μg). The blots were probed with antibodies to His-tag and GST.(H) p47 binds to FTCD in Golgi membranes. Salt-washed Golgi membranes were first incubated with either GST-tagged p47wt, p47(E86K, E88K) or p47(F253S). The isolated membranes were solubilized, and then GST-tagged p47wt/mutants and their binding proteins were recovered with glutathione-beads. The bound proteins were assayed by Western blotting with antibodies to FTCD and GST.
Figure 2(A) FTCD directly binds to p97. GST-tagged p97 (2.1 μg) was incubated with His-tagged FTCD (0.80 μg), and then isolated on glutathione beads. The blots were probed with antibodies to FTCD and GST.(B) Nucleotide dependency of the binding of FTCD with p97. FTCD (0.80 μg) and GST-tagged p97 (2.1 μg) were incubated in the presence of the indicated nucleotide (1 mM). The blots were probed with antibodies to FTCD and GST.(C) FTCD binds to the C-terminal region of p97. GST-tagged p97 fragments were incubated with either FTCD or p47. The blots were probed with antibodies to FTCD and p47.(D) p97 also binds to the CD domain of FTCD. GST-tagged p97 (2 μg) was incubated with either His-tagged FTCD(1-326) (FT, 0.88 μg) or His-tagged FTCD(327-541) (CD, 0.62 μg). The blots were probed with antibodies to His-tag and GST.(E) p97(V722K, E724K) shows very low binding affinity to FTCD. GST-tagged p97wt/mutant ( 2.0 μg) was incubated with the indicated His-tagged proteins (FTCD, 2.0 μg; p47, 0.5 μg). The blots were probed with antibodies to p47, FTCD and GST.(F) FTCD(R382A), which lacks binding ability to polyglutamates, shows a very low binding affinity to p97. GST-tagged p97 (1.4 μg) was incubated with His-tagged FTCDwt/mutant (0.53 μg). The blots were probed with antibodies to His-tag and GST.(G) p97 binds to FTCD in Golgi membranes. Salt-washed Golgi membranes were incubated with either GST-tagged p97wt or p97(V722k, E724K). The isolated membranes were solubilized, and then GST-tagged p97wt/mutants and their binding proteins were recovered with glutathione-beads. The bound proteins were assayed by Western blotting with antibodies to FTCD and GST.
Figure 3(A) Immunofluorescence staining of FTCD and GM130, a Golgi marker. HepG2 cells were fixed, permeabilized, stained with a monoclonal antibody to FTCD, a polyclonal antibody to GM130 and DAPI, and observed by confocal microscopy. Scale bar = 10 μm.(B) Depletion of FTCD by FTCD siRNA duplexes. HepG2 cells were either mock transfected with water or transfected with two distinct siRNA duplexes specific to FTCD. After incubation for 48 hrs, the cells were analyzed by Western blotting with antibodies to FTCD and α-tubulin.(C) The Golgi in cells which were depleted of either FTCD or p47. HepG2 cells, which were treated with either FTCD siRNAs, p47 siRNA or mock for 48 hrs, were fixed and stained with a monoclonal antibody to GM130. Scale bar = 10 μm. (D) The results of quantification of (C). The values are from five sets of independent experiments. Results are expressed as the mean±SD (n=5), with 100 cells counted in each group of each set. Asterisks indicate a significant difference at P<0.01 compared with the mock-treated cells (Bonferroni method). (E) Representative EM images of the Golgi in HepG2 cells treated with either mock or FTCD siRNAs for 42 hrs. Scale bar = 0.5 μm. (F) The results of quantification of (E). Golgi membranes in HepG2 cells were classified into cisternae, vesicles and tubules, and counted as previously described (Shorter & Warren, 1999). The results are shown as the mean±SD of five sets of independent experiments, with 15-20 cells counted in each group in each independent experiment. An asterisk indicates a significant difference (P<0.05) compared with the mock-treated cells in each category (Bonferroni method).
Figure 4(A) The localization of FTCD in mitotic cells. Mitotic HepG2 cells were collected by flushing and cultured on coated coverslips for 10-40 mins. The cells were fixed, stained with a monoclonal antibody to FTCD and polyclonal antibodies to GM130 and α-tubulin, and observed by confocal microscopy. Panels d, h, and l indicate the images of α-tubulin and the other panels show the distribution of FTCD or/and GM130. Scale bar = 10 μm. Arrowheads indicate the colocalization of FTCD and GM130.(B) The effects of FTCD siRNA treatment on Golgi reassembly at the end of mitosis. HepG2 cells were treated with either FTCD siRNA (FTCD siRNA 2 duplex) or mock for 48 hrs before the collection of mitotic cells. The collected mitotic cells were cultured for 35 mins and then fixed. The cells were stained with a polyclonal antibody to GM130 and a monoclonal antibody to α-tubulin, and observed by confocal microscopy. Panels a-p display representative images. Scale bar = 10 μm.(C) Rescue experiments in FTCD siRNA-treated HepG2 cells. The indicated HA-tagged FTCDs, which were insensitive to FTCD siRNA, were expressed 25 hrs after the treatment of cells with FTCD siRNA2. The cells were further cultured for 23 hrs and then used for the collection of mitotic cells. The collected mitotic cells were cultured for 35 mins and fixed. The cells were stained with a monoclonal antibody to α-tubulin (panels g and h) and polyclonal antibodies to GM130 and HA (panels a-f). Scale bar = 10 μm.(D) The cells at cytokinesis in (B) and (C) were analyzed. The results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiment. Asterisks indicate a significant difference (P<0.01) compared with the mock-treated cells (Bonferroni method).
Figure 6(A) Anti-FTCD antibodies inhibit p97/p47-mediated Golgi membrane fusion. Golgi fragments were mixed with the indicated components (p97, 60 μg/ml; p47, 30 μg/ml; p37, 24 μg/ml; p115, 40 μg/ml) together with either anti-FTCD or anti-p115 antibodies. Results are expressed as mean+SD of five sets of independent experiments; 0% and 100% represent the buffer only (25.9% in cisternal membranes) and p97/p47 with no antibodies ('+ none', 48.8%), respectively. Asterisks indicate a significant difference at P<0.01 compared with the no antibody groups ('+none') (Bonferroni method).(B) p47(E86K, E88K), which lacks binding affinity to FTCD, does not function in p97/p47-mediated Golgi membrane fusion. Golgi fragments were incubated with p97 (60 μg/ml) and p47wt/mutant (30 μg/ml). Data are shown as the mean+SD of five sets of independent experiments; 0% and 100% represent the buffer only (27.3% in cisternal membranes) and p97+p47wt ('+p47wt', 50.9%), respectively. Asterisks indicate a significant difference at P<0.01 compared with the group in which p47wt as well as p97 were added ('+p47wt') (Bonferroni method).(C) p97(V722K,E724K), which lacks binding affinity to FTCD, does not function in p97/p47-mediated Golgi membrane fusion. Golgi fragments were incubated with the following components: p97wt/mutant, 60 μg/ml; p47, 30 μg/ml; p37, 24 μg/ml; p115, 40 μg/ml. Data are shown as the mean+SD of five sets of independent experiments; 0% and 100% represent the buffer only (29.5% in cisternal membranes), and p47+p97wt ('+p97wt'. 57.5%), respectively. An asterisk indicates a significant difference at P<0.01 compared with the group in which p97wt as well as p47 were added ('+p97wt') (Bonferroni method).(D) Panels a-e show representative EM images. Scale bar = 0.5 μm.
Figure 9(A) FTCD located in mitochondria causes mitochondria aggregation during mitosis. Mitotic cells were collected by flushing from HeLa Tet-off cells in which either FTCDwt-HA-MAO or FTCD(R382A)-HA-MAO had been induced for 24 hrs. The mitotic cells were further cultured for 4 hrs to enable complete cytokinesis. Cells in which FTCDwt-HA-MAO was induced for 28 hrs without synchronization were used as the asynchronous control group. Cells were fixed and visualized with a monoclonal antibody to mitochondria and a polyclonal antibody to HA. Panels a-f display representative images. Scale bar = 10 μm. (B) The results of quantification of (A). Results are shown as the mean±SD of seven sets of independent experiments, with 100 cells counted in each group in each independent experiment. An asterisk indicates a significant difference at P<0.01 compared with the others (Bonferroni method). (C) A single living cell expressing FTCDwt-HA-MAO was tracked during mitosis. Mitotic cells were collected by flushing from the HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO, and cultured in the absence or presence of DOX. The cells were stained with MitoTracker at the 22 hr time point and confocal images of 30-40 cells were randomly taken at the 23 hr time point. From the 23 hr to 27 hr time point, cells were tracked in the bright field every 30 mins. At the 27 hr time point, confocal images were taken of the 21-38 cells that were successfully tracked. Panels a-j display representative images. Asterisks indicate the tracked cells. Scale bar = 10 μm. (D) The results of quantification of (C). The experiment was repeated five times and each line shows the average percentage of an independent experiment. The percentages of cells that underwent mitosis are as follows: 41.5 % in the DOX(-) group and 54.7 % in the DOX(+) group. (E) FTCD located in mitochondria causes mitochondria aggregation at the end of mitosis. Mitotic cells were collected from the HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO and cultured in the absence or presence of DOX as in (C). Cells were stained with MitoTracker at the 23hr time point and observed by confocal microscopy from the 24 hr to 26 hr time point. Panels a-t display representative images. Scale bar = 10 μm.(F) The results of quantification of (E). Cells were classified by their cell cycle phases into the following three categories: prometaphase/metaphase, anaphase/telophase and cytokinesis. Results are shown as the mean±SD of three sets of independent experiments, with 10-14 cells counted in each group in each independent experiment. Asterisks indicate a significant difference at P<0.01 compared with the DOX(+) group and with the prometaphase/metaphase category (Bonferroni method).
Figure 2A, Heat map of all reported cancer blood metabolites (each dark blue mark denotes that the metabolite was reported to be increased or decreased) in various cancer types, illustrating that most individual studies report only a small subset of all previously measured metabolites. It is typically not described whether the metabolites that were not reported (white "empty" space) were not measured, or measured but not reported. Labelings: X-axis (top): ribbon colour code, denoting the cancer type (right; indicated by red arrow); X-axis (bottom): cohorts, arranged from 1 to 71; Y-axis (right): all 1,206 metabolites reported in at least one of the studies; Y-axis (left): ribbon colour code, denoting the metabolite class (amino acids, carbohydrates, etc; "other" refers to all other metabolites than the listed classes).B, Vote counting of cancer blood metabolites (reported in at least six cohorts) showed consistently deregulated metabolites. Blue bars: decreased metabolites; red bars: increased metabolites. * (asterisk) in front of the name of the metabolite indicates a statistical trend (p < 0.1); red arrowheads denote metabolites mentioned in the main text.C,D, Volcano plot of bloodmetabolites (C) and tissuemetabolites (D), reported in at least six cancer studies, with the vote counting score on the X-axis and the -log10 adjusted p-value on the Y-axis. Cyan indicates deregulated metabolites that show a trend (p < 0.1; for blood, corresponding to metabolites marked with * in panel 2B; for tissue, corresponding to metabolites marked with * in Figure 3) or statistical significance (p < 0.05; above black dashed horizontal line); red indicates metabolites with a p-value > 0.1. A subset of metabolites is annotated (see Table EV4 (blood) and EV5 (tissue) for full annotation).
Figure 3Vote counting of cancer tissue metabolites (reported in at least six cohorts) showed consistently deregulated metabolites. Blue bars: decreased metabolites; red bars: increased metabolites. * (asterisk) in front of the name of the metabolite indicates a statistical trend (p < 0.1); red arrows denote key metabolites mentioned in the main text (see Table EV5 for full annotation).
Figure 1Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence of H676 cells treated with the indicated compounds for 24h at 1 μM. Scale bars, 5μm. Data are representative of n=3 biological replicates. (C) Western blot images (top) and TRF1 protein levels (bottom) of h676 cells treated with the indicated compounds for 24h at 1 μM. Data are representative of n=3 (PLKi, HSP90i and RTKi) and n=4 (AuroraI, mTOR, CDKi, Docetaxel, Gemcitabine, ERKi, MEKi) biological replicates.
Figure 2Representative images (left) and percentage (right) of cells presenting 1 or more γH2AX and RAP1 colocalizing foci (TIFs) upon treatment of CHA9-3 lung cancer cells with the indicated compounds. White arrowheads point to colocalization of γH2AX and RAP1. Scale bars, 5μm. Data are representative of n=6 (DMSO) and n=3 (mTORi, PI3Ki, RTKi, MEKi, ERKi, HSPO90i, CDKi, Docetaxel) biological replicatesRepresentative images (left) and percentage (right) of γH2AX-positive cells per field in DMSO or compound treated patient-derived h676 GSCs. Scale bars, 50μm. Data are representative of 6 (DMSO) and 3 (mTORi, Docetaxel, ERKi, MEKi, RTKi, HSP90i, Gemcitabine, CDKi) biological replicatesQuantification of multitelomeric signals (MTS) in h676 GSC metaphases upon treatment with the indicated compounds. Representative images of the qFISH in the metaphases (left). Multitelomeric signals are indicated by arrowheads. Scale bars, 1μm. Data are representative of n=31 (DMSO), n=18 (mTORi), n=11 (MEKi) and n=24 (RTKi) biological replicates. Data are represented as mean ± SEM. Significant differences using unpaired t‐test are indicated by *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 3Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.
Figure 41 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of mouse ERK2 kinase in the presence of 5μCi [γ-32-P]ATP. The mixture was resolve by SDS-PAGE followed by autoradiography.1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of human b-Raf kinase (WT or V600E) in the presence of 5μCi [γ-32-P]ATP. The mixture was resolve by SDS-PAGE followed by autoradiography.1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of mouse MEK1 kinase in the presence of 5μCi [γ-32-P]ATP. The mixture was resolve by SDS-PAGE followed by autoradiography.1 μM of GST-TRF1 and 0.2 μM of mouse ERK2 kinase were incubated in the presence of ERK and MEK inhibitors.2 μM of GST-TRF1 and 0.1 μM of human b-Raf kinase were incubated in the presence of the bRaf inhibitors dabrafenib and vemurafenib.1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of human mTOR kinase1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of human mTOR kinase in the presence of the mTOR inhibitors rapamycin and Ku0063794Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 or TRF1 plus ERK2Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 plus bRAFWT or bRAFV600EPhosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 plus mTORRepresentative image (down) and quantification (up) of in vitro phosphorylation assays with the indicated GST-TRF1 wild-type or mutated forms in the presence of mouse ERK2 kinase.
Figure 5Western blot images of Trf1lox/lox MEFS with or without overexpression of eGFP-Trf1 WT or mutant alleles followed by Cre recombinase transduction.Quantification of eGFP-TRF1 inhibition in Trf1Δ/Δ MEFs transduced with eGFP-Trf1 WT or mutant alleles as indicated. Data are representative of n=15 biological replicatesGrowth curves of Trf1Δ/Δ MEFs transduced with eGFP-Trf1 WT or mutant alleles as indicated. Data are representative of n=5 biological replicatesWestern blot images of Trf1lox/lox MEFS with or without overexpression of eGFP-Trf1 WT or mutant alleles followed by Cre recombinase transduction. Quantification of eGFP-TRF1 inhibition in Trf1Δ/Δ MEFs transduced with eGFP-Trf1 WT or mutant alleles as indicated. Data are representative of n=5 biological replicatesWestern blot images of p53-/- MEFS with or without overexpression of eGFP-Trf1 WT or mutant alleles followed treatment with ERKi. Data are representative of n=2 biological replicatesRepresentative images (above) and percentage (bottom) of Telomeric and 53BP1 colocalizing foci (TIFs) per cells of p53-/- MEFS with or without overexpression of eGFP-Trf1 WT and the indicated mutants upon treatment with the ERKi. White arrowheads: colocalization of telomeric and 53BP1. Scale bars, 10μm.
Figure 6Western blot image (above) and quantification (bottom) of TRF1 protein levels upon genetic depletion of ERK1/2 in p53-/- MEF line. Data are representative of n=3 independent experiments.Representative images (above) and quantification (bottom) of TRF1 telomeric foci in ERK1/2 RNA interfered p53-/- MEFs. Scale bars, 5μm. Data are representative of n=2 independent experiments.Western blot image of eGFP-tagged and endogenous TRF1 protein levels upon genetic depletion of ERK1/2 in p53-/- MEF line.Representative images (above) and percentage (bottom) of telomeric and 53BP1 colocalizing foci (TIFs) per cell in p53-/- MEF with or without overexpression of eGFP-Trf1 WT and indicated mutants upon genetic depletion of ERK1/2. White arrowheads: colocalization of telomeric and 53BP1.Scale bars, 10μm.