EMBO/sd-character-level-ner huggingface dataset:
Figure 5(A) Autophagy was monitored in WT hippocampal neurons treated with the lysosome inhibitor's cocktail for 3 days by immunoblot analysis with anti-LC3 antibodies. As control DIV10 WT neurons were left untreated and monitored by LC3 blot over the three days period. The LC3-II levels quantitation was shown. (B) Proteasome activity was measured in treated and control untreated WT neurons using pZsProSensor-1 vector (green). Cells were co-stained with anti SMI-32 (blu). Proteasome activity was quantified by measuring the green fluorescence (inversely correlated with proteasome activity) in 10 different cells (taken from 4-5 coverslips for each group) and expressed as fold to WT. (C) Activation of calpain 2 and caspase-3 proteolytic systems was evaluated by WB measurement of the protein levels of calpain 2 and caspase-3 (both full-length and activated cleaved forms of ~17 KDa) in treated and control untreated WT neurons. Arrow indicated the full-length caspase-3 protein. (D) After 3 days-treatment cell lysates from treated and control untreated WT neurons were immunoblotted with α-synuclein and CSPα and protein levels were quantified. (E) Treated and control untreated WT neurons were subjected to α-synuclein (green) and LAMP1 (red) confocal analysis. Enlarged merge images are also shown. Co-localization was quantified using the MCC coefficient (ImageJ) and displayed as % (MCC x 100) of α-synuclein co-localizing with LAMP1 (means ± s.e.m. from 15 different images taken from 4-5 coverslips for each group). Cells were also co-stained with anti SMI-32. (F) Treated and control untreated WT neurons were subjected to CSPα/SMI-32 co-immunofluorescence. CSPα synaptic puncta present in a neurite tract of 10µm is showed in a representative enlarged image. Quantification of CSPα synaptic puncta was calculated from 30 different enlarged images taken from 4-5 coverslips for each group. (G) SDS-resistant complex levels in treated and control untreated WT neurons were evaluated by immunoblotting analysis of non-boiled samples with VAMP2 or SNAP-25 antibodies. The amounts of SNARE complexes were quantified.
Figure 1(A) Huh7.5 cells were pre-incubated with neutralizing antibody to ACE2 and SARS-CoV-2 pseudovirus was pre-incubated with patient isolated mAb COVA1-18, COVA1-21 and COVA2-15 (10 µg/mL) or UF heparin (250 IU/mL) for 30 min at 37°C. SARS-CoV-2 pseudovirus alone or with blocks was added to the cells for 4h at 4°C and binding was determined by ELISA. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using ordinary one-way ANOVA with Tukey multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001(n=3) (B) Heparan sulfates were removed from Huh7.5 cells by enzymatic treatment with heparinase III for 1 hour at 37°C, then washed, and exposed to SARS-CoV-2 pseudovirus for 4h at 4°C. Treated and untreated cells were subsequently lysed and binding was determined by ELISA. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using unpaired Student's t-test with Welch's correction. *P≤0.05, **P≤0.01(n=3) (C) Flow cytometry analysis of cell surface expression of heparan sulfates (HS) in control transduced cells or upon CRISPR/Cas9-mediated EXT1 KO (EXT1-/-). (D) Control and EXT1-/- XG1 cells were exposed to SARS-CoV-2 pseudovirus or SARS-CoV-2 pseudovirus pre-treated with 250 IU/mL UF heparin for 30 min at 37°C. After incubation for 4h at 4°C, cells were lysed and binding was measured by ELISA. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using two-way ANOVA with Dunnett's multiple-comparison test. *P≤0.05, **P≤0.01 (n=3).
Blockade of EGFR induces accumulation of the autophagy protein LC3 around the parasite, vacuole-lysosome fusion and killing of T.Figure 6A, HBMEC were incubated with AG1478 (1 µM) 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy at 2 and 24 h. B, Human RPE cells transfected with either EGFR or control siRNA were challenged with T. gondii followed by examination of monolayers by light microscopy at 24 h. C, Mouse bone marrow-derived macrophages were incubated with AG1478 and challenged with T. gondii. Monolayers were examined by light microscopy at 2 and 24 h. D, Parental CHO and CHO-EGFR cells were challenged with RH T. gondii. Monolayers were examined microscopically 2 h or 24 h post-challenge. E, mHEVc-LC3-EGFP cells transfected with control siRNA or EGFR siRNA were challenged with T. gondii-RFP. Monolayers were examined by fluorescence microscopy 5 h post-challenge to determine the percentage of endothelial cells with LC3 accumulation around the parasite. F, HBMEC cells treated with or without AG1478 were challenged with either T. gondii-YFP. Expression of LAMP-1 was examined by fluorescent microscopy 8 h post-challenge. The percentages of endothelial cells with LAMP-1 accumulation of around the parasite were determined. G, H, mHEVc cells transfected with Beclin1 siRNA (G) or Atg7 siRNA (H) were transfected with EGFR siRNA or treated with or without AG1478 followed by challenge with T. gondii. Monolayers were examined by light microscopy 24 h post-challenge. Results are shown as the mean ± SEM and are representative of 3 independent experiments.
Figure 2(A) Representative images of COS7 cells expressing COP1-3xFLAG (green) co-transfected with empty vector, GFP, or the indicated GFP-TRIB1 constructs (red). Cells were stained with anti-FLAG (Sigma) and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm. (B) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of COP1-3xFLAG in (A). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance relative to GFP-COP1 with empty vector was calculated using the Student t test (*p < 0.01; **p < 0.001; ***p < 0.0001). (C) Representative images of COS7 cells expressing GFP-COP1 (green) co-transfected with empty vector, TRIB1-FLAG, TRIB2-FLAG, or TRIB3-FLAG (red). Cells were stained with anti-FLAG (Sigma) and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm. (D) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 in (C). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance relative to GFP-COP1 with empty vector was calculated using the Student t test (ns, not significant; ***p < 0.0001; ****p < 0.00001).
Figure 7(A) Maximum intensity projection of video acquired 10 min after inoculation of P. falciparum sporozoites visualizes trajectories of parasites, engaging with CD31-labeled vascular endothelia (magenta). Scale bar, 50 μm. See movie EV9. (B) Displacement of sporozoites 5 to 60 min after inoculation. Data is displayed in box and whisker plots with whiskers showing the 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal lines show the median at each time point. Percentage values indicate the fraction of tracks displacing over 75 microns. (C) Apparent speed of sporozoites 5 to 60 min after inoculation. Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal lines show the median speed at each time point. For panels B-C, data shown originates from the analysis of 3-4 videos per time point and a varying number of tracks were processed for each time point after inoculation: 5 min (157 tracks), 10 min (568 tracks), 20 min (286 tracks), 30 min (171 tracks), 60 min (101 tracks). No motile sporozoites were observed 120 min after inoculation. (D) Proportion of motile and non-motile sporozoites was manually counted and is displayed as percentage of sporozoites observed 5 min after inoculation. The data shown was obtained from one complete imaging session over 120 min after intradermal injection of sporozoites. (E) Sporozoite track straightness, the ratio of displacement to track length of P. falciparum sporozoites moving in mouse dermis, human skin ex vivo, and human skin graft, at 5-10 min post intradermal injection. Data is displayed in box and whisker plots with horizontal line showing the median and the whiskers extending to the minimum and maximum values. Comparisons showed statistically significant differences in track straightness between the human skin graft and both mouse skin (****p=0.0001) and human skin ex vivo (****p<0.0001) using Tukey's multiple comparisons test. A varying number of tracks were processed for each environment: mouse skin: 17 videos, 822 tracks; human skin ex vivo: 12 videos, 761 tracks; human skin graft: 9 videos, 718 tracks. (F) P. falciparum sporozoites engage with and enter blood vessels in human skin graft. Maximum intensity projection shows trajectories of moving P. falciparum sporozoites (green), engaging with human CD31-labeled vascular endothelia (magenta). Scale bar, 25 μm. See movie EV10. Graph shows lymphatic and blood vessel entry events of P. falciparum sporozoites in human skin graft. Combined invasion events observed at 5-20 min after intradermal injection in n number of videos. To view a blood vessel invasion event P. falciparum in human skin graft,
Trypsin enhances lineage B entry in various cell linesa, Primate cells, b, human cells or c, bat cells were infected with VSV-particles pseudotyped with the lineage B chimeric spike panel.Figure 2:Primate cells, were infected with VSV-particles pseudotyped with the lineage B chimeric spike panel. Pseudotypes were either left untreated or incubated with trypsin before addition to the cells. Luciferase was measured and normalized to particles produced without pseudotype. Shown are the data for 3 replicates. human cells were infected with VSV-particles pseudotyped with the lineage B chimeric spike panel. Pseudotypes were either left untreated or incubated with trypsin before addition to the cells. Luciferase was measured and normalized to particles produced without pseudotype. Shown are the data for 3 replicates. bat cells were infected with VSV-particles pseudotyped with the lineage B chimeric spike panel. Pseudotypes were either left untreated or incubated with trypsin before addition to the cells. Luciferase was measured and normalized to particles produced without pseudotype. Shown are the data for 3 replicates.
Gαq signaling acts through calmodulin to induce P-EMT Fura-2 Ca2+ measurements after addition of 5μM CNO (Top) or 10μM ATP (Bottom) in murine PDA cells expressing a Gαq-DREADD.Figure 4Fura-2 Ca2+ measurements after addition of 5μM CNO (Top) or 10μM ATP (Bottom) in murine PDA cells expressing a Gαq-DREADD. Blue or red line indicates average of n=22 cells. ±SD. Tracings are representative of experiments performed twice in 2 independent cell lines. Representative 20X brightfield images of Gαq-DREADD-expressing cells 48h after addition of CNO (Left). Scale bar = 100μm. MFI of surface E-cadherin in cells expressing a Gαq-DREADD after treatment with DMSO, 5μM CNO, or 2.5μM ionomycin (Right). Both experiments performed in triplicate in 2 independent cell lines. Statistical analysis by ANOVA (**, p<0.01; ±SD). MFI of surface E-cadherin in CnB knockout lines after 48h treatment with DMSO, 2.5μM ionomycin, or 10ng/ml TGFβ. Experiment was repeated in two different murine PDA cell lines in triplicate. Statistical analysis by ANOVA (***, p<0.001; ****, p<0.00001; ±SD). MFI of surface E-cadherin of murine PDA cells pretreated with 20μM W7 for 24h and then DMSO, 2.5μM ionomycin, or 20μM W7 with 2.5μM ionomycin for 24h. Experiment was repeated in two different murine PDA cell lines in triplicate. Statistical analysis by ANOVA (**, p<0.01; ***, p<0.001; non-significant (NS); ±SD). MFI of surface E-cadherin of shRNA non-targeting (shNT) (Left) or shCAMK2B (Right) after 48h treatment with DMSO, 2.5μM ionomycin, or 10ng/ml TGFβ. Experiment was repeated in two different murine PDA cell lines. Statistical analysis by ANOVA (***, p<0.001; ****, p<0.00001; ±SD).
Fig.Figure 4A: Ubc9 D100A enzymatic activity can be recovered by limited amount of DTT. 21 nM Aos1/Uba2 and 75 nM Ubc9 were incubated with H2O2 for 30 min prior addition of 65 nM each YFP-SUMO1 and CFP-RanGAPtail diluted in DTT-free buffer. The final DTT concentration in the reaction was 6 µM. Prior to addition of ATP, extra amount of DTT was added at indicated final concentration. B: Both Uba2~Ubc9 disulfide formation and reduction are accelerated with Ubc9 D100A.100 nM E1 and 1 µM E2 were incubated in presence of 1 mM H2O2. One hour after H2O2 addition, 500 µM reduced glutathione was added. Samples were taken at indicated time points and the Uba2~Ubc9 disulfide was monitored by immunoblotting against Uba2. Lower panel: Quantification of the ratio between crosslinked Uba2 vs total Uba2
a) Confocal microscopy of HeLa cells were treated with anti-CD95 antibody for 5 h or left untreated.figf5(a) Confocal microscopy of HeLa cells were treated with anti-CD95 antibody for 5 h or left untreated. Cells were stained with anti-Atg5 and anti-VDAC antibodies. In untreated cells, there was only little evidence for colocalization of Atg5 and VDAC. In anti-CD95 antibody treated cells some of the Atg5 colocalized with VDAC. The images of colocalization were prepared using Imaris software. Numerical analysis was performed on 10 cells in each group and the correlation coefficient calculated. The error bars represent the s.e.m. (b) Subcellular fractionation of blood neutrophils that were left untreated to induce spontaneous apoptosis. After 4 h incubation, cleavage of Atg5 was evident and both full-length and truncated Atg5 were present in the cytosol. After 8 h incubation, some 24K Atg5 but not full-length Atg5 was found in the mitochondrial fraction. Mitochondrial association of truncated Atg5 coincided with the release of cytochrome c into the cytosol. The same data were observed in staurosporine-induced apoptosis of Jurkat cells (data not shown). C, cytosol; M, mitochondria. (c) Cytochrome c release assay. Truncated and full-length recombinant Atg5 were added to isolated mitochondria and incubated at 37 °C for 1 h. Supernatants and mitochondrial pellets were then analysed for cytochrome c by immunoblotting. Truncated Bid (tBid) was used as a positive control for mitochondrial permeabilization. Truncated Atg5 released cytochrome c in a concentration-dependent manner, indicating mitochondrial permeabilization. Full-length Atg5 had only little effect in this system. (d) HeLa cells were transfected with 24K and 33K Atg5 using lentiviral gene transfer for 24 h and analysed for caspase-3 cleavage. GAPDH expression demonstrates equal loading. (e) Jurkat cells were treated with staurosporine for 5 h or left untreated. Staurosporine induced Atg5 cleavage and both full-length and truncated Atg5 were present in total lysates. Truncated but not full-length Atg5 was detected in Bcl-xL immunoprecipitates obtained from apoptotic Jurkat cells. No evidence for physical interaction between Atg5 and Bax was obtained. The lower panels demonstrate that both Bax and Bcl-xL were enriched following immunoprecipitation. The asterisk indicates P 0.05. The scale bars represent 10 μM.
Figure 4(A) Schematic diagram of human KLF4 functional domains and strategy for engineering a series of KLF4 deletion mutants. (B) Identification of amino acid stretch 411- 441 on the repression domain of KLF4 as a segment mediating the interaction with PARP1 in 293T cells.. (C) Schematic diagram of human KLF4 functional domains and strategy for engineering a set of KLF4 deletion mutants. (D). Identification of amino acid stretch 411- 441 on the Zinc 2 domain of KLF4 involved in mediating its PARylation modification in 293T cells.. PARylation for Flag tagged KLF4 deletion mutants were analyzed, respectively, by pulldown using PAR antibody followed by Western blotting using antibody against Flag. (E) Dissection of the PARylation region of KLF4 into three zinc finger motif-containing domains, Zinc 1, Zinc 2 and Zinc 3. While no effect was observed on Zinc 1 and Zinc 3 domains, deletion of Zinc 2 domain led to significant attenuation of KLF4 PARylation. (F) Schematic diagram of human PARP1 functional domains and strategy for engineering a series of PARP1 deletion mutants. (G) Identification of amino acid stretch 829-1014 on the C-terminal segment of PARP1 to be involved in mediating its interaction with KLF4 in 293T cells.
Figure 1(A) H1299 cells were transfected with Myc-β-catenin, CK1α and MDMX mutants. Phosphorylation of β-catenin S45 was determined by western blot. Co-expression of MDMX blocked the increase of pS45 level. MDMXSG (W200S/W201G) and MDMX-1-300 were defective for CK1α binding. (B) GST-β-catenin-1-200 was incubated with purified FLAG-CK1α, FLAG-MDMX and ATP at indicated ratios. Phosphorylation of β-catenin S45 was determined by western blot. MDMXS289A contains mutation of CK1α phosphorylation site. (C) Kinetic characterization of CK1α inhibition by MDMX. Reaction velocity as a function of β-catenin peptide substrate and increasing MDMX concentrations at 100 µM ATP and 2 nM CK1α. Data were fitted to the Michaelis-Menten equation. The resulting parameter values are shown in the table. (D) Reaction velocity as a function of ATP and increasing MDMX concentrations at 800 µM peptide substrate and 2 nM CK1α. Data were fitted to the Michaelis-Menten equation.
Figure 4(A-H) Lateral images of the PHS fixed at 36 hpf (A-B, E-F) and 42 hpf (C-D, G-H) in lyve1b:EGFP fish that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red). These show a decrease in the number of Prox1-positive PHS ECs in the ccbe1 morpholino-injected (B, D) and sFLT4 mRNA-injected embryos (F, H) compared with control morpholino-injected (A, C) and uninjected embryos (E, G). At 42 hpf, ccbe1 morpholino-injected embryos (D) and sFLT4 mRNA-injected embryos (H) lack the PHS-LP domains present in the control embryos (C, G; large arrow heads). (I-J) Quantitation of the percentage of PHS ECs that are lymphatic progenitors relative to the total number of PHS ECs in control morpholino-injected embryos at 36 hpf (n = 27) and 42 hpf (n = 17) and ccbe1 morpholino-injected embryos at 36 hpf (n = 36) and 42 hpf (n = 12) (I) or in uninjected embryos at 36 hpf (n = 27) and 42 hpf (n = 21) and sFLT4 mRNA-injected embryos at 36 hpf (n = 26) and 42 hpf (n = 17) (J). (K) Quantitation of the total number of PHS ECs in control morpholino-injected embryos at 36 hpf (n = 27) and 42 hpf (n = 17) and ccbe1 morpholino-injected embryos at 36 hpf (n = 36) and 42 hpf (n = 12) or in uninjected embryos at 36 hpf (n = 27) and 42 hpf (n = 21) and sFLT4 mRNA-injected embryos at 36 hpf (n = 26) and 42 hpf (n = 17) (L-O) Lateral images of the FLS (L, N; dotted line) or dorsolateral CCV region from where the FLS normally sprouts (M, O; dotted line) fixed at 36 hpf in lyve1b:EGFP embryos that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red). These show no change in the number of Prox1-positive lymphatic progenitors in the ccbe1 morpholino-injected (M) and sFLT4 mRNA-injected (O) embryos compared with control morpholino-injected (L) and uninjected (N) embryos. (P) Quantitation of the percentage of dorsolateral CCV ECs that are lymphatic progenitors relative to the total number of dorsolateral CCV ECs in control morpholino-injected (n = 26), ccbe1 morpholino-injected (n = 27), uninjected (n = 28) and sFLT4 mRNA-injected embryos (n = 32). (Q) Quantitation of the total number of dorsolateral region CCV ECs in 36 hpf embryos that were uninjected or injected with control morpholino (n = 26), ccbe1 morpholino (n = 27), or sFLT4 mRNA (n = 32). (R-U) Dorsolateral images of the VA-L fixed at 54 hpf in lyve1b:EGFP larva that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red). These show that lymphatic progenitor formation still occurs within the VA-L in ccbe1 morpholino-injected (S) and sFLT4 mRNA-injected embryos (U), with Prox1 expression resembling that of controls (R, T).
Figure 5A Schematic of A-485 inhibition of the histone acetyl transferase p300/CBP, which is recruited by NF-κB, and of JQ1 inhibition of BET bromodomains, which recruit the positive transcription elongation factor (P-TEFb). B Change in enrichment of histone H3 and AcH3 after treatment with 300 nM A-485 for 4 hours measured by ChIP-qPCR and shown as % input (non-IP control) normalized to the uninhibited control for each gene. Data are presented as mean ± SD for two or four biological replicates. C Bar graphs of mean mRNA level for basal condition, 1h TNF, 1h TNF + 4h pretreatment with 300nM A-485, and 1h TNF+62.5nM JQ1 cotreatment measured by smFISH for Tnfaip3 (left) and Tnf (right). Cells were combined from two replicates (Tnfaip3 A-485, JQ1; Tnf A-485) or one replicate (Tnf JQ1). Error bars indicated bootstrapped 95% CIs. Samples with non-overlapping CIs are significant. D Change in enrichment of total and Serine-5-phosphorylated RNAPII after 20ng/mL TNF treatment for 1 hour with 62.5nM JQ1 measured by ChIP-qPCR and shown as % input (non-IP control) normalized to the uninhibited control for each gene. Data are presented as mean for two biological replicates. E E-F Burst size (E) and burst frequency (F) parameter fits from the two-state model for the same conditions in (C) measured by smFISH for Tnfaip3 (left) and Tnf (right) for the data described in (C). Error bars indicated bootstrapped 95% CIs. Samples with non-overlapping CIs are significant. G-H Probability density of mRNA distributions measured by smFISH for 20 ng/ml TNF for 1h (blue) versus 1h TNF + 4h pretreatment with 300nM A-485 (green) (G) and 1h TNF (blue) versus 1h TNF+62.5nM JQ1 cotreatment (yellow) (H) for Tnfaip3 (left) and Tnf (right).