EMBO/sd-character-level-ner huggingface dataset:
Figure 4Study 4, tumor-bearing male mice receiving AR-42 (10 mg/kg, oral gavage; n=9), TFM-4AS-1 (10 mg/kg, subcutaneous; n=10), Combination of AR-42 and DHT (10 mg/kg oral gavage and 3 mg/kg subcutaneous, respectively; n=10), Combination of AR-42 and TFM-4AS-1 (10 mg/kg, both, n=9) or Vehicle (n=6) and tumor free mice receiving Vehicle (n=6) were treated daily for 12 days starting 6 days after cell injection. A) Terminal body weights corrected for tumor mass according to the Materials and Methods compared to tumor-free controls (mean±SD). Study 4, tumor-bearing male mice receiving AR-42 (10 mg/kg, oral gavage; n=9), TFM-4AS-1 (10 mg/kg, subcutaneous; n=10), Combination of AR-42 and DHT (10 mg/kg oral gavage and 3 mg/kg subcutaneous, respectively; n=10), Combination of AR-42 and TFM-4AS-1 (10 mg/kg, both, n=9) or Vehicle (n=6) and tumor free mice receiving Vehicle (n=6) were treated daily for 12 days starting 6 days after cell injection. B) Terminal gastrocnemius (mean±SD) and C) quadriceps muscle mass (mean±SD) compared to tumor-free controls. Study 4, tumor-bearing male mice receiving AR-42 (10 mg/kg, oral gavage; n=9), TFM-4AS-1 (10 mg/kg, subcutaneous; n=10), Combination of AR-42 and DHT (10 mg/kg oral gavage and 3 mg/kg subcutaneous, respectively; n=10), Combination of AR-42 and TFM-4AS-1 (10 mg/kg, both, n=9) or Vehicle (n=6) and tumor free mice receiving Vehicle (n=6) were treated daily for 12 days starting 6 days after cell injection. D) grip strength (mean±SD) compared to tumor-free controls. Statistics: V, T, A indicate significant differences versus tumor-bearing vehicle-treated, tumor-bearing TFM-4AS-1-treated and tumor-bearing AR-42-treated groups, respectively. p values provided in Appendix Table S4A-D, Tukey's multiple comparison test.
Matrin3 interacts with PTB via a PRI motifSchematic representation of Matrin3.Figure 2A Schematic representation of Matrin3. Matrin3 is composed by two zinc finger (ZF) domains, two tandem RNA recognition motifs (RRM), as well as a N‐terminal nuclear export signal (NES) and a C‐terminal nuclear localization signal (NLS). A PRI motif is localized between the first ZF and the first RRM, and the sequence is aligned with the sequence from the two functional PRIs from Raver1. Conserved PRI residues are in bold. B FLAGimmunoprecipitation of Matrin3 and Raver1, both with wild‐type and with PRI mutated, and FLAG‐MS2 as a negative control. The immunoprecipitated complex was separated in a SDS-PAGE and subjected to Western blot using antibody against PTB which showed interaction to wild‐type Matrin3 (lane 2) and Raver1 (lane 4). The input was also analysed by Western blot with antibodies against PTB as a loading control and against the FLAG tag to ensure equal expression of proteins. C The 20‐residue Raver1 491-511 (lane 1) and the Matrin3 346-365 (lane 3) peptide fused to MS2 were transcribed and translated in vitro (Input) and then pulled down with GST‐PTB or with GST‐SXL as a control. Effects of single mutation of the Matrin3 PRI GILPPP to GIAPPP were also tested (lane 4).
Figure 5Schematic representation of different domains of DBN1 (ADF-H: ADF homology domain, AB1: Actin-binding region 1, AB2: Actin-binding region 2, P: Proline-rich region) [44]. The numbers represent the amino acid positions of the denoted domains. Three independent gRNAs targeting different exons (3, 5, 7) of DBN1 were used to generate DBN1 KO cells. Successful targeting of DBN1 was confirmed by sequencing the PCR amplicons surrounding the locus. For example, in clone 3.3, there was a single base insertion in both DBN1 alleles (T in the one, G in the other) to cause disruption of protein expression through the introduction of a premature stop codon. The absence of DBN1 protein in the targeted clones was further verified by immunoblotting of the whole cell lysate. GAPDH is the loading control. One targeted clone from each gRNA (1.16, 2.18, 3.3) including the non-targeted clone 2.19 and WT control were serum starved for 48 h to induce ciliogenesis. Subsequently, cilia length was measured. The cilia from DBN1 KO cells were significantly longer than those from WT or non-targeted controls. Three independent experiments; N = 150 cilia for each experiment and condition. Mean ± SD. **** P≤ 0.0001. Overexpression of DBN1, DBN1S142A and DBN1S142D in RPE1 WT cells. Three independent experiments, N = 150 cilia for each experiment and condition. Mean ± SD. ** P≤ 0.01; *** P≤ 0.001; ns: not specific.  The phospho-inhibitory (S142A) version of DBN1 rescued the elongated cilia of DBN1 KO cells. Three independent experiments, N = 150 cilia for each experiment and condition. Mean ± SD. * P≤ 0.05; *** P≤ 0.001; ns: not significant. Pairwise comparison was done with unpaired student's t-test and the p-value was written in the figure. Global comparison was done with one-way ANOVA and the p-values were marked with stars.  The overexpressed WT DBN1 was hyper-phosphorylated in cells as detected by phospho-specific DBN1pS142 antibodies. The DBN1pS142 antibodies did not detect the overexpressed DBN1S142A or DBN1S142D.  Immunoblot analysis confirming the successful siRNA depletion of CDK5 and indicating hypo-phosphorylation of DBN1S142 in response to CDK5 depletion The DBN1pS142/GAPDH ratio from (H) was densitometrically measured. Three independent experiments. Mean ± SD. * P≤ 0.05 Depletion of CDK5 decreased the cilia length in WT cells. It restored cilia length in hCDC14APD cells. Three independent experiments; N = 150 cilia for each experiment and condition. Mean ± SD. **; P≤ 0.01; **** P≤ 0.0001; ns: not significant. CDK5 mediated cilia length decrease can be completely blocked by expression of the phospho-mimetic DBN1S142D in hCDC14APD cells. Three independent experiments; 150 cells per experiment and condition. Mean ± SD; ns = not significant; **** P≤ 0.0001.
Induction of autophagosomes by depletion of TAB2 or TAB3.Figure 3(A, B) Detection of autophagic GFP-LC3+ puncta. HeLa or U2OS cells stably expressing GFP-LC3 were transfected with siRNAs targeting TAK1, TAB1, TAB2 or TAB3 or with a control siRNA (siUNR). One day later, the subcellular localization and abundance of GFP-LC3 or immunostained TAB2 or TAB3 was determined by epifluorescence microscopy. Representative images are shown in (A) (HeLa cells) and quantitative results (mean values±s.d., n=3; *P0.01 versus siUNR‐transfected cells) are depicted in (B) (U2OS cells). (C) Lipidation of LC3 induced by TAB2 or TAB3 knockdown. Representative immunoblots showing the conversion of non‐lipidated LC3 (LC3‐I) to its lipidated variant (LC3‐II) as well as SQSTM1/p62 protein levels are shown. GAPDH levels were monitored to ensure equal loading. (D, E) Quantification of autophagosomes and autophagolysosomes by transmission electron microscopy. Representative images of HeLa cells transfected with siUNR or with TAB2‐ or TAB3‐targeting siRNAs are shown in (D), and quantitative results are depicted in (E) (mean values±s.d., n=3; *P0.01 versus siUNR‐transfected cells). (F) Epistatic analysis of the effects of TAB2 and TAB3 depletion on autophagy. HeLa cells stably expressing GFP-LC3 were transfected with siRNAs specific for TAB2 or TAB3 and/or with cDNAs coding for full‐length HA‐tagged TAB2 (HA-TAB2) or TAB3 (HA-TAB3). Twenty‐four hours later, the frequency of cells exhibiting >5 GFP-LC3+cytosolic puncta (GFP-LC3VAC cells) was determined. Results are mean values±s.d. (n=3; *P0.01 versus siUNR‐transfected cells). (G) U2OS cells stably expressing FYVE-RFP were transfected with siUNR, or with siRNAs specific for TAB2, TAB3, VPS34, Beclin 1 (BCN1) and TAK1, in the indicated combinations. Forty‐eight hours later, the percentage of cells with RFP-FYVE+ puncta cells was determined. Results are mean values±s.d. (n=3; *P0.01 versus siUNR‐transfected cells).
Figure 4A) Barplot showing the percentage of significant genes in the muscle modules. Next to each module name we report the total number of genes that it comprises. B) Barplot showing the percentage of significant genes in the blood modules. Next to each module name we report the total number of genes that it comprises. C) Matrix with the values of the cross-correlation between blood and muscle module eigengenes. To enhance visibility, only correlations with absolute value above 0.5 are shown.
a, b, Immunoblot of +/−KRas cells derived from independent tumours probed with PGC1a (a) and VDAC1 (b) antibodies.f2a, b, Immunoblot of +/−KRas cells derived from independent tumours probed with PGC1a (a) and VDAC1 (b) antibodies. Numbers indicate different independent tumours. c, d, In vivo immunofluorescence for CD44 (green) and PGC1a (red) (c), and CD44 and VDAC1 (red) (d) in +/−KRas tumours (×60). e, Oxygen consumption of +/−KRas cells (n = 7). f, In vivo mitochondrial potential of +/−KRas tumours (n = 3); representative flow cytometry of two tumours, as control uncoupler CCCP was added to acquired −KRas. g, TEM micrographs representing mitochondrial morphology (×25,000). h, ATP levels of +/−KRas cells in response to oligomycin (Oli) or dimethylsulphoxide (DMSO; Ctrl) (n = 4). i, ECAR response of +/−KRas cells to glucose, oligomycin and 2-deoxy-d-glucose (2DG) (n = 4). j, Fold change of glycolytic intermediates in +/−KRas cells (n = 4). Ala, alanine; BPG, bisphsphoglycerate; DHAP, dihydroxyacetone phosphate; F6P, fructose-6-phosphate; F(1,6) BP, fructose-1,6-bisphosphate; G6P, glucose-6-phosphate; G1P, glucose-1-phosphate; Ga3P, glyceraldehyde- 3-phosphate; G3P, glycerol-3-phosphate; Lac, lactate; PEP, phosphoenolpyruvate; 3PG, 3-phosphoglycerate; Pyr, pyruvate. k, l, Glucose uptake (k) and lactate production (l) of +/−KRas cells (n = 3). m, Isotopomer distribution for alpha-ketoglutarate (aKG) following steady-state tracing with uniformly carbon-13-labelled substrates (n = 3). Data are mean ± s.d.
a–c, Y.f5a-c, Y. enterocolitica colony forming units (c.f.u.) in human and murine macrophages infected for 6 h (20 m.o.i.). Data in a are pooled from 4 independent cohorts of 3 or 4 donors per genotype, total n = 16 (non-risk, WT), 15 (T300A, risk). Data in b represent 3 independent experiments, n = 4 mice. Data in c represent 3 independent experiments, n = 6. d, TNF-α, IL-1β and IL-6 levels in culture media of WT and T316A macrophages infected with 20 m.o.i. Y. enterocolitica. Data represent 2 independent experiments, n = 4 mice. e, Ileum and colon histology (haematoxylin and eosin staining) of WT mice. Images are representative of 3 (PBS) and 8 (Y. enterocolitica) treated male mice. f, Measurement of cytokine mRNA transcripts in mesenteric lymph nodes. Data are pooled from 2 independent experiments of 3 mice (PBS) and 7 mice (Y. enterocolitica) per experiment. Transcripts are normalized to Gapdh (2−Dgr;Ct). g, Y. enterocolitica c.f.u. in mesenteric lymph nodes of WT and T316A knock-in mice 48 h following oral gavage. Data represent 3 independent experiments, n = 9 male mice.
Interaction of TREM2-TMH with the signaling partner DAP12.Figure 2(a, b) Overlay of 2D-[1H,15N]-TROSY spectra of 2H,15N-labeled TREM2-TMH in DPC micelles (black) and in complex with (a) unlabeled DAP12 (red) or (b) TREM2-TMH lacking its positively charged lysine residue (K186A, red). (c) Chemical shift perturbations (CSPs) between the two spectra in panels (a) or (b). The α-helical secondary structure as determined for wild-type TREM2-TMH is indicated above and the position of the K186A mutation is labeled in green. (d) NOE-derived lowest-energy structure of TREM2-TMH in complex with disulfide-bridged dimeric DAP12 shows one continuous α-helix. (e) The experimental structure of TREM2-TMH was docked to a previously determined structure of DAP12 in complex with NKG2C (Call et al., 2010). The positive charge in K186 is engaged in a salt bridge with D50 of DAP12. This charge complementarity together with hydrophobic interactions stabilize the complex and lead to a straight α-helical conformation in TREM2-TMH. D50 of the second DAP12 monomer is not in direct contact with TREM2, similar to the DAP12-NKG2C complex (f) For TREM2-K186A-TMH a ~7% increase in α-helical secondary structure can be detected by CD spectroscopy. (g) A continuous α-helical secondary structure in the kink region (aa 189-192) was probed by sequential amide NOE contacts in TREM2-K186A-TMH. Red dots indicate the chemical shift of intra-residual (i) amide resonance and red broken lines the inter-residual connectivity (i±1). (h) Removal of the positive charge (K186A) leads to a very similar structure of the TREM2-TMH as in its complex with DAP12.
Figure 6Serial sections of human tissue. Representative immunohistochemistry of GSII (top; spasmolytic polypeptide-expressing metaplasia [SPEM] marker and neck cells) counterstained with hematoxylin (blue) and eosin (pink). Representative staining of ATF3 (bottom) counterstained with eosin (pink). Tissue was taken from regions adjacent to gastric adenocarcinoma. Scale bars left - 50μm right - 20μm. Arrowheads indicate strongly expressing ATF3 cells and the corresponding region in the same gland labeled with GSII in a serial tissue section. GSII at the base of gastric glands in the body of the stomach indicate transition to SPEM.
Fig.Figure 2The Ca2+ signal elicited by frustrated phagocytosis was significantly reduced in Ned-19 (10 μM)-treated WT and in all TPC-KO BMDMs; maximum Ca2+ amplitude, expressed as a percentage of WT control (n = 314-641 cells). Phagocytosis of opsonized 3-µm (B and C) and 6-μm (D) beads was inhibited by Ned-19 (B) and in BMDMs from Tpcn-/- mice compared to WT (n = 134-280 cells). (E) Phagocytosis of 3-µm beads was unchanged in MCOLN1-/- BMDMs, but was impaired using 6-µm beads (F), n = 89-157. Heterologous expression of mouse TPCs tagged with TagRFP-T (in red) rescues the phagocytic defect of Tpcn1-/- and Tpcn2-/- BMDM, but a pore-dead TPC2-mutant (N257A) does not, n = 39-94. Images taken 40 min post- 3-μm bead addition. Raising cytosolic Ca2+ with ionomycin (1 μM) rescues the Tpcn1-/- and Tpcn2-/- phagocytic defect, whilst UTP (100 μM) and ML-SA1 (50 μM) only weakly rescue (n = 84-192 cells).
Physical and genetic interaction between Aub and the CCR4-NOT complex (A-B"") Immunostaining of wild-type (A-A"") or nos-Gal/UASp-CCR4-HA (B-B"") germaria with anti-Aub (red), and anti-CCR4 or anti-HA (green).Figure 3(A-B'') Immunostaining of wild-type (A-A'') or nos-Gal/UASp-CCR4-HA (B-B'') germaria with anti-Aub (red), and anti-CCR4 or anti-HA (green). GSCs are shown in (A-A''). White arrowheads in (B-B'') indicate cytoplasmic accumulation of CCR4-HA colocalized with Aub in GSCs. (C) Co-immunoprecipitation (IP) of NOT1, NOT3 and CCR4 with GFP-Aub in ovaries. Wild-type (WT, mock IP) or nos-Gal4/UASp-GFP-Aub (GFP-Aub) ovarian extracts were immunoprecipitated with anti-GFP, either in the absence or the presence of RNase A. Western blots were revealed with anti-GFP, anti-NOT1, anti-NOT3 and anti-CCR4. Inputs are extracts prior to IP. (D) Co-IP of NOT1 and NOT3 with GFP-Aub or GFP-AubAA in ovaries. Wild-type (WT, mock IP), nos-Gal4/UASp-GFP-Aub (GFP-Aub) or nos-Gal4/UASp-GFP-AubAA (GFP-AubAA) ovarian extracts were immunoprecipitated with anti-GFP in the presence of RNase A. Western blots were revealed with anti-GFP, anti-NOT1 and anti-NOT3. Inputs are extracts prior to IP. (E-G) Genetic interaction between aub and twin in Vasa self-renewal. Immunostaining of germaria with anti-DNA (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. Examples of aubHN2/+, twinDG24102 and aubHN2/+; twinDG24102 germaria are shown. White arrowheads indicate GSCs; the white arrow indicates GSC loss. Scale bar: 10 μm in A-B'' and E-G. (H) Quantification of mutant germaria with no GSC in 3, 7 and 14 day-old females of the genotypes shown in (D-F). The number of scored germaria (n) is indicated. *** p-value <0.001 using the χ2 test.
Figure 6(A-D) ChAPs are differently dependent on each other in terms of binding at the TGN. Binding kinetics of GFP-tagged Chs6 (A), Bud7 (B), Bch2 (C) and Bch1 (D) at the TGN in the presence (WT) or absence of the other ChAPs (). The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.
Figure 2(A) Soft or stiff 4T1 or MCF-7 cells were isolated by the microfluidic chip, and then seeded (500 cells) in 90 Pa soft 3D fibrin gel for 3 days. The percentage of colonies formed was calculated and the colony size was recorded. Scale bar, 100 μm. Data information: Two-tailed Paired Student's t test The data represent mean ± SD. n = 3 independent experiments in (A). (B) The soft or stiff 4T1 or MCF-7 cells (100 cells) were injected into the mammary fat pads of NSG or BALB/c mice. Twelve weeks later, the tumor formation was recorded. n = 8-10. Data information: Two-tailed Paired Student's t test The data represent mean ± SD. (C) The different number of 4T1 cells were injected into the BALB/c mice. n = 10. Data information: Two-tailed Paired Student's t test The data represent mean ± SD. (D) The tumor-forming capacity from primary xenografts (F1) and tumors passaged into secondary (F2) and tertiary (F3) recipients induced by injecting 100 soft 4T1 or B16 cells into the BALB/c or C57BL/6 mice . n = 10. Data information: Two-tailed Paired Student's t test The data represent mean ± SD. (E) Stiff or soft B16 or MP-1 cells (100 or 10 cells) were injected into the NSG mice by tail veil. n = 8-12. Data information: Two-tailed Paired Student's t test The data represent mean ± SD. (F) NSG mice were intravenously injected with 100 soft or stiff B16 or MP-1 cells. Eight weeks later, the lung metastasis was analyzed by H&E staining. The metastasis index was defined as the percentage of total metastatic nodule area to the total lung area based on the calculation from 10 slides. Scale bar, 0.5 mm. n = 3 (for B16) or 5 (for MP-1) mice with metastatic tumor. Data information: Two-tailed Paired Student's t test The data represent mean ± SD.
Figure 3(A) Schematic overview of single molecule force spectroscopy (SMFS) experiments using full-length trimeric Spike coupled to an atomic force microscopy (AFM) cantilever tip and surface coated murine Clec4g-Fc or CD209c-Fc. Arrow indicates pulling of cantilever. (B) Representative force traces showing sequential bond ruptures in the SMFS experiments. Measured forces are shown in pico-Newtons (pN). (C) Experimental probability density function (PDF) of unbinding forces (in pN) determined by SMFS (black line, measured data). The three distinct maxima fitted by a multi-Gaussian function reveal rupture of a single bond (blue dotted line), or simultaneous rupture of 2 (red dotted line) and 3 (green dotted line) bonds, respectively. (D) SMFS-determined binding probability for the binding of trimeric Spike to Clec4g and CD209c. Data are shown as mean binding probability ± SD of single, double, triple or quadruple bonds (technical replicates, N=4). (E) High speed AFM of single trimeric Spike visualizing the real-time interaction dynamics with lectins. Top panel shows 5 frames of trimeric Spike alone imaged on mica. Middle and bottom panels show 5 sequential frames of trimeric Spike/Clec4g and trimeric Spike/CD209c complexes, acquired at a rate of 153.6 and 303 ms/frame, respectively. Association and dissociation events between lectin and Spike are indicated by white and red arrows, respectively. The blue dotted ellipses display the core of the complexes showing low conformational mobility. Color schemes indicate height of the molecules in nanometers (nm). Volumes of single trimeric Spike, trimeric Spike/Clec4g and trimeric Spike/CD209c complexes are indicated, as well as numbers of lectins bound to trimeric Spike, averaged over the experimental recording period.
Figure 1A. Immunohistochemical analysis of SHARPIN expression in the human mammary gland. Cross-section of a mammary duct (upper panel) and magnification of the marked area (lower panel). SHARPIN-positive luminal (grey arrow) and stromal cells (red arrow), and the approximate position of the basal lamina (dashed red line) are indicated. Scale bar represents 50 μm. B. Western blot analysis of SHARPIN protein expression in isolated primary mammary epithelial cells (MECs) and mammary stromal fibroblasts (MSFs). CDH1 and vimentin were used as markers of epithelial and stromal cell lineages, respectively. GAPDH served as a control for protein loading. C. FACS-based isolation of mouse mammary gland basal epithelial cells (LinnegCD24intICAM-1hi), mature luminal epithelial cells (LinnegCD24hiICAM-1neg), luminal progenitor cells (LinnegCD24hiICAM-1int), and stromal cells (LinnegCD24neg). D. Quantitative PCR analysis of SHARPIN mRNA expression in cell populations isolated in (C) (mean ± SEM, n = 3 - 5).
Figure 1(A) Representative results of Western blot analysis of RAD21, E-cadherin, and VIM are shown for luminal and basal-like breast cancer cell lines. The images were analyzed using ImageJ (National Institutes of Health, USA). Protein levels from the Western blots were quantified and normalized relative to α-tubulin. Bars represent the mean ± S.D for four independent experiments. (B) Immunofluorescence staining for VIM (green) or E-cadherin (green) and RAD21 (red) in a panel of MCF7 or HCC1143 cells. (C) The level of RAD21 mRNA in breast cancer cell lines. Values were normalized relative to 18S transcription. The plotted data are the average for three independent experiments. Bars represent the mean ± S.D. (D) Stabilities of the RAD21 transcript and protein in MCF7 or HCC1143 cells. Cells were treated with 10 µg/mL actinomycin D for 0, 3, 6, 9, 12, and 24 h, after which total RNA and proteins were extracted. c-Myc and 18S were used as the controls.
Figure 4A Luciferase assay of NF-κB activation in HEK293T cells transfected with WT or mutant Mtb PknG. The graph shows the mean ± SEM of three independent biological replicates, n = 3. *P < 0.05, **P < 0.01. Data were analyzed according to one-way analysis of variance (ANOVA) using GraphPad Prism 7. B, qPCR analysis of Tnf mRNA in primary human monocyte-derived macrophages. Cells were infected with WT, ΔpknG, ΔpknG:pknG, ΔpknG:pknG K181M, ΔpknG:pknG E190A, or ΔpknG:pknG K181M, E190A Mtb strains at an MOI of 1 for 0-24 hours. The fold changes obtained from each sample were normalized to those of GAPDH. The graph shows the mean ± SEM of three independent biological replicates, n = 3. *P < 0.05, **P < 0.01. Data were analyzed according to one-way ANOVA using GraphPad Prism 7. C qPCR analysis of Il6 mRNA in primary human monocyte-derived macrophages. Cells were infected with WT, ΔpknG, ΔpknG:pknG, ΔpknG:pknG K181M, ΔpknG:pknG E190A, or ΔpknG:pknG K181M, E190A Mtb strains at an MOI of 1 for 0-24 hours. The fold changes obtained from each sample were normalized to those of GAPDH. The graph shows the mean ± SEM of three independent biological replicates, n = 3. *P < 0.05, **P < 0.01. Data were analyzed according to one-way ANOVA using GraphPad Prism 7. D Survival of Mtb strains in primary human monocyte-derived macrophages treated as in B and C. The graph shows the mean ± SEM of three independent biological replicates, n = 3. *P < 0.05, **P < 0.01. Data were analyzed according to one-way ANOVA using GraphPad Prism 7.