EMBO/sd-character-level-ner huggingface dataset:
CCB02 prevents proliferation of cells with centrosome amplification.Figure 3(A) 14 day CCB02 treatment of MDA-MB-231, HCC827-GR, Calu-6 and MCF10A (+Dox, extra centrosomes) mostly eliminated cells with extra centrosomes. At the end of the treatment, cells were fixed and stained for centrosomes. Bar graph shows the percentage of cells displaying two- and more than two centrosomes. 'Vehicle' at the bar graph indicates percentages of cells with extra centrosome Note that percentages of cells containing more than two centrosomes are significantly reduced upon CCB02-treatment (open bars). Data represent mean ± SEM of three independent experiments. (N)=3, (n=70). p values were obtained using unpaired t test **P < 0.001, ***P < 0.0001 (B) CCB02 treatment prevents centrosome clustering in HCC827-GR cells. Activated centrosomes (arrows) fail to cluster leading to multipolar mitosis. Cells were stained with Cep152 (green), CPAP (magenta) and microtubules (α-tubulin, red). Scale bar, 2µm, Insets, 0.5µm. Time-lapse images show the effect of CCB02-mediated centrosome activation in cell cycle progression of MCF10A (+Dox, extra centrosomes) (C cells. White arrows indicate centrosome dots. Red arrows (in CCB02-treated cells) indicate activated centrosomes with enhanced microtubule intensity compared to vehicle treated cells. Note, centrosome activation, declustering and prolonged multipolar mitosis occur only with CCB02 treatment. Cartoons of a cell with extra centrosomes below to the panel (C) represent various events occurring upon vehicle or CCB02 treatment. Scale bar, 2µm. Bar graphs at right to panel (C quantifies mitotic duration (ii), quantifies microtubule intensity prior to mitosis (iii) and depicts relative events occurring in cells treated with vehicle or CCB02 (iv) At least 100 cells were analyzed in each condition. (N)=3. Error bars, mean ± SEM. Unpaired t-test **P < 0.001, ***P < 0.0001 Time-lapse images show the effect of CCB02-mediated centrosome activation in cell cycle progression o HCC827-GR (D) cells. White arrows indicate centrosome dots. Red arrows (in CCB02-treated cells) indicate activated centrosomes with enhanced microtubule intensity compared to vehicle treated cells. Note, centrosome activation, declustering and prolonged multipolar mitosis occur only with CCB02 treatment Bar graphs at right to pane (D) quantifies mitotic duration (ii), quantifies microtubule intensity prior to mitosis (iii) and depicts relative events occurring in cells treated with vehicle or CCB02 (iv). At least 100 cells were analyzed in each condition. (N)=3. Error bars, mean ± SEM. Unpaired t-test **P < 0.001, ***P < 0.0001
Sustained proteasome dysfunction inhibits autophagosome formation.Fig 3(A-D) Primary cultures from D. melanogaster embryos expressing GFP-Atg8a under the control of Dmef2-Gal4, stained with Phalloidin (actin) in red. Inset are magnifications of areas denoted by dotted line. (A) In untreated control cultures, GFP-Atg8a localizes throughout the musclecytoplasm and nuclei. (B) Addition of rapamycin (Rap) for 12 hrs results in the formation of small GFP-Atg8a labeled punctae. (C) Addition of chloroquine (CQ) for 12 hrs results in the accumulation of large GFP-Atg8a labeled vesicles that localize around the nucleus and between the myofibers. (D) Addition of both CQ and Rap for 12hrs, triggers the formation of greater numbers of enlarged vesicles in the musclesthan either drug alone. (E) Knockdown of the core autophagy gene, Atg1, prevents the formation of GFP-Atg8a vesicles in CQ+Rap treated cultures. (F) Quantification of the mean ratio of GFP-Atg8a punctae area to total muscle area per well (n = 4). Error bars indicate SEM with **p<.01; ***p<.001. (E) Control white RNAi muscles starved and treated with CQ accumulate GFP-Atg8a punctae, but very few colocalize with ubiquitin (Ubi). (F) Knockdown of Rpn1 in starved + CQ muscle strongly inhibits the formation of GFP-Atg8a punctae. The remainder colocalize with ubiquitin. (G) Quantification of the mean GFP-Atg8a punctae area and Mander's overlap coefficient for GFP-Atg8a and Ubi for genotypes shown in E and F. Error bars indicate SEM for n = 8 ventral longitudinal muscles from individual animals, with p-values for Rpn1 RNAi relative to white RNAi control (***p<.001). (H, I) Autophagosome number is reduced, but autophagic flux is not significantly altered in starved Rpn1 vs. white RNAi muscles expressing GFP-mCherry-Atg8. (J) Flux measurement is shown on the right as the ratio of mCherry punctae to GFP-mCherry (n = 6). SEM is indicated. (K) Anti-Atg8a Western blot of third instar muscle lysate from Dmef2-Gal4; UAS-RNAi animals. In fed white RNAi control, endogenous Atg8a appears as a single band (Atg8a-I). 6 hrs starvation induces lipid modification of Atg8a and the appearance of a second band (Atg8a-II). Knockdown of Atg1 or Rpn1 inhibits Atg8a modification, indicating reduced autophagy levels.
a, b) HeLa cells were transfected with plasmids encoding GFP, CFP–IRGMa, GFP–IRGMc or GFP–IRGMd, and after 24 h (a) or 48 h (b), stained with MTR.figf8(a, b) HeLa cells were transfected with plasmids encoding GFP, CFP-IRGMa, GFP-IRGMc or GFP-IRGMd, and after 24 h (a) or 48 h (b), stained with MTR. Percentages of positively transfected cells that were also MTR+ were then quantified. (c, d) Cells were transfected as in a, b, and after 24 h (c) or 48 h (d) the percentages of positively transfected cells that were also rounded by morphology were quantified. (b) HeLa cells transfected with plasmids encoding GFP or GFP-IRGMd were stained with 7-AAD after 48 h and percentages of GFP+ cells that were also 7-AAD+ were quantified.
a) Constitutively active RagA and RagB stimulate S6K phosphorylation.figf2(a) Constitutively active RagA and RagB stimulate S6K phosphorylation. 200 ng of each mammalian RagA, RagB, RagC or RagD construct was co-transfected with HA-S6K (20 ng) into HEK293 cells. Their corresponding dominant-negative mutants (RagAT21N, RagBT54N, RagCS75N, RagDS76N) and constitutively active mutants (RagAQ66L, RagBQ99L, RagCQ120L, RagDQ121L) were also tested. Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated. (b) RagA has a dominant role over RagC in regulating S6K phosphorylation. Each Rag construct (200 ng) indicated was co-transfected with HA-S6K (20 ng). The different Rag mutants used in the transfection are indicated at the top of each lane. (c) Rag regulates TORC1 but not TORC2 activity. Each indicated Rag construct (200 ng) was co-transfected with either HA-S6K (20 ng), Myc-4EBP1 (20 ng) or of GST-Akt (100 ng). Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated. Full scans of blots are provided in Supplementary Information, Fig. S6.
a) Western blot analysis of biomarker modulation in the HCT116 cell line.figf5(a) Western blot analysis of biomarker modulation in the HCT116 cell line. HCT116 cells were treated with the indicated doses of inhibitors for 8 h (top) or 24 h (bottom), and protein cell extracts were resolved on SDS-PAGE gel. Filters were probed with the indicated antibodies. C, control; B, bortezomib (0.01 μM); T, thapsigargin (0.1 μM); Mg, MG132 (0.1 μM). (b) NMS-873 compound-mediated stabilization of Mcl-1 in live cells. Top, confocal time-lapse images of live stable, inducible T-REx-293 cells expressing a bicistronic construct of mCherry-BimS(2A) ΔC (red) with eGFP-Mcl-1 (green) after doxycycline treatment. Cells were treated with cycloheximide (CHX, 50 μg ml−1) ± 2.5 μM of NMS-873 for 6 h. Bottom, total intensity of eGFP-Mcl-1 and mCherry-BimS(2A) ΔC was measured for time-lapse series shown at top. A decrease in eGFP-Mcl-1 and mCherry-BimS(2A) ΔC total intensity was calculated for two different concentrations of indicated compounds and normalized to DMSO control. WT, wild type. Scale bar, 10 μm. (c) High-content analysis of poly-Ub and CHOP induction upon treatment with VCP inhibitors. HeLa cells were treated with increasing doses of the indicated compounds. Cells were then fixed and immunostained with the indicated antibodies. Cell numbers and mean fluorescence intensity (MFI) were determined using the ArrayScan platform. Data were collected in triplicate (n = 3), and error bars represent s.d. AU, arbitrary units. (d) VCP and proteasome inhibitors induce cell killing in distinct cell targets. IC50 data are plotted as log10 ratio, calculated according to the following equation: log10 ratio = log10 (IC50 cell line/mean IC50 of all cell lines). Data shown are representative results of one of three independent experiments.
Figure 4A) Fluorescent micrographs of pachytene germ-cell nuclei from animals expressing indicated fluorescent proteins. Scale bar, 2μm. B) 3D render of one set of GFP and TagRFP foci from panel A. Scale bar, 0.5μm. C) Germlines dissected from animals expressing the indicated proteins were fixed and stained with α-FLAG and α-HA antibodies, followed by secondary antibodies conjugated with Alexa 488 and CF430, respectively. Pachytene germ-cell nuclei were imaged with a Leica TCS SP8 microscope. Scale bar, 2μm. D) Quantification of the degree to which fluorescent signals from the indicated fluorescent proteins colocalize. See Fig. EV3A and Materials and Methods for details. For each group, 27 granules from 3 animals (3 cells per animal, 3 granules per cell) were quantified. Black line indicates mean of the degree of colocalization in each group. E) Germlines dissected from animals expressing the indicated proteins were fixed and stained with α-GFP and α-HA or α-FLAG antibodies, followed by secondary antibodies conjugated with Alexa 488 and CF430, respectively. Pachytene germ-cell nuclei were imaged with a Leica TCS SP8 STED 3X microscope using 592nm to selectively deactivate fluorophores. Bottom panel. GFP::ZNFX-1 and HA::TagRFP::ZNFX-1 expressing animals were crossed and germlines of trans-heterozygous progeny from this cross were fixed and stained and imaged by STED as described above. Scale bar, 0.5μm. F) Degree of colocalization of indicated fluorescent proteins in STED images was determined as described in Materials and Methods. For each group, 12 granules from 3 animals (4 granules per animal) were quantified. Black line indicates mean of the degree of colocalization in each group.
LAPTM4B inhibits EGF‐stimulated EGFR endosomal sorting A–D Control or LAPTM4B siRNA‐transfected MDA‐MB‐231 cells were starved, pretreated or not with chloroquine for 2 h, stimulated with 100 ng/ml EGF for 15 min, washed, and chased for indicated time periods before fixation for costaining of EGFR (red) with EEA1 (A, green) or LAMP1 (C, green).Figure 3A-D Control or LAPTM4B siRNA‐transfected MDA‐MB‐231 cells were starved, pretreated or not with chloroquine for 2 h, stimulated with 100 ng/ml EGF for 15 min, washed, and chased for indicated time periods before fixation for costaining of EGFR (red) with EEA1 (A, green) or LAMP1 (C, green). Quantification of the average percentages of EGFR signals colocalized with EEA1 (B) and LAMP1 (D) at indicated time points; mean + SD; n = 3; *P 0.05, **P 0.01, ***P 0.001, one‐tailed t‐test. E MDA‐MB‐231 cells were starved, stimulated with 100 ng/ml EGF for 15 or 120 min, fixed, and costained for EGFR and LAPTM4B, followed by fluorescence microscopy. Cells with higher and lower LAPTM4B expression were marked with arrows and arrowheads, respectively.F The amounts of total EGFR staining in individual cells in (E) were plotted against LAPTM4B levels at 15 min and 120 min, respectively. Note: for LAPTM4B quantification, the non‐specific nuclear staining was not included. Trend lines and Pearson's correlation coefficients are shown. AU, arbitrary unit.
a,b) RIPK2 bone marrow chimeras were analyzed at day 2 after PR8 infection for contribution to elevated IL-18 production by using IL-18 immunohistochemistry (a) and IL-18 ELISA (b) of whole lung homogenates.figf4(a,b) RIPK2 bone marrow chimeras were analyzed at day 2 after PR8 infection for contribution to elevated IL-18 production by using IL-18 immunohistochemistry (a) and IL-18 ELISA (b) of whole lung homogenates. WT, wild type; t.p., total protein. In a, blue arrows denote IL-18+ macrophages, and red arrows denote IL-18+ bronchial epithelium. Original magnification, ×200. (c) Flow cytometry analysis of NK cell counts in Ripk2−/− and wild-type mice. (d) Analysis of wild-type and Ripk2−/− NK cells for IFN-γ production on a per cell basis. (e) Accumulation of T and B cells in lungs. (f) Analysis of CD8+ T cells for IFN-γ and CD44 expression in wild-type and Ripk2−/− mice. *P 0.05, **P 0.01 (one-way ANOVA with Tukey's post-hoc test (b) and unpaired two-sided Student's t-test (d,f)). Data are representative of two experiments (a,b,f) or three experiments (c-e) (mean ± s.e.m.; n = 6-8 mice per experiment).
Sequential dual-color imaging of synaptic Ca2+-influx and glutamate release.Figure 2IHCs were patch-clamped in ruptured-patch configuration with 800 μM Rhod-FF in the patch pipette, and simultaneously imaged for Ca2+-signals or glutamate release by spinning disc microscopy. Mean ΔF images of Rhod-FF and iGluSnFR signals in response to a voltage ramp and a 50-ms-long step depolarization, respectively. The synapse marked with * on the overlaid image is further analyzed in the following panels. (P: pillar side, M: modiolar side; Scale bar: 2 μm) Voltage command (top), corresponding whole-cell Ca2+-influx (middle) and the functional fluorescence responses (bottom) (bandstop filtered at 33.3 Hz) from Rhod-FF and iGluSnFR, respectively. A modified Boltzmann function (see Materials and Methods, R2 = 0.81) was fitted to a Rhod-FF fluorescence trace in response to a voltage ramp (C-C'). iGluSnFR-AUC was calculated per depolarization voltage and used for a Boltzmann fit (D', R2 = 0.92). The voltage of half-maximal activation (V1/2) and the dynamic range (10-90%) of synaptic Ca2+-influx and glutamate release were calculated from the fits and depicted as circle and bar in C' and D', respectively. The obtained fits from a Ca2+ "hotspot" (C') and from glutamate release (D') were plotted against each other in a voltage range from -57 mV to -17 mV in 1 mV increments. A power function was fitted to the 25% of the maximum iGluSnFR-AUC (R2 = 0.99) to obtain the m-estimate. (ruptured patch-clamp, 10 mM intracellular EGTA, 5 mM [Ca2+]e) (See also Fig EV4)
Figure 5(A) Experimental schemes LacI-CENP-B or LacI control (baits) were expressed into CENP-AEA/- lacO-tetO U-2OS cells. Immunostaining against CENP-C or CENP-A (preys) was performed following LacI-CENP-B transfection in untreated, IAA treated and IAA wash-out (WO) conditions. (B) Representative immunofluorescence images showing CENP-C recruitment at the LacO array in the indicated conditions. LacO array is displayed in the insets. (C) Bar plot showing frequency of CENP-C (orange) or CENP-A (green) recruitment to LacO array in the indicated conditions. Each dot represents one independent experiment (>20 cell analyzed for experiment). (D) Quantification of CENP-C (orange) or CENP-A (green) protein levels at the LacO array in the indicated conditions normalized to protein intensities in non-treated conditions at the mCherry-LacI-CENP-B-LacO array using CENP-A or CENP-C antibody. Each dot represents one independent experiment (>20 cell analyzed for experiment). Error bars represent SEM from 3 independent experiments. (E) Representative immunofluorescence images showing CENP-A recruitment to ectopic mCherry-LacI-CENP-B in the indicated conditions. LacO array is shown in the inset. (F) Quantification of the frequency of CENP-C recruitment to LacO arrays by different LacI constructs. Error bars represent SEM from 5 independent experiments. (G) CENP-B immunoblot following GST pulldown experiments using GST tagged CENP-C (1-727) as bait and with the indicated proteins as preys.
Fig.Figure 5A, BiFC (split-YFP) analysis of HOs and phytochrome. Strains (SChS29-33) were grown over night at 28°C minimal medium with 2% threonine and 0.2% glucose. MitoTracker Red CMOXRos was used for mitochondrial staining. The mitotracker signal was observed in the RFP and the split-YFP signal in the YFP channel of the fluorescent microscope. Pictures in both channels were overlaid (merge). The left pictures show the same hyphae in differential interference contrast (DIC). The scale bar represents 10 µm. B, HoxA was expressed in E. coli 20 h at 15°C. Purification was performed with the Strep-tag system. HoxA was analyzed by size exclusion chromatography. C, Co-expression of the HOXs along with the photosensory domain of A. nidulans FphA (PGP) over night at 20°C. PGP was purified via the Strep-tag system and the eluate concentrated (upper panel). The lanes were loaded as follows: negative control before induction (-), 20 h after induction (20 h) crude extract (CE), supernatant (S), pellet (P), flow through (F) and eluate (E). The fractions were analyzed by Western Blot using the Anti-His antibody (lower panel).
Figure 2A Bacterial two-hybrid assay reveals an interaction between SiaB and SiaC. The recombinant strains harboring different proteins were separately streaked on nonselective and dual-selective media (3-amino-1, 2, 4- triazole + streptomycin). NA represents the empty vector pBT or pTRG. The strain expressing LGF2 and Gal11P were used as positive controls. B Pull-down assays confirmed the interaction between SiaB and SiaC. Cell lysates of P. aeruginosa containing pMM67EH-siaC-Flag were incubated with GST or GST-SiaB individually, and protein complexes were captured by glutathione beads. C SPR measurements of SiaC binding at varying concentrations to SiaB. SiaC specifically interacted with SiaB with a KD of 40.1 nM. Shown are measured binding responses (black) and curve fits to a 1:1 interaction (red). Plots are representative from two experiments with similar results. RU, response units; KD, dissociation constant. D SiaB-mediated regulation of biofilm formation is dependent on SiaC. The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down). E Bacterial two-hybrid assayreveals an interaction between SiaC and SiaA PP2C-like domain. NA represents the empty vector pBT or pTRG. The recombinant strains harboring different proteins were separately streaked on nonselective and dual-selective media (3-amino-1, 2, 4- triazole + streptomycin). The strain expressing LGF2 and Gal11P were used as positive controls. F Pull-down assays confirmed the interaction between SiaC and SiaA. Cell lysates of P. aeruginosa containing pMM67EH-siaA-Flag were incubated with GST or GST-SiaC individually, and protein complexes were captured by glutathione beads. G Pull-down assays confirmed the interaction between SiaC and SiaA PP2C-like domain. Cell lysates of P. aeruginosa containing pMMB67EH-siaA386-Flag were incubated with GST or GST-SiaC individually, and protein complexes were captured by glutathione beads. H The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).
a) Ba/F3 cells expressing myrPI(3)K-ER, myrPKB-ER, FOXO3(A3)-ER or FOXO4(A3)-ER were stimulated with 4-OHT for 4 h (myrPI(3)K-ER, myrPKB-ER) or 8 h (FOXO3(A3)-ER and FOXO4(A3)-ER) and microarray analyses were performed.figf1(a) Ba/F3 cells expressing myrPI(3) K-ER, myrPKB-ER, FOXO3(A3)-ER or FOXO4(A3)-ER were stimulated with 4-OHT for 4 h (myrPI(3) K-ER, myrPKB-ER) or 8 h (FOXO3(A3)-ER and FOXO4(A3)-ER) and microarray analyses were performed. Shown are the fold changes relative to control cells for glutamine synthetase (GS), Mxi1 and Pink1. Data are represented as mean values of one experiment performed in quadruplicate. (b) Ba/F3 cells expressing either myrPI(3) K-ER or myrPKB-ER were cytokine starved overnight and stimulated with 4-OHT. Relative mRNA levels of glutamine synthetase were analysed using quantitative rtPCR. Data are represented as mean ± s.e.m. normalized for B2M (n=4). *P0.05, **P0.01. (c) Ba/F3 cells expressing either myrPI(3) K-ER or myrPKB-ER were cytokine starved overnight and stimulated with 4-OHT. Cell lysates were analysed for protein levels of phospho-FOXO3 (T32), glutamine synthetase, p27 and actin. Shown are representative blots (n=4). (d) Ba/F3 cells expressing either FOXO3(A3)-ER or FOXO4(A3)-ER were stimulated with 4-OHT in the presence of mIL-3. Relative mRNA levels of glutamine synthetase were analysed using quantitative rtPCR. Data are represented as mean ±  s.e.m. values normalized for B2M (n=3). **P0.01. (e) Ba/F3 cells expressing either FOXO3(A3)-ER or FOXO4(A3)-ER were stimulated with 4-OHT in the presence of mIL-3. Cell lysates were analysed for protein levels of glutamine synthetase, p27 and actin. Shown are representative blots (n=3). (f) Wild-type Ba/F3 cells were cytokine starved overnight and stimulated with mIL-3. Cell lysates were analysed for protein levels of phospho-FOXO3 (T32), glutamine synthetase, p27 and actin. Shown are representative blots (n=4). (g) Wild-type Ba/F3 cells were incubated with LY294002 in the presence of mIL-3. Cell lysates were analysed for protein levels of glutamine synthetase, p27 and actin. Shown are representative blots (n=3). (h) FOXO1,3,4−/− MEFs and wild-type MEFs were incubated with LY294002 (10 μM) for the indicated times. Cell RNA was isolated and relative mRNA levels of glutamine synthetase were analysed using quantitative PCR. Data are represented as mean values normalized for B2M (n=2). Uncropped images of blots are shown in Supplementary Fig. S7.