EMBO/sd-character-level-ner huggingface dataset:
Tpl2[D270A] mutation promotes phagosome maturation independently of MAP kinase activationA-F.Figure 2Experiments were performed using murine BMDMs. (A) Intra-phagosomal proteolysis in WT, Nfkb1[SSAA] (Nfkb1SSAA/SSAA) and Nfkb1[SSAA]/Tpl2[D270A] BMDMs was monitored as Fig 1B (n = 4 wells). (B) Intra-phagosomal acidification in WT, Nfkb1[SSAA] and Nfkb1[SSAA]/Tpl2[D270A] BMDMs was monitored as in Fig 1C (n = 4 wells). Data information: One representative experiment out of three shown. Error bars and shaded areas represent SEM. **** P < 0.0001. Panels Paired Mann-Whitney t-test; all differences relative to WT are ****. Experiments were performed using murine BMDMs. (C) Cell extracts from LPS-stimulated Nfkb1[SSAA] and Nfkb1[SSAA]/Tpl2[D270A] BMDMs were immunoblotted for the indicated antigens. Experiments were performed using murine BMDMs. (D) Intra-phagosomal proteolysis in WT and inhibitor-treated BMDMs (n = 4 wells) was monitored as in Fig 1B. BMDMs were pre-treated with 0.1 μM PD0325901 (10 min) to inhibit MEK1, pre-treated with 1 μM VX-745 (1 h) to inhibit p38α and pre-treated with 10 μM C34 (1 h) to inhibit TPL-2. (E). Intra-phagosomal acidification in WT and inhibitor-treated BMDMs (see (D) for conditions) was assayed as in Fig 1C (n = 4 wells). Data information: One representative experiment out of three shown. Error bars and shaded areas represent SEM. **** P < 0.0001. Paired Mann-Whitney t-test; all differences relative to WT are ****. Experiments were performed using murine BMDMs. (F) Cell extracts from LPS-stimulated, inhibitor-treated WT and Tpl2[D270A] BMDMs were immunoblotted for the indicated antigens. 1 μM bafilomycin A1 was added to inhibit V-ATPases. Experiments were performed using human primary monocyte-derived macrophages. (G) Intra-phagosomal proteolysis in human macrophages was monitored following uptake of DQ Green BSA / AF594 latex beads. TPL-2 catalytic kinase activity was blocked by pre-treatment with 10 μM C34 for 1 h (n = 4 wells). (H) Intra-phagosomal acidification in human macrophages was monitored following uptake of BCECF-coupled latex beads (n = 4 wells). Data information: One representative experiment out of three shown. Error bars and shaded areas represent SEM. **** P < 0.0001. Paired Mann-Whitney t-test; all differences relative to WT are ****. Experiments were performed using human primary monocyte-derived macrophages. (I) Intra-phagosomal proteolysis in human macrophages was assayed as in Fig 2G. MAP kinase activity was inhibited by combinatorial pre-treatment with 0.1 μM PD0325901 (MEK1 inhibition) for 10 min and 1 μM VX-745 (p38 inhibition) for 1 h (n = 4 wells). (J) Intra-phagosomal acidification in human macrophages was monitored as in Fig 2H (n = 4 wells). Data information: One representative experiment out of three shown. Error bars and shaded areas represent SEM. **** P < 0.0001. Paired Mann-Whitney t-test; all differences relative to WT are ****. Experiments were performed using human primary monocyte-derived macrophages. (K) Cell extracts from LPS-stimulated, inhibitor-treated primary human macrophages were immunoblotted for p-ERK1/2, ERK1/2, p-p38, p38 and HSP90.
a) HeLa cells transfected for 24 h with GFP–Atg16L1 (green) were incubated with CellMask Orange plasma membrane stain for 5 min at 37 °C, after which they were imaged immediately (in an incubated chamber at 37 °C).figf7(a) HeLa cells transfected for 24 h with GFP-Atg16L1 (green) were incubated with CellMask Orange plasma membrane stain for 5 min at 37 °C, after which they were imaged immediately (in an incubated chamber at 37 °C). A time series after fusion of a CellMask Orange vesicle (red) with a GFP-Atg16L1 vesicle (green) is shown. Scale bar, 5 μm. A higher-magnification image showing co-localization (yellow) of GFP-Atg16L1 vesicle (green) with CellMask Orange-positive vesicle (red), indicated by arrows, is also shown in the top and bottom panels. (b, c) HeLa cells transfected with control, clathrin heavy chain, AP1 or AP2 siRNA for 72 h were subsequently transfected with GFP-Atg16L1 (green) together with the siRNA for the next 24 h. The cells were then incubated for 15 min with Alexa Fluor-555-conjugated cholera toxin subunit B (red) at 4 °C, followed by further incubation for 10 min at 37 °C, after which the cells were fixed for confocal analysis (b). The co-localization (yellow) of GFP-Atg16L1 (green) with cholera toxin (red) in control or clathrin heavy-chain knockdown is shown and is quantified in the graphs in c. n = 25 cells. Three asterisks, P = 0.0002 for clathrin heavy-chain knockdown, and P 0.0001 for AP2 knockdown. All error bars represent s.e.m. Scale bar, 5 μm.
Expression of microglial marker proteins characteristic for the homeostatic or disease associated state.Figure 2A. Immunoblot analysis of ApoE, CLEC7A, TREM2 and CD68 in lysates of acutely isolated microglia from Grn-/- and Grn+/+ mice (9 months of age, n=3 per genotype, female). IBA1 was used as loading control. B. Quantification of protein expression normalized to levels of Grn+/+ microglia (n=3). Data represent the mean ± SD. C. ELISA mediated quantification of sTREM2 in brain homogenates of Grn-/- and Grn+/+ mice Data are shown as mean ± SEM (n=3-6). D. ELISA mediated quantification of sTREM2 in serum of Grn-/- and Grn+/+ mice. Data are shown as mean ± SEM (n=4-13). E. Microglial expression of P2RY12 in cortical sections of wt, Grn-/-and Trem2-/- mice. (9 months of age, female) F. Quantification of P2RY12 positive microglia. Data are shown as mean ± SD (n=3 per genotype, female, except one male for Trem2-/-). G. Immunoblot analysis of secreted PGRN and ApoE (ApoEmed) in conditioned media and PGRN, ApoE, CLEC7A, P2RY12 and TREM2 in lysates of BV2 wild type (Grnwt) and PGRN deficient (Grnmut) cells. Soluble amyloid precursor protein (APPs) and calnexin were used as a loading control. H. Quantification of Immunoblots normalized to BV2 Grnwt levels (n=3-5). Data represent the mean ± SD. I. ELISA mediated quantification of sTREM2 in conditioned media of BV2 Grnwt and Grnmut cells. Data represent the mean ± SD J. Quantification of relative mRNA levels of Apoe, Clec7a, P2ry12 and Trem2 in BV2 Grnwt and Grnmut cells. Data represent the mean ± SD.
Figure 3A. Commonly and exclusively quantified proteins in liver cell types (hHSC: hepatic stellate cell, hHEP: hepatocyte, hKC: Kupffer cell, hLSEC: liver sinusoidal endothelial cell). B. Pair-wise correlation of the proteomes of the four primary cell types, with Pearson correlation coefficients noted. C. Circos plot representing proteins predicated to be co-localized in subcellular compartments. D. Cumulative protein abundance of liver cell types as a function of protein rank, indicating the total of the top ten abundant proteins and number of proteins that comprise the four quartiles. E. Schematic representation of the subcellular mass composition of an average hepatocyte. F. The bar plot shows the contribution of each organelle to total cellular protein mass, also accounting for unassigned proteins. Percentage of cytosol, mitochondria, nucleoli, plasma membrane and actin filaments is indicated.
Loss of Golgi microtubules does not affect RhoA activity or cell contraction Western blot showing the protein level of active RhoA, total RhoA in negative control and CAMSAP2 siRNA transfected hRPEs.Figure 3Western blot showing the protein level of active RhoA, total RhoA in negative control and CAMSAP2 siRNA transfected hRPEs. GAPDH is used as loading control. Bar chart shows quantification of protein levels normalized to GAPDH in each condition. Left: Representative live-cell fluorescence imaging of GFP-AHPH in negative control and CAMSAP2 siRNA transfected hRPEs. Scale bar: 20 μm. Right: Quantification of GFP-AHPH fluorescence intensity. Left: Representative immunofluorescent staining of pMLC (yellow) and DAPI (blue) in negative control and CAMSAP2 siRNA transfected hRPEs. Scale bar: 20 μm. Right: Quantification of pMLC fluorescence intensity. Below: Western blot showing the protein level of pMLC in negative control and CAMSAP2 siRNA transfected hRPEs. α-tubulin is used as loading control Left: Images showing collagen gel deformation at 0 h, 8 h and 24 h. HRPEs transfected with negative control, CAMSAP2 and AKAP450 siRNA were seeded in 1% collagen gel (yellow dashed lines). Scale bar: 5 mm. Right: Quantification of gel area.
Figure 4(A) Simulations display heterogeneous σV activation after lysozyme stress (N=100). Dashed vertical line marks point of stress addition (parameter L changed from 0 to 1) in simulation. (B) In simulations cells activate σV faster as stress levels (the value of the L parameter) are increased, as observed experimentally (N=100 simulations for each stress level: 0.5, 0.75, 1, 2, 4). (C) With increasing stress levels, the steady state level of σV activity increases, as observed experimentally. Bars correspond to the mean ± s.d. (N=100 simulations for each lysozyme stress level).
Figure 4A Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of freshly isolated CD8 T cells from wildtype (WT) or Peli1 KO (KO) mice (6 weeks old). Data are presented as a representative blot (left) and summary graphs of densitometric quantifications of the indicated proteins (presented as phosphorylated/total protein ratio) based on three independent experiments. B,C Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype or Peli1-KO OT-I CD8 T cells that were first rested on ice for 15 min and then stimulated in low-serum (1% FBS) medium with anti-CD3 and anti-CD28 for the indicated time periods. D,E Seahorse analysis of basal and maximal ECAR (D) or OCR (E) using wildtype or Peli1-KO OT-I CD8 T cells that were activated for 16 h with plate-bound anti-CD3 (1 μg/ml) plus anti-CD28 (1 μg/ml) in the presence of solvent control (DMSO), rapamycin (Rapa, 10 nM), or Torin 1 (100 nM). Data are presented as a representative plot (left) and summary graphs (right). F,G Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype or Peli1-KO OT-I CD8 T cells that were either not treated (-) or stimulated (+) for 1 h (F) or 24 h (G) with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) in the presence (+) or absence (-) of rapamycin (10 nM) and Torin 1 (100 nM). H qRT-PCR analysis of Ifng gene expression in naïve wildtype or Peli1-KO OT-I CD8 T cells that were either not treated (-) or stimulated for 6 h (+) with anti-CD3 plus anti-CD28 in the presence (+) or absence (-) or rapamycin. I-K Schematic of experimental design (I), tumor growth curve (J), and summary of day 15 tumor weight (K) of B6.SJL mice inoculated with B16-OVA melanoma cells (2 x 105) and adoptively transferred on day 7 with in vitro activated and rapamycin- or DMSO-treated wildtype or Peli1-KO OT-I CD8 T cells (4 x 105).
Netrin-3 is a therapeutic target in SCLC.Figure 7A. Analysis of the NP137 antibody (anti-netrin-1) epitope across netrin family members. B. Analysis of NP137 binding on netrin-2L, netrin-1; netrin-4 or netrin-G2 by bio-layer interferometry assays. Kd rhNTN1: 0.96 ± 0.30nM; Kd for rchNTN2: 1.53 ± 0.96nM C. NP137 inhibits NTN1/UNC5B or NTN-2L/UNC5B interaction, detected by Elisa assays. Here the recombinant proteins are the murine forms (Control IgG= mouse IgG2A, mNP137= murine Fc, UNCB=rat UN5H2) bars indicate SD, n=3. D. NMRI nude mice were engrafted with NCI-H82 cells by subcutaneous injection of 2 million cells. When the mean tumor volume reached approximately 80 mm3, animals were treated 3 x time/weekly by intra-abdominal injection of PBS; NP001 (Human IgG1, isotype); NP137 (anti-netrin1/3) during 20 days. n=10 animals/group (*p < 0.05, two-way Anova, Error bars indicate s.e.m.).F. Quantification of cell death (cleaved PARP) in H82 treated tumors in D. Tumors were analyzed 7 days after the first treatment (ie: 400mm3; n=3/group. U-test, bars indicate SD).
Figure 4(A-E) Control and Tg(bactin:igfbp1a)-overexpressing larvae were treated with MTZ from 3-4 dpf to ablate β cells, and analyzed at 6 dpf, after 2 days of regeneration. (A-B) Representative confocal images of control and Tg(bactin:igfbp1a)-overexpressing Tg(ins:dsRed);Tg(gcg:GFP);Tg(ins:Flag-NTR) larvae. A bihormonal glucagon- and insulin-expressing cell (gcg+;ins+) is indicated by an arrow. Scale bars: 20 μm. (C-D) Representative confocal images of control and Tg(bactin:igfbp1a)-overexpressing Tg(gcg:GFP);Tg(ins:Flag-NTR) larvae stained for Pdx1. Pdx1- and glucagon-expressing cells (pdx1+;gcg+) are indicated by arrows. Scale bars: 20 μm. (E) Quantification of gcg+;ins+ and pdx1+;gcg+ cells in the control and Tg(bactin:igfbp1a) group; P<0.001, P=0.0012; n=18 and 18 in the control groups, n=15 and 10 in the Tg(bactin:igfbp1a) groups, for the gcg+;ins+ and pdx1+;gcg+ quantification, respectively. (F-I) Lineage-tracing evidence supports α- to β-cell transdifferentiation. (F) Schema for temporal and spatial lineage tracing. loxP-mediated excision of the STOP-cassette permits heat-inducible expression of the stable fusion-protein H2B-GFP. Control and bactin:igfbp1a-overexpressing Tg(hsp:loxP-mCherry-STOP-loxP-H2B-GFP);Tg(gcg:CRE);Tg(ins:Flag-NTR) larvae were first heat-shocked at 3 dpf to label the glucagon-expressing cells with the stable fluorescent protein H2B-GFP, thereafter treated with MTZ from 3-4 dpf to ablate the β cells, and analyzed at 6 dpf after 2 days of regeneration. Note that the temporal control of the lineage tracing is mediated by a heat-shock. (F-I) Lineage-tracing evidence supports α- to β-cell transdifferentiation. (G) Quantification of insulin-expressing cells that originate from α-cells. Control larvae, n=13; bactin:igfbp1a larvae, n=16. P=0.0051. (F-I) Lineage-tracing evidence supports α- to β-cell transdifferentiation. (H-I) Representative confocal images at 6 dpf (after 2 days of regeneration) of control and bactin:igfbp1a-overexpressing larvae stained for insulin, glucagon, to-pro, and H2B-GFP. ins+;H2B-GFP+ cells are indicated by the arrowheads, and H' and I' show staining against only insulin and H2B-GFP. Moreover, most insulin+;H2B-GFP+ cells no longer express glucagon. Scale bars: 15 μm.
Figure 6(A, B) Distribution of mCherry-GFP-LC3 in HEK293-AT1 cells (without starvation) after treatment with control siRNA (A) or siRNA for PIP5Kγ (PIP5K1C). Scale bars: 20 µm. Note the massive tubulation of the LC3 compartment in some of the knocked-down cells (B, lower images, scale bar: 10 µm). (C) Comparison of Pearson coefficients from cells treated with control or PIP5Kγ RNAi. For statistical analysis the unpaired t-test was used (n = 50 and 45 cells for control a PIP5Kγ knock down groups, respectively; *** P< 0.0001). (D) Representative confocal images of live HEK293-AT1 cells expressing GFP-Rab7 treated with control or PIP5Kγ RNAi. Note the tubulation in the PIP5Kγ RNAi-treated cells. Scale bars: 10 µm.
Figure 8A-B. Profiles and heatmaps for normalized ATAC-seq read distribution averaged for all known mouse genes in control or 4-OHT-treated MSCs for reads below 120 bp (A, nucleosome-free) and between 120 and 250 bp (B, mononucleosomes). One of two replicates is shown. C-D. ATAC-seq read density profile for two biological replicates of control and treated MSCs for all mouse genes of the nucleosome-free (C) and mononucleosome (D) fragments. E-F. ATAC-seq read density profiles for nucleosome-free (E) and mononucleosome (F) fragments for differentially expressed genes. G. Box plots of mean read density (y-axis) at the gene body (bins 21-100 out of 100 between TSS - TES) for all genes and genes expressed at different levels. *** - p < 1e-10 (paired t-test). n = 2 biological replicates. Central band is mean, box is interquartile range, whiskers are minimum and maximum. H. Comparison of Hoechst 33342 binding to live control and 4-OHT-treated MSCs from different genotypes. Data are presented as the median fluorescence intensity ± SD, n = 4 biological replicates. P < 0.05 is shown (unpaired t-test).
Autoreactivity of iNKT cell requires both the IRE1α and PERK pathway A-B: qPCR analyses showing the expression (fold induction) of UPR signature genes compared to DMSO in murine and human macrophage cell lines J774.Figure 3qPCR analyses showing the expression (fold induction) of UPR signature genes compared to DMSO in murine macrophage cell lines J774.2 stimulated either with thapsigargin, tunicamycin or DMSO for 4 or 8 hours. Graphs show mean ± SEM from n=2 biological replicates (two technical replicates per biological replicate). qPCR analyses showing the expression (fold induction) of UPR signature genes compared to DMSO in human macrophage cell lines U937 stimulated either with thapsigargin, tunicamycin or DMSO for 4 or 8 hours. Graphs show mean ± SEM from n=2 biological replicates (two technical replicates per biological replicate). Bar graph represents percentage of IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with murine macrophage cell line (J774.2) treated either with thapsigargin or tunicamycin in the presence of IRE1α inhibitor (4μ8c) (black bar) or PERK inhibitor (GSK) (grey bar) or ATF6α inhibitor (Ceapin-a7) (red bar) respectively. Inhibition of IRE1α or PERK resulted in significant reduction in IL-2 secretion by iNKT hybridoma compared to controls. In total, n=2 biological replicates were performed (two and four technical replicates per biological replicate respectively). Graphs show mean ± SEM of each biological replicate. Immunoblots represents the knockdown efficiency for IRE1α and PERK protein in J774.2 cell line transduced with lentiviral particles carrying gRNA against the IRE1α or PERK gene. Bar graph represents percentage of IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with thapsigargin-treated murine IRE1α or PERK knockdown macrophage cell line (J774.2). Knockdown of IRE1α (black bar) or PERK (grey bar) resulted in significant reduction in IL-2 secretion by iNKT hybridoma. In total n=2 biological replicates were performed (three technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.
Inducible NAV3 decreases velocity and enhances persistence of mammary cell migration.Figure 1The results of oligonucleotide array screens of EGF- or control-stimulated MCF10A cells (Amit et al, 2007) were re-analyzed, and genes induced by > twofold were included in the Venn diagram. The heatmap presents a time series of genes induced upon EGF treatment. siRNAs specific to the indicated genes were used to treat MCF10A cells, and migration tracks were subsequently quantified (see Supplementary Fig S1A). Shown are means ± SEM of three experiments; 60 cells were analyzed in each case. The results of statistical analyses (two-tailed Student's t-test) are shown in Supplementary Table S1. Changes in NAV3 expression levels in response to EGF were extracted from DNA arrays or determined by means of quantitative PCR. Serum-starved MCF10A cells were treated with EGF (10 ng/ml) for the indicated time intervals, and cell extracts were subjected to immunoblotting (IB), as indicated. Shown are trajectories of MCF10A cells expressing either siCTRL or siNAV3 and migrated on collagen in the absence or presence of EGF. Red tracks indicate low persistence (< 0.3). See quantification of the data in Supplementary Fig S2D.Quantitative analysis of the mode of migration of NAV3-depleted cells. The distribution of step sizes was computed, normalized and presented as probability distribution functions (left panel). Log-log plots indicating modes of migration for each of the four conditions are shown (right panel). Note that only EGF-treated siCTRL cells exhibit a persistent walk (diagonal), whereas the other three asymptote, as expected for random walks.
Figure 7(A) AFM image of a control SLB prepared from LUVs with a mitochondrial-like lipid composition. The bilayer looks flat, without aggregates or defects. Scale bar, 1 µm.(B) AFM image of a SLB prepared from LUVs with a mitochondrial-like lipid composition pre-incubated with heat-activated Bax. The green arrows indicate the presence of membrane pores, which are heterogeneous in size and shape. The edges of the pores present protrusions corresponding to Bax clusters. Scale bar, 1 µm. (C) 3D AFM topography of a Bax arc (left) and ring (right). Both images reveal a circular dark hole that spans the lipid membrane (green). Bax molecules around the pore rim (magenta and white) protrude 3,97 ± 1,02 nm above the membrane plane, as confirmed by the height profiles shown below each image (corresponding to the white line in the 2D image insets). The topography of the arc structure reveals a pore only partially surrounded by Bax molecules, while lipids alone form the rest of the pore rim. Images are shown in a 42° tilted representation. (D) Quantitative analysis of the distinct structures found for activated Bax on SLBs. Data show the total number of structures in all the measurements.
Modulation of ROS by TIGAR correlates with the modulation of autophagy.Figure 4(A) U2OS cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h, with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments. (B) (Left panel) ROS levels in U2OS cells left untreated or treated with 0.5 or 1 mM of H2O2 for 24 h. ROS levels were measured by flow cytometry after DCF treatment. The results are expressed as the mean DCF fluorescence (and standard deviation) from three independent experiments. (Right panel) Quantitation of the percentage of GFP-LC3 puncta positive cells for cells treated as described above. Data are shown as the mean and standard deviation from three independent experiments. (C) Quantitation of the percentage of GFP-LC3 puncta positive cells. U2OS cells stably expressing GFP-LC3 were transfected with vector pCHER1A expressing the mCherry gene as control, or expression plasmids for Flag-tagged-TIGAR or HA-tagged-FBPase-2. After 48 h transfection, cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h, with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments. (D) Quantitation of the percentage of GFP-LC3 puncta positive cells. Cells were left untreated, exposed to nutrient starvation for 6 h with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)), AO2 (glutathione ethyl ester (4 mM)) or AO3 (ethyl pyruvate (4 mM)) for 24 h. U2OS cells stably expressing GFP-LC3 were transfected with vector pCHER1A expressing the mCherry gene as control, or expression plasmid for Flag-tagged-TIGAR. After 48 h transfection, cells were treated. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments. (E) (Left panel) Quantitation of the percentage of GFP-LC3 puncta positive cells. Cells were left untreated, exposed to nutrient starvation for 5 h or to metabolic stress for 18 h, with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h. U2OS cells stably over-expressing GFP-LC3 in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2. (Right panel) Basal or nutrient starvation-induced (5 h) ROS levels in U2OS cells in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2 with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h, measured by flow cytometry after DCF treatment. The results are expressed as the mean intensity of cell fluorescence (and standard deviation). * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).