EMBO/sd-character-level-ner huggingface dataset:
pre-existing, ER targeted mEOS3.Figure 2Left: Representative images of cells were transfected with super-folder GFP (sfGFP) treated with 250ng/mL Tunicamycin (Tm), 50nM Thapsigargin (Tg) and 250ng/mL Brefeldin-A (BFA) for 24 hours. Right: Quantification of the microscopy images of cells expressing ER-targeted sfGFP and the cytosolically localized mCherry. Data values are the mean ± SD of technical replicates (n=10) from 3 independent experiments (****p<0.0001). One-way ANOVA was applied for the statistical analysis through the GraphPad Prism 9 software. Scale bar 15μm. Subcellular protein fractionation of several ER resident proteins in HEK293T cells treated with the indicated concentrations of Tm or Tg for 16 hours using Digitonin (NP40 represents the membrane fraction extracted with NP40 Cell Lysis Buffer) protocols, representative western blots are showed. Subcellular protein fractionation of several ER resident proteins in HEK293T cells treated with the indicated concentrations of Tm or Tg for 16 hours using Digitonin (NP40 represents the membrane fraction extracted with NP40 Cell Lysis Buffer) or differential centrifugation protocols, representative western blots are showed. Quantification of the subcellular protein fractionation of several ER endogenous proteins in HEK293T cells treated with different concentrations of Tm and Tg for 16 hrs from panels B and C respectively. Biological triplicates, mean ± SD calculated using Prism 9 (GraphPad). Quantification of the subcellular protein fractionation of several ER endogenous proteins in HEK293T cells treated with different concentrations of Tm and Tg for 16 hrs from panels B and C respectively. Biological triplicates, mean ± SD calculated using Prism 9 (GraphPad). Mass spec analysis of soluble ER targeted glycoproteins in HEK293T cells treated with Tg and analyzed as described in materials and methods. Biological triplicates, Data analyses were carried using Cytoscape v3.8.0 for network representation (PMID: 14597658) with the Cytoscape Stringapp for enrichments (PMID: 30450911). statistics, were done using the default settings of the Cytoscape app. Mass spectrometry analysis of cytosolically located soluble ER glycoproteins in HEK293T cells treated with Tg compared to that found in human GBM tumor cells-derived cytosols.
Figure 4Pearson correlation between S1P (A-C) PSI days from admission and NLR in COV (n=111). Scatter plots and fitted regression line are shown in each figure. Exact p values or ****p < 0.0001 are reported. Pearson correlation between apoM (D-F) and PSI , days from admission and NLR in COV (n=111). Scatter plots and fitted regression line are shown in each figure. Exact p values or ****p < 0.0001 are reported.
a) Activation of mTOR activity with rheb increases aggregation and toxicity of mutant (Gln74; Q74) huntingtin.figf5(a) Activation of mTOR activity with rheb increases aggregation and toxicity of mutant (Gln74; Q74) huntingtin. COS-7 cells were cotransfected with a mammalian expression vector encoding rheb (or empty vector control) and mutant huntingtin for 48 h at a 3:1 mass ratio (rheb or empty vector to huntingtin) to ensure that almost all cells expressing huntingtin also expressed rheb. The percentages of EGFP-positive cells with aggregates or apoptotic nuclear morphology (as described previously) were markedly higher when rheb was overexpressed. Control cells express mutant huntingtin (green) without rheb. The middle panels show images captured at the same comparatively low gain to distinguish aggregates from soluble EGFP. The left panels show the same fields at a similar higher gain to indicate the total number of EGFP-positive cells (this shows that the effect seen in the middle panels is not due to loss of cells). Nuclear staining with DAPI (blue) is shown in the right panels. ***P 0.0001. Error bars represent s.e. for a representative triplicate experiment. (b,c) Treatment with rapamycin rescues huntingtin-induced degeneration in flies 2 d after eclosion. Photographs of ommatidia of flies expressing mutant (Gln120; Q120) huntingtin treated with DMSO or rapamycin from the larval stage into adulthood. More rhabdomeres are visible in the ommatidia of flies treated with rapamycin. This effect is significant (P 0.0001; Mann-Whitney U test). At 2 d after eclosion, there is no reduction in the number of visible rhabdomeres in wild-type untreated flies (data not shown).
Figure 2(A) Titration of fluorescence polarization of FITC-peptides (100 nM) spanning α11.2 aa 1906-1959 with purified MBP-β2AR C-terminus. Kd value was obtained by fitting a non-linear direct binding curve to Pep2. (B) IP of β2AR in the absence (Control) or presence of 10 μM peptides, as indicated. Pep2 specifically displaced α11.2 (top of IB) but not GluA1 (middle, same IB) from β2AR (bottom, same IB). Use of non-specific IgG (left lane) indicates specificity of coIPs.(C, D). For quantification, α11.2 and GluA1IB signals were normalized to β2AR signals (***p<0.001, ANOVA). (E) IP of β2AR in the absence (Control) or presence of 10 μM Pep2 or Pep2 with S1928 being phosphorylated (phPep2), which did not displaced α11.2.(F) α11.2 and GluA1 IB signals were normalized to β2AR signals (***p<0.001, ANOVA).
Figure 4A Immunofluorescence (IF) staining of PC3-LN4 cells following 6 h treatment with DMSO (Control), PIMi (PIM447, 3µM), AKTi (GSK690693, 5μM), and both inhibitors (AKTi + PIMi) and stained with anti-EDC3 and anti-DCP1a antibodies as well as DAPI. Data information: IF Images were subsequently merged using FIJI image analysis software. The yellow box shows a zoom of the merged image. Images were captured under 40x magnification; scale bar: 10µm. P-body number was counted by FIJI. Quantification of EDC3/DCP1a foci number from Control (Ctrl), PIMi, AKTi and the AKTi + PIMi combination in (A). Data information: The quantification data of (B are presented as mean +/- SEM (n = 30 cells/treatment group). p values<0.05, calculated using unpaired student t-test, are considered significant. p values>0.05 not significant (n.s.). The analyses done included multiple independent fields. C Immunofluorescence (IF) staining of PC3-LN4 EDC3 knock out cells following 6 h treatment with DMSO (Control), PIMi (PIM447, 3µM), AKTi (GSK690693, 5μM), and both inhibitors (AKTi + PIMi) and stained with anti-EDC3, and anti-DCP1a antibodies (Abs) as well as DAPI. Data information: IF Images were subsequently merged using FIJI image analysis software. The yellow box shows a zoom of the merged image. Images were captured under 40x magnification; scale bar: 10µm. P-body number was counted by FIJI. D Quantification of DCP1a foci number for Control (Ctrl), PIMi, AKTi, and AKTi + PIMi combination in (C). Data information: The quantification data of D) are presented as mean +/- SEM (n = 30 cells/treatment group). p values<0.05, calculated using unpaired student t-test, are considered significant. p values>0.05 not significant (n.s.). The analyses done included multiple independent fields.
a) P- and total MEK and ERK are enriched in APh in vivo.f2(a) P- and total MEK and ERK are enriched in APh in vivo. Immunoblots for indicated proteins in homogenate (Hom), APh, APL and Lys fractions from livers of fed mice, n=4. (b) EGF enhances enrichment of P-MEK and ERK in APh fractions in vitro. Immunoblots for indicated proteins in Hom, APh and APL fractions from NIH/3T3 cells in presence or absence of EGF (10 min). The bars represent mean±s.e.m. *P0.05, **P0.01; Student's t-test, n=3. (c) P- and total MEK and ERK localize on to the cytoplasmic/extra-luminal face of APh in vivo. Left; model depicting localization of MEK and ERK on the extra-luminal face of autophagic vesicles and the ability of trypsin to degrade extra-luminal MEK and ERK, and right; immunoblots for indicated proteins in APh fractions from mice livers untreated (−) or treated with increasing amounts of trypsin for 15 min. The bars represent mean±s.e.m. *P0.05, **P0.01, ***P0.001 compared with corresponding trypsin-untreated value; ANOVA-Bonferroni post hoc test, n=3. The p44, p42 forms of ERK, LC3-I and membrane-associated LC3-II, and 37 kDa and 25 kDa forms of cathepsin (Cath) B are indicated.
Figure 3Hi-C interaction matrices of the Control (EGFP)-KD and Piwi-KD OSCs at Chromosome 3L (40k resolution). Differential interaction heatmap for chromosome 3L (40k resolution), showing increased (red) and decreased (blue) interacting bins in log2 fold change upon Piwi-KD. Differential interaction heatmap for 16-19Mbp of chr3L shown in log2 fold change. piRNA target TE insertions are indicated at the bottom (green filled triangles), along with TADs defined using Hi-C interactions of Control (EGFP)-KD OSCs (black triangles). Differential intra-TAD interaction density score upon Piwi-KD (upper), centering TE insertions. Heatmap showing changes in the intra-TAD interaction score of each piRNA target TE insertion sites (lower). The results are shown for piRNA target TE and non-piRNA target TE insertions. Dot plot showing the correlation between the normalized density of indicated TEs per TAD (x-axis) and changes in intra-TAD interactions upon Piwi-KD (y-axis). Trend lines are in red. Spearman's rank correlation rho is indicated along with the p-value (algorithm AS 89). Dot plot showing the correlation between the normalized density of indicated TEs per TAD (x-axis) and changes in inter-TAD interactions upon Piwi-KD (y-axis). Trend lines are in red. Spearman's rank correlation rho is indicated along with the p-value (algorithm AS 89). Oligo-FISH images for changes in the intra-TAD interaction at the TAD described in (C), upon EGFP (control)- or Piwi-KD. Gray indicates DAPI staining for DNA, green and magenta indicates FISH signal at the different positions of TADs (TAD-1 and TAD-2). Details of TAD probe design is described in Fig EV2C. Scale bar, 5μm. Distance between two FISH signals is quantified at right, dot in the box plot indicates mean value; Boxplot whiskers, box, and central band show minimum, first quartile, median, third quartile, and maximum, respectively. P-values calculated with Mann-Whitney U test. Diagram illustrating the possible loss of the intra-TAD interaction for TADs harboring piRNA target TEs. Differential interaction heatmap for 16-19Mbp of chr3L shown in log2 fold change. TADs defined using Hi-C interactions of EGFP-KD (control, upper right) or Piwi-KD (lower left) OSCs are overlaid on the heatmap. Venn diagram displaying the number of TAD boundaries detected using EGFP- or Piwi-KD Hi-C interactions. Dot plot showing the correlation between the change in coverage of LADs for each TAD (x-axis), and changes in intra-TAD interactions upon Piwi-KD (y-axis). Trend lines are in red. Spearman's rank correlation rho is indicated along with the p-value (algorithm AS 89).
Figure 3(A) Representative images of TH+ dopamine neurons and fibers staining by immunofluorescence, in the SN and the striatum (Str) 6 weeks after CTL (AAV6-GFP); Cre (AAV6-GFP Cre); sh DCC (AAV6-GFP Cre + Sh-DCC) and shUNC5B (AAV6 GFP Cre + Sh-UNC5B) injection into the SN of Netrin-1fl/fl mice. (Scale bar, 2000 µm). (B) Quantification of TH fluorescent intensity in the SN (upper bar graph) and striatum (Str) of Netrin-1fl/fl mice (lower bar graph). Data are shown as mean + SEM. Statistical significance was determined by an unpaired t-test. N = 3 each group. Tukey post hoc analysis after one-way ANOVA *P < 0.05, **P <0.01, N.S., not significant. (C) DCC depletion mitigates netrin-1 depletion-induced apoptosis in Netrin-1fl/fl mice. TH-positive cell loss was assessed by TUNEL assay in the SN. Upper panel, TUNEL (red) and TH (Cy5-white) (Scale bar, 50 µm). Apoptotic index (bar graph, bottom panel) expressed as a percentage of TUNEL positive neurons out of the total number of TH-positive neurons. N = 3 each group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey test for multiple group comparison. *P < 0.05; P**< 0.01. (D) Immunoblot of SN lysates (upper panel) and corresponding quantification (bottom panel). Quantification of band intensity (bottom). Mouse N=2. N=3 independent immunoblot analysis. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05; P**< 0.01. (E) Motor behaviour tests at six weeks after treatment. N = 8 animals/group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey test for multiple group comparison. *P < 0.05; N.S., not significant.
Figure 4A) 3D reconstructions of immuno-labelled endogenous Ptc of wing discs in wild type condition and after RNAi expression for different ESCRT complex components. All RNAis' expression result in Ptc accumulation in large structures, located apically under Hrs (ESCRT-0 component) inhibition, and basally under inhibition of the ESCRT components I (Tsg101), II (Vps22) and III (Shrub). Graphs depicting signal values from apical to basal show a shift of higher intensity curves either towards acute apical or basal accumulation. B) Detailed confocal imaging of immuno-labelled endogenous Ptc localization after clonal Tsg101 down-regulation. The image shows abnormal accumulation of Ptc clusters in basal cytonemes emanating from receiving cells (arrows), since in wild type conditions Ptc visualization at basal membranes is not possible due to its rapid internalization and processing after Hh reception C) Ptc accumulation in these punctated structures (arrows) is also visualized on receiving cytonemes stabilized with the co-receptor Ihog-RFP and after Tsg101 inhibition. D) 3D reconstruction of Hh producing cytoneme network stabilized by Ihog-RFP shows that Ptc accumulation after Tsg101 treatment localizes above the signal sending cytoneme extensions (arrow).
Figure 8(A) Cellular protein extracts were isolated from immortalized patient-derived skin fibroblasts that were treated with the indicated concentrations of MG132, bortezomib, or carfilzomib. The samples were analyzed by reducing SDS-PAGE and Western blot. (B) Cellular fractions were prepared from immortalized patient-derived skin fibroblasts that were treated with the indicated concentrations of MG132, bortezomib, or carfilzomib. The samples were analyzed by reducing SDS-PAGE and Western blot. T, total; C, cytosol; M, mitochondria.
The GBS pigment causes fetal injury by NLRP3 inflammasome-dependent and NLRP3 inflammasome-independent mechanisms.Figure 7Female pregnant wild-type (CD-1, C57BL6) or NLRP3-deficient mice were injected in utero with 106-7CFU of GBS WT, ΔcovR, or ΔcovRΔcylE and monitored for preterm birth. Surgery and GBS inoculation for each pregnant mouse were performed independently. Data shown are representative of experiments with 6 animals per group for each GBS strain and two animals were used for saline controls.Scheme of pup numbering in utero and injection site between fetuses P1 and P2 is shown. In utero fetal death in wild-type CD-1mice due to infection with GBS WT, hyperhemolytic ΔcovR, and non-hemolytic ΔcovRΔcylE. Fetal death is represented by the number of dead fetuses/total number of fetuses obtained from six pregnant mice per group. 'n' indicates total number of pups (both live and dead); ****P < 0.0001, Fisher's exact test. H&amp;amp;E staining of uterine tissue. Open arrow indicates the presence of few mononuclear cells, whereas filled arrows indicate increased infiltration of inflammatory cells and necrotic debris. Fetal death due to infection with hyperhemolytic GBS ΔcovR and non-hemolytic ΔcovRΔcylE in WT C57BL6 and NLRP3 inflammasome-deficient mice; fetal death is represented by the number of dead fetuses/total number of fetuses obtained from 6 pregnant mice per group. 'n' indicates total number of pups (both live and dead); *P = 0.011, **P = 0.0015, ****P < 0.0001, Fisher's exact test. Fetal death due to ΔcovRΔcylE in WT C57BL6 and NLRP3KO mice was not significant and is indicated as NS; P = 0.31, Fisher's exact test. Female pregnant wild-type (CD-1, C57BL6) or NLRP3-deficient mice were injected in utero with 106-7CFU of GBS WT, ΔcovR, or ΔcovRΔcylE and monitored for preterm birth. Surgery and GBS inoculation for each pregnant mouse were performed independently. Data shown are representative of experiments with 6 animals per group for each GBS strain and two animals were used for saline controls.Bacterial burden in fetal pups and uterine horns from mice infected with the various GBS strains (n = 6/pup; of note, pups that were delivered preterm were excluded from CFU enumeration. Scheme of pup numbering is shown in (A). RUH and LUH indicate right uterine horn and left uterine horn, respectively. CFUs are not significantly different between any of the groups (ANOVA, P = 0.6, error bars ± SEM). Female pregnant wild-type (CD-1, C57BL6) or NLRP3-deficient mice were injected in utero with 106-7CFU of GBS WT, ΔcovR, or ΔcovRΔcylE and monitored for preterm birth. Surgery and GBS inoculation for each pregnant mouse were performed independently. Data shown are representative of experiments with 6 animals per group for each GBS strain and two animals were used for saline controls.IL-1β levels in GBS-infected tissues (placenta and fetus, n = 6/group) was measured by Luminex assay (*P = 0.025, ***P = 0.0002, ****P < 0.0001, Bonferroni's multiple comparison test following ANOVA). IL-1β levels was not significantly different in NLRP3KO mice infected with ΔcovR compared to NLRP3KO mice infected with ΔcovRΔcylEmice and is indicated as NS; P = 0.99.
Figure 3(A) Volcano plot represents DVL3 transcriptional targets identified by RNA-seq in MDA-MB-468 cells (NTC vs. shB DVL3 and EV vs. DVL3-WT, upper panel), and in MCF7 cells (NTC vs. shB DVL3 and EV vs. DVL3-WT, lower panel). In the upper panel, the green dots represent significant downregulated genes, the red dots represent significant upregulated genes and the orange dots represent DVL3-ChIP hits in MDA-MB-468 cells. In the lower panel, the blue dots represent significant downregulated genes, the red dots represent significant upregulated genes and the orange dots represent DVL3-ChIP hits in MCF7 cells. Gene expression levels were quantified using Fisher's Exact Test Signal Search in the DNASTAR ArrayStar software package. (B) Diagram showing the percentage of up- and down-regulated genes in the RNA-seq experiment comparing NTC vs. shB DVL3 (left panel) and EV vs. DVL3-WT (right panel) in MDA-MB-468 cells. (C) Diagram showing the percentage of up- and down-regulated genes in the RNA-seq experiment comparing NTC vs. shB DVL3 (left panel) and EV vs. DVL3-WT (right panel) in MCF7 cells. (D) Venn diagram showing overlapping between DVL3-bound genes and DVL3 transcriptional targets in MDA-MB-468 cells. A total of 32% of differentially expressed genes identified by RNA-seq comparing NTC and shB DVL3 were also DVL3 direct targets identified by ChIP-seq. From these, 69% were up-regulated and 31% down-regulated. A total of 36% of differentially expressed genes identified by RNA-seq comparing EV and DVL3-WT were also DVL3 direct targets identified by ChIP-seq. From these, 1% were up-regulated and 99% down-regulated. (E) Venn diagram showing overlapping between DVL3-bound genes and DVL3 transcriptional targets in MCF7 cells. A total of 3% of differentially expressed genes identified by RNA-seq comparing NTC and shB DVL3 were also DVL3 direct targets identified by ChIP-seq. From these, 87.5% were up-regulated and 12.5% down-regulated. A total of 5.6% of differentially expressed genes identified by RNA-seq comparing EV and DVL3-WT were also DVL3 direct targets identified by ChIP-seq. From these, 56.5% were up-regulated and 43.5% down-regulated.
Figure 3A, B. Schematics of the domain organization and secondary structure of Heh2, Snf7 and truncations with amino acid numbers. LEM is LAP2-Emerin-MAN1 (green), TM is transmembrane (black), MCHD is MAN1 C-terminal Homology Domain (brown), MIM is microtubule interacting motif (purple). The striped α-helix 5 represents the autoinhibitory helix. C. GST, GST-Snf7 and GST-snf7OPEN were immobilized on GT-resin and incubated with buffer(-) or recombinant heh2(1-308)-His6. Bound proteins separated by SDS-PAGE were visualized by coomassie stain and by Western blot with anti-His6 antibody and ECL detection (WB; top panel). Middle panel shows indicated cropped region of gel where contrast has been increased. Numbers on side of gel show position of molecular weight (MW) markers. D. In vitro transcription translation (IVT) reactions generating radiolabeled (35S) truncations of Heh2 (inputs) were incubated with bead-bound GST, GST-snf7OPEN or GST-Snf7 before washing, elution and detection of bound proteins by autoradiography (top panel) or coomassie staining (bottom). E. As in C except with GST-Chm7 constructs. Upper panel shows western blot (WB) with anti-His6 antibody. *chm7-NTD is also expressed with a His6 tag. Bottom panel is coomassie stained. F. As in E except with MBP-Snf7.
a) Each of the compounds induced LC3-II in primary neurons, as determined by western blotting.figf4(a) Each of the compounds induced LC3-II in primary neurons, as determined by western blotting. At 5 μM but not 0.5 μM, the compounds significantly increased LC3-II/GAPDH (b) and LC3-II/LC3-I (c) ratios (nonsignificant (NS), P > 0.05; 0.05 *P 0.01, **P 0.01; F = 13.74 (in b) and 9.74 (in c), one-way ANOVA with Dunnett's post-test). Error bars represent s.e.m. Values were pooled from five independent experiments. (d) Optical pulse labeling of LC3-Dendra2 in rodent primary cortical neurons. Scale bars, 25 μm. (e) The change in red fluorescence intensity over time was used to calculate LC3-Dendra2 half-life, as in Figure 2 (DMSO, R2 = 0.7333; Beclin, R2 = 0.6968, FPZ, R2 = 0.8752; MTM, R2 = 0.8447; NCP, R2 = 0.8357; *P 0.0001 for global trend, F = 12.6, extra sum-of-squares F-test). Error bars represent s.e.m. ( . (f) The median single-cell half-life of LC3-Dendra2 was significantly reduced by beclin expression or treatment with 0.5 μM NCP, MTM or FPZ (*P 0.01, **P 0.001, Kruskal Wallis statistic = 52.56, Kruskal-Wallis with Dunn's test). (g) Probability density plot of LC3-Dendra2 half-life measurements from individual neurons treated with vehicle control (DMSO, n = 140), NCP (n = 128), MTM (n = 91), FPZ (n = 77) or those expressing beclin (n = 101). n*, P = 6 × 10−4; m*, P = 1 × 10−10; f*, P = 5 × 10−9; b*, P = 2 × 10−3; two-sided Kolmogorov-Smirnov test). Colored hash marks represent values from individual neurons. Values were pooled from eight wells per condition, with experiments performed in triplicate.