EMBO/sd-character-level-ner huggingface dataset:
Figure 6(A) Q-PCR (Taqman) analysis of LIF expression in BCE cells and bovine pericytes (bPericyte). Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOV was used as statistical test. (B) Bovine LIF ELISA of BCE cells and bovine pericyte. Cells were cultured for 48 hrs before harvesting the medium. Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOV was used as statistical test. (C) LIFR expression in BCE cells and bovine pericyte by qPCR (Taqman). Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOV was used as statistical test. (D) Image of FACS selection of CD13+/CD45- and CD31+/CD45- cells. (E) Immunostaining for CD31, CD13 and DAPI inin isolated primary mouse cells before and after FACS selection. (F) Mouse LIF expression in FACS-selected cells. Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOV was used as statistical test. (G) Human choroidal EC (HCEC) and mouse retinal EC (MREC) were treated with LIF 10 ng, LIF 100 ng for 30 min. (PBS was used as control). STAT3 phosphorylation was detected by western blot. (H) H&E staining of LIFR and CD31 in mouse eye sections. The magnified images of the dashed line boxes, a and b, are shown on the right panel. Box a shows LIFR staining in retina and box b showed LIFR staining in RPE/choroid layer. (I) Whole mount staining of LIFR and CD31 in mouse retina. The fluorescent staining for CD31 (green) and LIFR (red) is shown. White arrows indicate the co-staining of CD31 and LIFR on retinal ECs.
Figure 3(A) ssDNA repair efficiency of the rev1∆ mutant treated with MMS only in G1 as determined from the maximal and final percentages of cells with Rad52 foci during the time course (right panel). Cells were synchronized in G1 and treated with 0.033% MMS for 1 hour; after that the MMS was inactivated with 2.5% sodium thiosulfate and cells were released into fresh medium in the absence of DNA damage. The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (middle panel). A scheme on the left shows the experimental set up followed in this (MMS in G1 only) and the other experiments (MMS in S/G2). An asterisk represents a blocking DNA lesion for the replicative polymerase. Data information: The mean and SEM of 4 independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively. (B) Effect of the rev1∆ and rev3∆ mutations in spontaneous and MMS-induced uSCE. The frequency of spontaneous and MMS-induced recombinants was determined from colonies grown in the absence or presence of 0.01% MMS. The mean and SEM of 4 independent fluctuation tests are shown. (C-D) ssDNA repair efficiency of the rad5∆ and rev1∆ mutants as determined from the maximal and final percentages of cells with Rad54 foci during the time course (right panels). Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (left panels). Data information: The mean and SEM of independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively.
Figure 3Correlation between IL-6 and maximal body temperature during hospitalization. Correlation between IL-6 and C-reactive protein (CRP). Correlation between IL-6 and lactate dehydrogenase (LDH). Correlation between IL-6 and D-dimer. Correlation between IL-6 and ferritin. Levels of baseline IL-6 in patients who received glucocorticoids, human immunoglobulin, high flow oxygen inhalation or mechanical ventilation during hospitalization versus patients who did not. Error bars, SEM.
Reverse transcription in nuclear clusters of infected macrophages The left panel shows the timeline of drug exposure experiments.Figure 5The left panel shows the timeline of drug exposure experiments. (A-D) Images show vRNA and vDNA in an infected ThP1 cell (MOI 20) for each of four experimental conditions. See Appendix Fig. S15-S17 for images of larger regions. (A,B) ThP1 cells were exposed to NVP for 48 h (A) or 72 h after infection (B), then NVP was washed out, and cells were fixed for click chemistry 24 h later. (C) Cells were exposed to PF74 for 72 h after infection, then fixed for click chemistry. (D) Cells were exposed to NVP for 24 h after infection, then NVP was washed out, and cells were exposed to PF74, before being fixed for click chemistry 24 h later. (E,F,H) Scatter plots show vDNA and vRNA intensities in colocalizing clusters with Spearman correlation r and associated p-values. Replicates of the four experiments yielded similar results. (G) qPCR measures DNA synthesis (left) and nuclear import (right) at 72 h p.i. in absence of drug treatment, or after exposure to NVP or PF74. Bars and error bars define mean and standard deviation, respectively. Samples were analyzed in triplicate and two biological replicates were performed.
Kdm3b-KO cells retain 5hmC at pluripotency-associated locations where it should be resolved in reprogramming a.Figure 5Quantification of percent Nanog-GFP positive cells of WT or Kdm3b-KO pre-IPSC clones expressing Nanog, Tet1CD, or Nanog+Tet1CD after 15 days of AA+2i or AA+2i with 4 or 5µM of UNC0638. Left - Heatmap of K-mean clustered 5hmC patterns in WT pre-iPSC with 5hmC profile of Kdm3b-KO pre-iPSC treated with AA+2i for 0, 2 or 10 days. Middle - Metaplot of 5hmC signal on day 10 between WT (orange) and Kdm3b-KO (black) for each cluster. Y-axis scale is in normalized coverage data (1x106/Total Count). Right - Illustration of 5hmC pattern in WT and Kdm3b-KO during AA+2i treatment. Representative 5hmC profiles during AA+2i conversion of WT and Kdm3b-KO pre-iPSC. Scheme for identifying genes with both misregulated 5hmC and expression. Heatmap of two-fold misregulated 5hmC and two-fold differential expression between WT and Kdm3b-KO after 10 days of AA+2i treatment (607 genes). Peaks are sorted by 5hmC signal in WT. RNA seq values represent a log2-fold change of day 10 over DMSO for either WT or Kdm3b KO pre-iPSCs. Box plot of expression for genes that are two-fold misregulated 5hmC and expression between WT and Kdm3b-KO on day 10 (607 genes). Gene ontology for genes that are two-fold misregulated 5hmC and expression between WT and Kdm3b-KO on day 10.
Figure 3A Genome-wide mapping through high density genotyping. The structural genomic variation and extended regions of homozygosity (loss of heterozygosity [LOH]) over the entire genome is shown for individuals II.1 to II.5. The homozygosity segment shared by the four affected siblings in chromosome 3 is indicated with an arrow. B This region is represented in more detail, where extended regions of homozygous genotypes in contiguous biallelic polymorphisms are highlighted with a green background. C The 14.6 megabase region of homozygosity that is shared by all affected siblings is highlighted by vertical red lines in (B) and genes within this genomic region are shown. Note that POGLUT1 gene is underlined. D Chromatograms reveal a homozygous T-to-G substitution (black boxes and arrowheads) in the four affected family members, while a heterozygous G/T in the healthy sibling. E Schematic structure of the humanPOGLUT1protein, which contains a signal peptide (SP), a CAP10 domain, and a Lys-Asp-Glu-Leu (KDEL)-like endoplasmic reticulum retention signal. D233E is located in the CAP10 domain (arrow), close to the predicted ERD catalytic motif (asterisk). Alignment of amino acids flanking the mutated aspartic acid from POGLUT1 orthologs indicates evolutionary conservation of D233 in vertebrates (open arrowhead). Black boxes denote similarity of amino acid residues.
Figure 5(A) Euc181-183 binds to Slx5 in Y2H assays. Y2H assay performed as in Fig. 4A. Serial dilutions were spotted for each plasmid combination and cells were grown at 30°C for 2 days. The Slx5-RING∆ construct is deleted for the Slx5 C-terminus from beginning of the RING-domain (∆aa 488-619). See also Fig. EV3B. (B) Euc1 binds Slx5 in vitro. Recombinant purified GST or GSTEuc1 were used in GST pull-down assays to co-precipitate recombinant 6HisSlx5. (C) Euc1 binds to Slx5 in vivo. Cell lysates from an euc1∆ strain expressing the indicated 3FLAGEuc1 constructs from plasmids (under EUC1 promoter) were subjected to immunoprecipitation using anti-FLAG beads. IP-eluates were probed by WB with Slx5, SUMO and Euc1 antibodies, inputs were probed with Slx5, FLAG and Dpm1 antibodies (top to bottom). Note that the Euc1-Slx5 interaction is independent of the Slx8 ligase activity (slx8-CD, lanes 5-8). (D) Euc1 Slx5-binding mutants (SBM1, SBM2) affect Euc1-Slx5 interaction in Y2H assays. AD-Euc181-183 constructs harboring mutations in the region required for Slx5 binding were probed for interaction with BD-Slx5 constructs as described in Fig. 4A. Mutations: SBM1: aa 139-143 ENQKK>ANAAA, SBM2: aa 162-165 KEVF>AAAA. Serial dilutions were spotted and cells were grown at 30°C for 2 days. See Appendix Fig. S6A for expression levels. (E) Euc1-SBM constructs show defective Slx5 binding in vivo. Mutations described in (D) were introduced into full-length Euc1 constructs (with or without the K231R mutation) and FLAG IPs were performed as described in (C). WB analysis showed strong Slx5-binding defects for the SBM1/SBM2 and SBM1+2 constructs (top panel, immunoprecipitated Slx5). WBs were probed as in (C). (F) The Slx5 middle domain (Slx5-Md) is required for interaction with Euc1. Y2H assays with AD-Euc181-183 and BD-Slx5 constructs. Slx5-Md: aa 201-335, Slx5-Md∆: ∆aa 201-338. Serial dilutions were spotted and cells were grown at 30°C for 4 days. See also Fig. EV3C. (G) Euc1-SBM constructs show defective binding to the Slx5-Md. Y2H assay with the indicated constructs as in (D). See Appendix Fig. S6A for expression controls. (H) Schematic representation of Euc1, Slx5 and Slx8, domain features and interactions. Domains and protein lengths are drawn to scale, numbers below each bar denote amino acid positions. The mapped interaction between Euc1 and Slx5 is indicated by an arrow. CC: coiled-coil domain, SBM: Slx5-binding mutant, S: SUMO, SIM: SUMO interacting motif.
Figure 2Mitochondrial translational products were labelled with [35S]methionine and anti-FLAG immunoisolation was performed as described in Fig. 1D. Eluates were analyzed by SDS-PAGE and digital autoradiography. Total 1.5 %, Eluate 100 %. Solubilized mitochondria from MITRAC15FLAG- or TIM21FLAG-expressing cells were subjected to anti-FLAG immunoisolation and analyzed by 2D-BN/SDS-PAGE and western blotting. MITRAC15FLAG isolation: Total 0.1 %, Eluate 100 %. TIM21FLAG isolation: Total 0.2 %, Eluate 66 % (TIM21FLAG decoration), 33 % (MITRAC15, ACAD9, MITRAC12, COX1 decoration). Solubilized mitochondria from MITRAC15FLAG- or C12ORF62FLAG- expressing cells were subjected to anti-FLAG immunoisolation and analyzed by SDS-PAGE and immunoblotting. Total 0.4 %, Eluate 100 % (MITRAC15FLAG isolation), 70 % (C12ORF62FLAG isolation). Solubilized mitochondria from ACAD9FLAG-or MITRAC15FLAG- expressing cells were subjected to anti-FLAG immunoisolation and analyzed by SDS-PAGE and immunoblotting. Total 0.5 % (ACAD9FLAG isolation), 0.4 % (MITRAC15FLAG isolation) Eluate 100 %. Asterisk (*) indicates TIM21 signal. ACAD9/ACAD9FLAG could not be separated with the used gel system, both proteins are detected with ACAD9 antiserum.
Gene sets associated with ATRA sensitivity.Figure 11Gene sets associated with ATRA sensitivityUsing the microarray and RNA-seq data associated with the breast cancer cell lines, two ATRA score-associated gene lists ranked for their variable importance were generated. Upper Panels: The gene-expression results of the first 100-ranking genes in the RNA-seq (left) and microarray (right) datasets were used to perform a cluster analysis of the breast cancer cell lines according to the gene-expression profiles. Data are expressed using a log2 scale of the expression signal intensity after normalization of the data across the different cell lines. The genes marked in red are present in both the microarray and the RNA-seq gene sets. The cell lines marked in red are those belonging to the ATRA score T1 group and are sensitive to ATRA, while the ones marked in blue belong to the T3 group and are refractory to the retinoid. The left dark blue lines indicate the genes with higher levels of constitutive expression in the ATRA-sensitive cell lines, while the light blue lines indicate the genes with higher levels of basal expression in the ATRA-refractory cell lines. Lower Panels: The box plots show the enrichment score (single sample Gene Set Enrichment Analysis, ssGSEA) of the microarray (left) and RNA-seq (right) gene sets in the TN (patients 9, 22, 23, 31, 50) and Lum (patients 13, 18, 27, 36, 41, 44, 55, 60, 61, 62, 64) tumors cultured in the absence of ATRA for 48 h. The P-values of the enrichment are indicated.
Figure 7(A) Diagram showing the experimental procedure for the evaluation of the effect of Palbociclib on the autoinflammatory disease in Trex1-/- mice. (B-F) Representative H&E staining of the histological changes in heart, stomach, tongue and thigh muscle tissues harvested from mice of indicated groups (B). Scale bar, 200 μm. The quantification of histological score was shown in (C-F). Mann-Whitney U-test was used for statistical analysis of two independent groups. Values represent means ± SEM, each symbol indicates the value of 1 mouse from n = 5 mice per group. *, P<0.05; **, P<0.01; ns, not significant. (G-I) qRT-PCR measurement of transcripts of Ifnb1(G), Cxcl10 (H) and Il6 (I) in mouse stomach tissues harvested from mice of indicated groups. The data shown are mean ± SEM of n=5 mice from 1 of 3 biological replicates with similar results. Each symbol indicates the value of 1 mouse from n = 5 mice per group. Statistical significance was determined by one-way ANOVA test followed by Tukey's multiple comparisons post-hoc test between all groups. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant.
Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.f5Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.p., n=3) for 10 days. Low-fat diet-kept mice (n=5) of same age were used as a negative control. (a,b) Levels of LC3-II from 1% Triton X-100-insoluble fraction of livers were analysed by immunoblotting (a) and quantified (b). (c,d) Frozen sections of indicated livers were subjected to LC3/LAMP1 immunostaining and DAPI counterstaining (c). Co-localization between LC3 and LAMP1 was quantified (d). Boxed areas in fluorescence images are magnified in right-most panels (c). Scale bar, 5 μm (c). All data are shown as mean±s.e.m. ***P0.001 (Student's t-test). Molecular weight markers are indicated in kDa.
Figure 1A, Schematic of chemical synthetic vulnerability screens. Clinical related drugs (n=21) were combined with BI2536 to treat BEAS-2B-KRAS cells harboring a KRASG12V allele. Cell viability was determined 72 h post-treatment, with combination index (CI) calculated by CalcuSyn software: CI＞1, antagonism; 1＞CI＞0.8, synergy; CI＜0.8, strong synergy Drug combinations leading to strong synergy (CI＜1) in BEAS-2B-KRAS were further tested in KRAS wild-type BEAS-2B cells, where CI＜1 indicates toxicity and CI＞1 safety. B, The Fa-CI plot of drug combinations (candidate drug plus BI2536) in KRAS-mutant (BEAS-2B-KRAS) and wild-type (BEAS-2B) cells. The data are mean values of two independent experiments (n=2) where all drugs are used at their IC50 determined in BEAS-2B-KRAS cells. Note that combination treatment with AZD4547 and BI2536 exhibit the leading therapeutic efficacy and safety profile, manifested by the greatest synergy (CI=0.25) in BEAS-2B-KRAS while the two drugs are antagonistic in BEAS-2B cells (CI>1). C, Dose-response curves of BEAS-2B and BEAS-2B-KRAS cells treated for 72 hours with inhibitors of FGFR (AZD4547, BGJ398) and PLK1 (BI2536, BI6727), alone or combination. Values of Fa and CI are shown underneath, with CI<1.0 indicating synergistic effect. Data were from three independent experiments (n=3), error bar: SD D, BEAS-2B and BEAS-2B-KRAS treated with the indicated drugs, alone or combination for 72 hours, were cultured in drug-free medium for additional 7-21 days. Surviving cells after the treatment were fixed and visualized by crystal violet staining. E, Apoptotic assay of BEAS-2B and BEAS-2B-KRAS cells treated with vehicle (DMSO), AZD4547 (2.5 µM) and BI2536 (5 nM), alone or in combination for 72h. Data are presented as mean ± SD (n=3). *p<0.05 and ***P<0.001 by two-way ANOVA with Tukey's multiple comparisons test.
Rac1 is associated to Rab11-positive recycling endosomes.Figure 1A-C: JurkatT cells were transfected with control, FIP3.1 or FIP3.2 siRNA oligonucleotides. A: Intracellular distribution of endogenous Rab11 and Rac1 was assessed by immunofluorescence. Jurkat T cells were transfected with control, FIP3.1 or FIP3.2 siRNA oligonucleotides. A: Intracellular distribution of endogenous Rab11 and Rac1 was assessed by immunofluorescence.A, E, G: Three-dimensional (3D) confocal images were post-treated by deconvolution. A 0.4-µ-thick medial stack is shown. The pericentrosomal vesicular compartment is zoomed at the bottom right-hand corner.B, F, H: Population analysis of the co-localization volume between Rab11 and Rac1 within the percentriolar vesicular compartment. Each dot represents one cell. Horizontal bars represent the mean ± SEM, Mann-Whitney test was used. Images are representative of three experiments. Bar, 5 µm C: FIP3 expression was analyzed by Western blot. Quantification of bands was performed with ImageJ and normalized to a non-specific band of 35 kDa used as loading control. D: Schematic representation of FIP3 structural features: proline-rich region, EF-hand domain, coiled-coil domain (CC), Arf binding domain (ABD) and Rab binding domain (RBD). E, F: Jurkat cells were transfected with expression vectors encoding GFP, GFP-FIP3-WT or GFP-FIP3-I738E. Endogenous Rac1 was detected by immunofluorescence. G: Jurkat cells were transfected with expression vectors encoding GFP, Rab11WT-GFP, or Rab11Q70L-GFP (constitutively active) or Rab11S25N-GFP (dominant negative) mutants. Endogenous Rac1 was detected by immunofluorescence.A, E, G: Three-dimensional (3D) confocal images were post-treated by deconvolution. A 0.4-µ-thick medial stack is shown. The pericentrosomal vesicular compartment is zoomed at the bottom right-hand corner.B, F, H: Population analysis of the co-localization volume between Rab11 and Rac1 within the percentriolar vesicular compartment. Each dot represents one cell. Horizontal bars represent the mean ± SEM, Mann-Whitney test was used. Images are representative of three experiments. Bar, 5 µm
Figure 5(A-D) Cluster analysis of proteins enriched at telomeres lacking POT1 using STRING. The largest identified clusters are colored in red (mitosis-associated proteins, (A), green (recombination and DNA damage response proteins, (B), purple (RNA-binding proteins, (C), and dark blue (chromatin and DNA remodelers, (D). Line thickness indicates the strength of data support.
Figure 6(A) Typical examples of single EPSCs. EPSC amplitude (M18WT: 2.98 ± 0.76 nA, n=11; M18Y473D: 0.042 ± 0.018 nA, n=11, M18Y473D/P335A: 2.02 ± 0.67 nA, n=7, Kruskal-Wallis test, p < 0.0004). Evoked and spontaneous release in autaptic munc18-1 null hippocampal neurons expressing M18Y473D, M18Y473D/P335A or wild-type Munc18-1 as control.(B) Typical traces of spontaneous release. Evoked and spontaneous release in autaptic munc18-1 null hippocampal neurons expressing M18Y473D, M18Y473D/P335A or wild-type Munc18-1 as control.(C) mEPSC frequency (M18WT: 8.23 ± 2.29 Hz, n=11; M18Y473D: 0.096 ± 0.056 Hz, n=8, M18Y473D/P335A: 2.18 ± 1.02 Hz, n=11, Kruskal-Wallis test, p = 0.2390), mEPSC amplitude (M18WT: 16.7 ± 1.0 pA, n=11; M18Y473D: 21.6 ± 5.2 pA, n=10, M18Y473D/P335A: 14.8 ± 1.2 pA, n=6, ANOVA, p = 0.1465) and decay time (M18WT: 2.53 ± 0.29 ms, n=11; M18Y473D: 3.79 ± 0.71 ms, n=10, M18Y473D/P335A: 2.85 ± 0.36 pA, n=6, Kruskal-Wallis test, p < 0.0003). Evoked and spontaneous release in autaptic munc18-1 null hippocampal neurons expressing M18Y473D, M18Y473D/P335A or wild-type Munc18-1 as control.(D) Depression of EPSC amplitude during a 10 Hz train. Inset shows first 4 pulses normalized to the first EPSC.
Figure 5Cortical extracts from rTg4510-Mapt0/0 brains had significantly less phospho-tau (CP13, PHF1, 12E8) than rTg4510 extracts. Compared to WT mice, both transgenic tau lines had high levels of phospho-tau. Mean±SEM, n=3 mice/group. Representative images of gallyas silver stained aggregated tau in cortices from 12-month-old mice unravel stunning differences in the degree of tau pathology in rTg4510 compared to rTg4510-Mapt0/0 mice. Mean±SEM, n=3 mice/group. Higher magnification images of silver (12 month-old) and Thioflavine-S (9month-old) stained cortices show mature tangles (white arrows in Thio-S stain) in rTg4510 and rTg4510-Mapt0/0 mice; enhanced pathological changes such as neuritic tau accumulation and neuropil vacuolation around NFTs are found only in rTg4510 mice. Stereological counting revealed similar numbers of cortical NFTs between in rTg4510 and rTg4510-Mapt0/0 mice at 9 and 12 month of age. Because of the pronounced neuronal death only in rTg4510 mice, the percentage of tangle-bearing neurons was ≈1.6 to 1.8-fold higher higher in 9 and 12-month old rTg4510 mice. Mean±SEM, n=3 sections/mouse, 3 mice/group. Immuno-FISH for huTau mRNA (green) and phospho-tau (PHF1, red) shows obvious differences in the distribution of neurons in cortex layer II/III: 9-month-old rTg4510 mice had more neurons filled with NFT-like phosphorylated tau (PHF1+, red), and rTg4510-Mapt0/0 mice had significantly more huTau mRNA positive neurons (FISH+). rTg4510-Mapt0/0 mice had also more neurons still expressing both PHF1 and huTau mRNA (PHF1+ FISH+), suggesting a reduced neurotoxicity of P301Ltau expression in rTg4510-Mapt0/0 mice. Mean±SEM, n=3 sections per mouse, 3 mice/group. For all panels: two-tailed Student's T-test and one-way ANOVA with Bonferroni for multiple comparison, ns not significant, scale bars 100 μm. Immuno-FISH showing EC neurons having both misfolded somatic tau (Alz50, red) and human tau mRNA (green; white arrows) in rTg4510-Mapt0/0 but rarely in rTg4510 mice. n=3 sections, 2 mice/group. Scale bars 50 μm.