EMBO/sd-character-level-ner huggingface dataset:
Figure 8Wild type mouse cortical neurons depleted of Nprl3 or Tsc2 with shRNA had mitochondria that were insensitive to amino acids (R+L) or insulin, respectively Wild type mouse cortical neurons depleted of Nprl with shRNA had mitochondria that were insensitive to amino acids (R+L Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Knockdown efficiency was monitored by western blotting of phosphorylated ribosomal S6 protein, a downstream target of the mTORC1-S6K pathway. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test Wild type mouse cortical neurons depleted o Tsc2 with shRNA had mitochondria tha were insensitive t insuli Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Knockdown efficiency was monitored by western blotting of phosphorylated ribosomal S6 protein, a downstream target of the mTORC1-S6K pathway. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test (D Human fibroblasts deficient in Tsc2 (TSC2 Δexons1-14) were imaged before and 30 minutes after insulin addition. Lack of Tsc2 made mitochondria insensitive to insulin Cultures were serum-starved in Hank's balanced salt solution for 2 hours before stimulation with insulin or amino acids, and were imaged before and 30 minutes after stimulation. Each colored line refers to a single field of view containing 2-5 cells, and each pair of solid and dotted lines of the same color refers to the same field of view Statistical analyses were performed using Student's two-tailed unpaired t-test (E) Re-expression of human Tsc2 rescues mitochondrial responses to insulin. Cultures were serum-starved in Hank's balanced salt solution for 2 hours before stimulation with insulin or amino acids, and were imaged before and 30 minutes after stimulation. Each colored line refers to a single field of view containing 2-5 cells, and each pair of solid and dotted lines of the same color refers to the same field of view. Re-expression of the Flag-WT Tsc2 was corroborated by indirect immun­o­fluorescence (IF). Statistical analyses were performed using Student's two-tailed unpaired t-test
Figure 4(A) Immunofluorescence analysis of expression of Hnf4α, A6, Sox17 and Krt19. The nuclei were counterstained with DAPI. EHBD, extrahepatic biliary duct.(B) Higher magnification of the regions demarcated by the white rectangles in panel A. Scale bar: 25 m.
Figure 1(A) WT and CITK -/- embryonic (E14.5) mousecerebral cortex was stained for γ-tubulin (green) and DNA (gray). The ventricular plane is marked by a red dashed line, and the spindle axis of apical progenitors is indicated by a white dashed line. The angle between these two lines represents the mitotic angle. Scale bars = 5 μm.(B) Quantification of vertical divisions of apical progenitors in WT and CITK -/- mice. n= 3 per each genotype. (C) Pregnant CITK +/- females, crossed with CITK +/- males, were injected with BrdU at E13.5; 24 hours later (E14.5) WT and CITK -/- embryonic mouse cerebral cortex were fixed in 4% PFA, cryosectioned and stained for Ki67 (red), BrdU (green), Tbr2 (blue) and DAPI (gray). The different cortical regions are indicated: CP=cortical plate; IZ= intermediate zone; SVZ= subventricular zone; VZ= ventricular zone. Scale bars = 10 μm. White arrows in the inset indicate two Ki67- cells positive for BrdU and Tbr2. (D) Quantification of cell cycle exit (ratio of cells BrdU+/ Ki67−) in the proliferative regions (VZ/SVZ) and in the neuronal layers (IZ and CP) of sections prepared as in panel C (n=4 per each genotype). (E) Sections prepared as in C were stained for TUBB3 (TuJ) to reveal post-mitotic neurons and with DAPI. Red arrowheads indicate apoptotic neurons (defined by pyknotic nuclei). Quantification of neuronal distribution in VZ-SVZ versus total neurons population is showed on the right. Scale bars = 10 μm. (F) Quantification of the percentage of Tbr2-positive cells in the BrdU-positive population of the same sections analyzed for panel D (n=4 per each genotype). (G) Neuroblasts of wild type and dck alleles immunostained for α-tubulin and Miranda. Note that whereas in wild type cells there is a tight coupling of the mitotic spindle with the polarity axis, in dck alleles the spindle show a more oblique orientation respect to Mira crescent. Scale bar = 5 μm.(H) Distribution of spindle angle amplitude (°) in neuroblasts of wild type and ck alleles (n=58).
Figure 4A. Photomicrographs of the MCC cell line PeTa after 6 days of 100 nM GSK-LSD1 or DMSO treatment. Scale bar, 100 μm. B. Metagene plots showing the average CUT&RUN signal for H3K4me1, H3K27ac, and H3K27me3 for differentially expressed genes after 6 days of LSD1i or DMSO treatment. kb, kilobase; TSS, transcription start site; TES transcription end site. C. ATAC-seq peak profile around TSS of differentially expressed genes after 6 days of LSD1i or DMSO treatment. kb, kilobase; TSS, transcription start site D. Volcano plot showing the -log10 (adjusted p-value) and log2 fold-change (log2FC) for transcripts detected by RNAseq analysis of PeTa cells treated with 100 nM GSK-LSD1 or DMSO for 6 days. Significantly down-regulated genes (FDR≤0.05; log2FC≤-1) involved in mitotic cell cycle (GO:0000278) are highlighted in blue. E. Upstream promoter motif enrichment of the downregulated genes from the dataset in Fig 4D. Top-5 enriched motifs are depicted. TF, transcription factor. D. Volcano plot showing the -log10 (adjusted p-value) and log2 fold-change (log2FC) for transcripts detected by RNAseq analysis of PeTa cells treated with 100 nM GSK-LSD1 or DMSO for 6 days. Significantly down-regulated genes (FDR≤0.05; log2FC≤-1) involved in mitotic cell cycle (GO:0000278) are highlighted in blue. E. Upstream promoter motif enrichment of the downregulated genes from the dataset in Fig 4D. Top-5 enriched motifs are depicted. TF, transcription factor.
Goblet cell mucin accumulation correlates with diminished ROS production.Figure 6(A) Colonic epithelial cell spheroids from p22phox−/−, Atg5VC and wild‐type mice. Wild‐type spheroids were treated with vehicle (control), 10 μM DPI (NADPH oxidase inhibitor) or 100 μM Dynasore. Intracellular ROS in live cells were labelled by 5 μM DCF. Bars=100 μm. (B) Quantification of fluorescent DCF normalized to control (n=3 independent experiments/group; 30 spheroids quantified/experiment). (C) Quantification of average area of cytoplasmic mucin from wild‐type mice treated with 3% NAC as compared to controls (n=6-7 mice/group). Error bars indicate s.e.m. *P0.05; **P0.01; ****P0.0001 as determined by the Student's t‐test. (D) TEM images of colonic epithelial monolayers treated with cerium chloride. The presence of electron dense precipitate is indicative of ROS production. Bar=500 nm.
Figure 10A Relative MtNPF expression following treatment with 5mM KNO3 Data information: expression shown is relative to Ubiquitin; *p< 0.05, Student's t-test; biological replicates: n= 3 (3 pooled root organs in each replicate) ±SEM. B Changes in NO3- and Cl- content (mol/g dry weight) of NO3- starved plants after NO3- provision Data information: , *p< 0.05, Student's t-test; biological replicates: n=4 ±SEM. C Data from 'B' expressed as NO3- to Cl- ratios Data information: expression shown is relative to Ubiquitin; *p< 0.05, Student's t-test; biological replicates: n= 3 (3 pooled root organs in each replicate) ±SEM. D Relative MtNPF expression following treatment with 100mM NaCl Data information: expression shown is relative to Ubiquitin; *p< 0.05, Student's t-test; biological replicates: n= 3 (3 pooled root organs in each replicate) ±SEM. E Relative MtNPF expression following treatment with 5mM KNO3 +100mM NaCl Data information: expression shown is relative to Ubiquitin; *p< 0.05, Student's t-test; biological replicates: n= 3 (3 pooled root organs in each replicate) ±SEM.
Figure 1Candidate modifiers are categorized here based on their known functions in gene expression. Fly genes are listed in the left columns, while their human homologs are listed in the right columns. Disruption of genes highlighted in dark blue strongly suppressed (CGG)90-EGFP toxicity. Disruption of genes highlighted in light blue weakly suppressed (CGG)90-EGFP toxicity. Disruption of genes highlighted in light red enhanced (CGG)90-EGFP toxicity selectively. Disruption of genes highlighted in dark red enhanced the toxicity of both (CGG)90-EGFP and GMR-GAL4 (these were toxic independent of the repeat.) All other genes are displayed in white. The methyl-7-guanosine (m7G) cap, eIF4F complex, 43S pre-initiation complex (PIC), and ribosomes are indicated.
Figure 2A Yeast two hybrid analysis showing the interaction between MPK3/6 and ARR12. Yeast cells were plated on selection medium: SD-LW (-Leu/Trp) or SD-LWHA (-Leu/Trp/His/Ade) supplemented with AbA (Aureobasidin A) and X-α-gal (5-bromo-4-chloro-3-indolyl-b-d-galactopyranoside). MPK3/4/6 coding sequences were fused with the GAL4 DNA-binding domain (Bait proteins), and ARR1/10/12 coding sequences were fused with the GAL4 activation domain (Prey proteins). B BiFC analysis of interaction between MPK3/4/6 and ARR1/10/12. Partial yellow fluorescence protein (YFP) constructs were fused to MPK3/4/6 or ARR1/10/12. The fusion constructs were co-expressed transiently in Arabidopsis protoplasts cells. The interaction between MPK3/4/6 and ARR1/10/12 were visualized by fluorescence microscopy. The percentages labelled in the images mean the probability of present cell in cells observed. Scale Bar, 50 μm. C In vivo Coimmunoprecipitation assay of MPK3/4/6 and ARR1/10/12. MPK3/4/6-YFP and ARR1/10/12-MYC were co-expressed in Arabidopsis protoplast cells. Protein extracts (Input) were immunoprecipitated with GFP Trap magnetic agarose beads. Immunoblots were performed with anti-MYC antibody to detect ARR1/10/12 and with anti-GFP antibody to detect MPK3/4/6.
Figure 4Effects of KYA1797K on the interaction and phosphorylation of β-catenin and pan-RAS. WT- or MT-HCT116 cells were treated with 20 µM of KYA1797K in the presence of MG132. WCLs were immunoprecipitated with pan-RAS antibody and subjected to IB analyses Effects of Wnt3a-CM treatment on the formation of β-catenin destruction complex and phosphorylation of β-catenin and RAS in HEK293 cells treated with L-CM or Wnt3a-CM in the presence of MG132 for 8 hr. WCLs were immunoprecipitated with β-catenin or pan-RAS antibodies and subjected to IB analyses IB analyses were performed to detect β-catenin and pan-RAS in DLD1 cells cotransfected with FLAG-β-catenin-WT or -MT and Myc-KRAS-WT or -2A for 24 hr and then treated or transfected with 20 µM of KYA1797K or HA-GSK3β for 24 hr, respectively
Figure 3Schematic representation of the sequences of the DNA target and sgRNAs. The PAM is shown in yellow. The nontarget DNA strand was labeled with Cy5. The matched and mismatched sequences in sgRNAs are shown in purple and blue, respectively. Representative gel shows DNA cleavage by SaCas9 guided by partially matched sgRNAs.
Figure 6A Bimolecular fluorescence complementation tests using in combination PAP8ΔcTP-NY (P8Δc-NY) with PAP5ΔcTP-YC (P5Δc-YC) or PAP8ΔcTP-YC (P8Δc-YC) with PAP5ΔcTP-NY (P5Δc-NY); PAP10-RFP (P10-RFP) was used as internal positive control for transfection; the ratio in grey depicts the number of green-fluorescent cells over red-fluorescent cells. Arrowheads indicate nuclei. See Appendix Fig. S6 for control experiments; transgenes expressed under CaMV35S promoter. B Overlay of 1H-15N correlation 2D NMR spectra of free 15N-labelled PAP8 alone (blue) or in complex with PAP5 (red). Grey areas depict changes of signals in the PAP8 spectrum. C 15N-Filtered Diffusion Ordered Spectroscopy-NMR measurements to PAP8. Exponential decay curves of PAP8 in absence or in presence of MBP-PAP5 are shown in red and black respectively. The units on the y-axis are normalized values of the integrals of the signal measured in the amide proton region.
4D7 disrupts interaction with TpoR and downstream signalling A.Figure 2A. Cell extracts blotted for phospho-STAT5, total STAT5, phospho-ERK, total ERK and actin from TF-1 TpoR cells after incubation with 10 or 20 µg/mL 4D7 or IgG for 4 or 8 hours, in presence of TPO. The last line indicates TPO withdrawal. B. Similar experiment using TPO-independent TF-1 TpoR CALRdel61 cells. C. Similar experiment using TPO-independent TF-1 TpoR CALRdel52 cells at 8 hours. D. Similar experiment using PBMNCs from CALRdel52 PMF primary cells at 8 hours. Additionally, cells were treated with 280 nM of ruxolitinib as a positive control. E. Fraction of apoptotic sub-G0 population of TF-1 TpoR CALRdel61 cells after 48 hours of 4D7 or IgG treatment (n=3 biological replicates, bars represent standard deviation for 3 replicates, normalised to IgG, with a students unpaired t-test used to determine statistical significance). F. Western blot showing caspase 3 cleavage occurring within 24 hours of TPO withdrawal in TPO dependent TF-1 TpoR cells. An increase in cleaved caspase 3 is observed after 48 hours of treatment with 20 µg/mL 4D7 in TF-1 TpoR CALRdel61 and TF-1 TpoR CALRdel52 cells. G. Western blot of TpoR immunoprecipitation under non-reducing conditions showing associated CALR 50 kDa monomers and 100 kDa dimers (red arrowheads) present only in TF-1 TpoR CALRdel61 disrupted by 8-hour treatment with 20 µg/mL 4D7 but not PBS or 20 µg/mL IgG. CALR monomers & dimers are detectable by polyclonal anti-wild type CALR or anti-mutant CALR monoclonal antibodies. Red arrowheads, detected mutant CALR protein; brown arrowheads, detected wildtype CALR protein, asterisk, non-specific bands.
Combined MEK1/2-PLK1 inhibition specially kills basal-like breast cancer lines.Figure 6(A) Heat map showing relative cell viability of a panel of human breast cancer lines treated single or in combination with AZD6244 (1 µM) and BI2536 (2.5 nM) inhibitors and cell viability was determined after 6 days. DMSO treated control was used to calculate percentage of cells affected by individual or combination treatment. NN: near normal. Heat map represents data derived from three independent experiments. (B) Immunoblot analysis was performed on MDA-MB-157 and SUM159PT cells treated with single or in combination with AZD6244 and BI2536 inhibitors after 96 hours and cleaved PARP and Caspase-3 were determined along with CEP55, MYC and both phosphorylated and total ERK1/2. COX-IV as a loading control (left panels). Percentage of sub-G1 population identified using propidium iodide staining and quantified by FACS following single and combination treatment with AZD6244 and BI2536 inhibitors after 96h. Graph represents the mean±SEM of two independent experiments (middle panels). Representative images of colony forming capacity at 14 days determined using crystal violet staining in cells treated with single and combination inhibitors (middle panels). Combination index (CI) values calculated through CompuSyn software (right panels) where 1 indicative of additive (no interaction), >1 indicative of antagonistic, and <1 indicative of synergistic. (C) Percentage of mitotic outcomes determined using time-lapse microscopy of MDA-MB-231 cells treated with single or in combination drugs of BI2536 (2.5 nM) and AZD6244 (1 µM). Graph represents the mean±SEM of two independent experiments. For each experiments n=50 cells were counted per condition. (D) Immunoblot analysis was performed to determine the expression and activity of mitotic and apoptotic regulators upon single and combination treatment with AZD6244 and BI2536 inhibitors as indicated time points in both MDA-MB-231 and SUM159PT cells. Levels of phospho-MEKT286 and dephosphorylation of phospho-CDK1Y15 served as markers of CDK1 activation/mitotic entry. COX-IV as a loading control.
a, GFP–MAP1LC3B foci after RNAi and/or rapamycin treatment in U2OS cells.figf5a, GFP-MAP1LC3B foci after RNAi and/or rapamycin treatment in U2OS cells. Scale bar, 20 µm.
Figure 5A-C) Gene ontology analysis for rhythmic proteins was carried out using Gene ontology enrichment analysis and visualisation Significantly rhythmic WT proteins were compared against background (all proteins identified in the experiment), and the top non-overlapping GO Biological Process terms shown, sorted according to FDR q-value. Fold enrichment is annotated on each bar (A). The same GO analysis was carried out comparing proteins rhythmic that were rhythmic in CKO cells (B) and for proteins that were rhythmic across both genotypes (C). D-E) From one time-course experiment, ions, cytosolic proteins and total protein were extracted in parallel samples. The presented experiment is representative of 3 separate time-course experiments that were carried out (N=3). Representative experiment shown (n=4 technical replicates for [K+], n=3 technical replicates for the other traces). Blue lines highlight the antiphasic relationship between oscillations in cytosolic protein and potassium concentration. Mean±SEM, p-values from RAIN, red lines are fits by a damped cosine compared with a straight line (null hypothesis). Parallel PER2::LUC recordings were also performed and plotted as a phase marker. F) Examples of key ion transporters are shown, as detected in the proteomics experiment. P values show the results of an F test comparing fits of damped cosine against straight line. All proteins except WT NKCC1 had RAIN p-values <0.05. G-H) Relative amplitudes of cytosolic protein (G) and potassium concentrations oscillations (H) in D) and E) were greater in CKO compared to WT (Student's t test with Welch correction, mean±SD, *p≤0.05, **p≤0.01).
Beclin and PtdIns 3‐kinase form a complex.Figure 1(A) Cell lysates prepared from HeLa cells were solubilized with Triton X-100 and subjected to immunoprecipitation using preimmune sera (lane 1) or anti-Beclin antibodies (lanes 2 and 3). The immunoprecipitates were incubated with phosphatidylinositol, [γ-32P]ATP and 60 μM cold ATP in the presence of Mn2+ (lanes 1 and 2) or Mg2+ (lane 3) for 5 min at 30°C. (B) Immunoprecipitates of anti‐Beclin antibodies prepared as described in (A) were assayed for PI 3‐kinase activity in the presence of wortmannin at concentrations of 0 nM (lane 1), 1 nM (lane 2), 10 nM (lane 3), 100 nM (lane 4). The labeled lipids were extracted and separated by thin layer chromatography, followed by detection by autoradiography using a bioimaging analyzer BAS2000 (Fuji Film). (C) Cell lysates prepared from HeLa cells were solubilized with Triton X‐100 and incubated with protein A‐immobilized anti‐Beclin (lane 2) or anti‐PtdIns 3‐kinase antibodies (lane 4). As controls, preimmune sera of the respective antibodies (lanes 1 and 3) were used. Retained proteins were separated by SDS-PAGE and detected by immunoblotting with anti‐PtdIns 3‐kinase (upper panels) and anti‐Beclin (lower panels) antibodies.
NSMCE2 is essential for early embryonic developmentScheme illustrating the Nsmce2 genetrap allele (Nsmce2GT) used in this study.Figure 2Scheme illustrating the Nsmce2 genetrap allele (Nsmce2GT) used in this study. Comparison of the wt and mutant NSMCE2 protein. Note that the trapped protein lacks most of the C‐terminal region including the SUMO ligase domain. Brightfield microscopy of 2.5 dpc embryos obtained from crosses of Nsmce2GT/+ mice. The right panel is of the same 2 embryos shown on the left panel, 36 h after being cultured in vitro. The arrow indicates embryos that showed asymmetric cell sizes when isolated, which degenerated upon culture. These embryos were identified as Nsmce2GT/GT through genotyping. Examples of the three genotypes found at Mendelian frequencies (see Appendix Table S1) on 2.5 dpc embryos. Examples of the type of nuclei found on Nsmce2GT/GT and Nsmce2+ (+/+ or +/GT) 2.5 dpc embryos, 12 h after in vitro culture. Mutant embryos were identified due to the absence of NSMCE2 signal (green). DAPI was used to stain DNA (blue). White arrows indicate examples of cells with condensed chromosomes or irregular shapes, which are hardly seen in wild type embryos.
Figure 5A. Heat map of proteins identified by LC-MS/MS analysis as differentially secreted in CM of normal fibroblasts (P-NF#1 and P-NF#2), CAFs with low level of hMENA∆v6 expression (P-CAFs low#44 and #49), CAFs with high level of hMENA∆v6 expression (P-CAFs high #67 and #36). The boxed hMENA∆v6 associated signature represents proteins exclusively present in the CM derived from CAFs high. Color code is shown in the upper left corner. Co-variance = 0.2, q value = 0.1 (10% false discovery rate), adjusted P value = 0.0074542. N = 36. The values of mass spec identified for each protein were plotted in log2 scale. B. Quantification of GAS6 secretion levels, as detected by ELISA, in the CM of P-NF (#1), P-CAFlow (#44 and #49) and P-CAF high (#67 and #36). Data are presented as the mean ± SD of two biological replicates. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. ***P<0.001. C. Real time qRT-PCR analysis of the relative GAS6 mRNA expression level in NFs (P-NF#1), P-CAF low and P-CAF high, as described above. Data are presented as the mean ± SD of three replicates. Statistical analysis was performed with one-way ANOVA P=0.001, followed by Bonferroni's multiple comparison test. *P< 0.05, **P<0.01. D. Real time qRT-PCR analysis of the relative GAS6 mRNA expression level in PANC-1 and P-CAF (n=3), showing a low GAS6 expression in PANC-1 cells. Data are presented as the mean ± SD. P values were calculated by two‐sided Student's t‐test. ***P<0.001. E. Boxplots showing the mRNA expression of GAS6 in normal lung fibroblasts (white) (n=15) versus primary NSCLC fibroblasts (light blue) (n=15) (GSE22862 data set). In each boxplot the median value (horizontal line), 25th-75th percentiles (box outline), and highest and lowest values within 1.5x of the inter-quartile range (vertical line) are shown. Statistical significance was calculated by Mann-Whitney U-test (two-sided) (P=0.0208). F, G. Real time qRT-PCR analysis of P#138 CAF (F) and L#189 CAF (G) transfected with control siRNA (siCNT) or hMENA(t) siRNA (sihMENA(t)) as representative cases. The siRNA‐mediated knock‐down of hMENA/hMENA∆v6 resulted in a significant reduction of GAS6 mRNA expression levels compared to siCNT cells (set as 100). Data are presented as the mean ± SD of three replicates. P values were calculated by two‐sided Student's t‐test **P<0.01, ***P<0.001. H. Quantification of matrigel invasion assay of PANC-1 cultured for 48 h with DMEM (culture medium), or conditioned medium (CM) derived from control siRNA P#106 CAFs (siCNT P-CAF-CM), GAS6 siRNA (siGAS6 P-CAF-CM), hMENA(t) siRNA (sihMENA(t) P-CAF-CM), hMENA(t) siRNA plus rGAS6 (sihMENA(t) P-CAF-CM +rGAS6). Number of invaded PANC-1 cells after 48 h of treatment was measured by counting 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one-way ANOVA P=0.004, followed by Bonferroni's multiple comparison test. *P< 0.05, **P<0.01.