EMBO/sd-character-level-ner huggingface dataset:
Figure 4A. Cells were treated with 273 kinase inhibitors (10 µM) from the Sellekchem library and with SA, and Sec24C localization in G3BP puncta (% of total G3BP area) was analyzed by automated microscopy under High content screening and is reported as a percentage of the control (cells treated with SA alone). Gray dashed line, mean of control; gray box, control standard deviation (± 18.2%); red line, threshold of positive hits; red box: positive hits. B. Enrichment Analysis of the positive hits; for each class of inhibitor, the enrichment (in percentage) of compounds falling into positive hits and the enrichment (in percentage) in the total number of compounds was calculated and expressed as a ratio. C. Evaluation of CDK kinase activity upon SA treatment. Western blot analysis of phosphoSer-CDK substrates in control, SA (300µM, 30 min), Dinaciclib (10 µM, 180 min) and SA+Dinaciclib (150 min Dinaciclib and 30 min SA). CDKs are hyperactivated upon oxidative stress and this activation is partially prevented upon CDK inhibition. Left, Western blot, right: Ponceau was used as loading control. Image, one representative experiment out of 3 independent replicates. D. Drug specificity from high (++++) to low (+) of the different CDKs as described by Selleckchem, and Sec24C recruitment to SGs as a percentage of control. E. Analysis of Sec24C recruitment to SGs in control and CDK1+2-KD HeLa cells. Different levels of CDK1+2 KD were achieved by transfecting cells with different amounts of siRNA for different times (2 nM 24 h; 5 nM 24 h; 5 nM 48 h; 20 nM 48 h) to give 50, 30, 20 and 10%, respectively, of the CDK1+2 levels in the control. CDK1+2 levels measured by qRT-PCR and Sec24C recruitment to SGs were evaluated from parallel cultures. Sec24C recruitment to SGs was performed by automated microscopy as described in (A) and is expressed as a percentage of mock (CTRL, set at 100%) ± s.d. N=8. F. Representative images of HeLa cells treated with the three best hits (Flavopiridol, SNS-032, Dinaciclib; 1µM 150 min) and then exposed to SA (300 µM, 30 min), followed by immunostaining for Sec24C and G3BP. Scale bar, 10 µm. G. HeLa cells were treated with Flavopiridol, SNS-032, or Dinaciclib for 150 min at the indicated concentrations, subsequently treated with SA (300 µM, 30 min), and then immunostained for Sec24C and G3BP and imaged by OPERA. BS-181, a specific CDK7 inhibitor, was used as negative control. Sec24C localization to SGs is expressed as a percentage of the control (cells treated with SA alone). Dashed red line, mean of control; yellow box, standard deviation of control (± 9.8%), N=12. H. Cells were treated as in (G) and immunostained for TRAPPC2 and eIF3. Scale bar, 10 µm. The graph shows the quantification of TRAPPC2 localization in SG puncta (mean intensity). Data are mean ± s.e.m. expressed as a percentage of TRAPPC2 signal in SGs after Flavopiridol, SNS-032 or Dinaciclib treatment compared to the control (cells treated with SA alone). N=3, three independent experiments, n=60-80 cells per experiment. ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.
Figure 7Schematic representation of the generation of chimeric embryos. Schematic representation of the induction of diapause and reactivation of chimeric embryos. E4.5 chimeric embryos stained for tdTomato and DAPI.   Quantification of donor cells' contribution in E4.5 blastocysts, Mann-Whitney test, data represent mean ± SD; Ronin fl/del n embryos = 26, Ronin del/del n embryos = 27.   Diapause embryos stained for tdTomato and DAPI.   Quantification of donor cells' contribution in diapause embryos, unpaired Student's t test, data represent mean ± SD, Ronin fl/del n embryos = 4, Ronin del/del n embryos = 12.   E5.5 chimeric embryos stained for tdTomato and DAPI.   Quantification of donor cells' contribution in E5.5 embryos, unpaired Student's t test, data represent mean ± SD, Ronin fl/del n embryos = 21, Ronin del/del n embryos = 15.   Reactivated E5.5 chimeric embryos stained for tdTomato and DAPI.   Quantification of donor cells' contribution in reactivated E5.5 embryos, Mann-Whitney test, data represent mean ± SD, Ronin fl/del n embryos = 17, Ronin del/del n embryos = 12.   E7.5 chimeric embryos stained for tdTomato and DAPI. Quantification of donor cells' contribution in E7.5 embryos, data represent mean ± SD, Ronin fl/del n embryos = 14, Ronin del/del n embryos = 8. E5.5 chimeric embryos stained for Podocalyxin, tdTomato, E-cad and DAPI. Ronin fl/del and Ronin del/del (day 4) cells grown in 3D culture conditions for 48 h and stained for apical polarity markers - Podocalyxin and Par6, as well as Phalloidin, E-cad and tdTomato. Quantification of Par6 positive and negative ESC clumps of Ronin fl/del (n = 44) and Ronin del/del (n = 13) cells grown in 3D culture conditions for 48 h.
Figure 7A, B Indicated VHH antibodies (1 µM) were incubated at room temperature or at 90°C for 5 min and centrifuged for 20 min at 20 000 g. The supernatants were diluted 50-fold (20 nM) and analyzed for RBD-binding by BLI. C Re5D06 produced in the indicated expression host was analyzed by differential scanning fluorimetry (DSF) with a temperature range from 20 to 95ºC (in the presence of SYBR Orange). Unfolding is measured as an increase in fluorescence (caused by the exposure of hydrophobic residues that bind the dye). Melting temperatures are defined as the inflection point of the melting curve before reaching the first melting peak. Stable proteins produce no melting peak. D The left two panels show surface representations of Re5D06 (magenta) and the thermostable variant R28 (yellow). The ribbon diagram (right) depicts an overlay of both nanobodies with changed hydrophobic core side chains shown as sticks. Note that these mutations close a large cavity originally present in Re5D06. E Thermostability of the Re5D06 variants R15 and R28, measured as in C. F Neutralization of SARS-CoV-2 by the thermostable Re5D06 variants R15 and R28 at the indicated concentrations. The neutralization experiment was performed G DSF measurements of additional hyperthermostable VHHs as in C.
Figure 7(A) Immunofluorescence analysis of spine in PGIS mice. Quantitative analysis of CaSR+ p-p65+ cells and CaSR+ p-Stat3+ cells number in pathological new bone formation of PGIS model. n = 5, one‐way ANOVA, Bonferroni post hoc. Data information: AF: Annulus fibrosus; GP: Growth plate; NB: New bone (B) Immunofluorescence analysis of pathological new bone of hind paws in DBA/1 mice. Quantitative analysis of CaSR+ p-p65+ cells and CaSR+ p-Stat3+ cells number in pathological new bone formation sites. n = 9, one‐way ANOVA, Bonferroni post hoc. CB: Cortical bone; NB: New bone (C) Immunofluorescence analysis of Achilles tendon enthesis compartment in SMTS model. Quantitative analysis of CaSR+ p-p65+ cells and CaSR+ p-stat3+ cells number in PCT. n=5, Student's t-test. UF: Uncalcified fibrocartilage; CF: Calcified fibrocartilage (D) Schematic presentation of multiple inflammatory cytokines from inflammatory cell up-regulate hyperstimulation of CaSR through NF-κB/p65 and JAK/Stat3 pathways. Hyperstimulation of CaSR induce osteogenic differentiation through PLCγ signalling pathway in osteogenic precursor cell to promote pathological new bone formation in AS.
Osteoblast-specific overexpression of the EGFR ligand Amphiregulin accelerates tumor formation in H2-c-fosLTR mice.Figure 5X-ray analysis of 6-months-old H2-c-fosLTR and H2-c-fosLTR/ColAREG littermates. Scale bars: 1cm Bone tumor number per mouse at 5-6 months of age (n=22 wt, 13 ColAREG) Quantification of tumor size in tibiae at 5-6 months of age (n=22 wt, 13 ColAREG) Alkaline Phosphatase (ALP) levels in the serum at endpoint (age = 5-6 months; n=29 wt, 19 ColAREG) c-fos mRNA expression levels in osteosarcomas of H2-c-fosLTR (n=11) and H2-c-fosLTR/ColAREG mice (n=14) Western Blot analysis of bone tumor protein lysates from H2-c-fosLTR and H2-c-fosLTR/ColAREG mice
chd7 regulates GABAergic neuron development via MAPK/ERK signalling.Figure 3A. Volcano plot showing each gene plotted according to its log2 fold change. All highly differentially expressed genes with p<0.05 are in orange with fold-change>1.5. Biological processes that are enriched in the differentially expressed genes. Molecular function that are enriched in the differentially expressed genes. D. ERK activation by phosphorylation is increased in mutants as shown by Western blot quantification (N=4; ***p<0.005; Student's t-test). E. pERK immunohistochemistry showing increased ERK activation in the mutant brain (N=3, n=8). F-G. Treatment with the ERK signalling inhibitor U0126 ameliorated the number of GABAergic neuron (n=10-15; **p<0.05; ****p<0.0001, one-way ANOVA). H. Treatment with the ERK signalling inhibitor U0126 rescues locomotor hyperactivity phenotype in chd7 mutant fish (n=8 for chd7+/+ and chd7-/-; n=16 for U0126 treated fish; ****p<0.0001 ; ns, not significant, one-way ANOVA).
Figure 8A. PHSL1-CDC20 ama1∆ cdc28-as2 cells were treated with 1Na-PP1 at t = 3 h in SPM to inhibit Cdc28-as2. At t = 5 h (left) or t = 9 h (right), Cdc28-as2 was activated by washing cells with conditioned SPM (cSPM) lacking 1Na-PP1 (washout). Top, time-lapse series from the imaging of Mpc70-GFP and RFP-tubulin. The weak, nuclear signal from Ndt80-GFP serves to confirm entry into meiosis. Middle, quantification of spindle formation and Mpc70 loading to SPBs. Bottom, quantification of control experiments. Cells were washed with cSPM plus 1Na-PP1 at t = 5 h (left) or t = 9 h (right) in SPM. B. TEM analysis of SPBs. PHSL1-CDC20 ama1∆ cdc28-as2 cells were treated with 1Na-PP1 at 3 h in SPM. At 9 h in SPM, cells were washed with cSPM containing (top) or lacking 1Na-PP1 (washout, bottom). TEM samples were collected at 13 h in SPM. Washout of 1Na-PP1 causes MP formation (P < 0.0001, Fisher's exact test). Scale bar, 0.2 μm. C. PHSL1-CDC20 ama1∆ cdc28-as2 control cells and cells containing spo13∆ or cdc5-as were treated with 1Na-PP1 at t = 3 h in SPM to inhibit Cdc28-as2. At t = 9 h in SPM, Cdc28-as2 was activated by washing cells with cSPM lacking 1Na-PP1 (washout). cdc5-as cells were washed with cSPM plus CMK to activate Cdc28-as2 and inhibit Cdc5-as. Top, time-lapse series from the imaging of Mpc70-GFP and RFP-tubulin. The weak, nuclear signal originates from Ndt80-GFP. Bottom, quantification of spindle formation and Mpc70 loading to SPBs. D. PHSL1-CDC20 ama1∆ cells containing SPC72, spc72-7, or spc72-7 plus cdc5-as were shifted from 24 to 36°C at t = 4.2 h in SPM (to inactivate Spc72-7, arrow heads) and treated with CMK at t = 6 h in SPM (to inhibit Cdc5-as). Top, time-lapse series from the imaging of SPBs (Cnm67-RFP) and Mpc70-GFP. Bottom, quantification of SPB separation and Mpc70 loading to SPBs. Inhibition of Cdc5-as in spc72-7 cells advances the time of Mpc70 loading by 119 min (95% CI, 76-162; P < 0.0001; Welch's t-test). Data are representative of two independent experiments.
Figure 2A) For each population (CD38- and CD33+ cells), Z stacks of two representative cells are shown in the left panel. Microtubules are shown in green, actin in magenta, centrosome in white and chromatin in blue. In the middle panel, inverted image the corresponding actin staining is shown. In the left panel, the corresponding 3D projection of microtubules is presented as inverted image. Scale bar 5 µm. B) Microtubules are tightly associated with the nucleus. Z stacks of representative CD38- and CD33+ cells are shown. Microtubules are shown in green, nucleus envelope (Sun2 immuno-staining) in magenta and chromatin in blue. Scale bar 5 µm. C) As schematized, centrosome-to-nucleus center distance (d) over nucleus center-to-nucleus convex envelope (R) ratio is calculated to extract relative position of the centrosome to the center. This parameter is significantly lower in myeloid progenitors (n=129) than in HSPC (n=200; 3 independent experiments; ****; p<0.0001. Mann-Whitney test) indicating that centrosome becomes internalized within an invaginated region upon differentiation. D) p150 glued associates with the nuclear membrane of HSPC (CD38-) and myeloid progenitors (CD33+) cells. Z stacks of representative cells are shown. Image contrast has been inverted. Stainings show DAPI, Sun2 and p150 glued . Scale bar 5µm. E) The dynein adaptor BicD2 associates with the nuclear membrane of HSPC (CD38-) and myeloid progenitors (CD33+) cells. Z stacks of representative cells are shown. Image contrast has been inverted. Stainings show DAPI and BicD2. Scale bar 5µm.
Figure 1Figure 1. Sample and analysis flowchartAll samples from TB patients and healthy controls were quality controlled and randomly assigned into a training and test set. Built classification models were tested in both the test set and the total dataset. External validation was performed on two independent blinded RT-PCR datasets, as well as on external microarray datasets.
Figure 3A. HEK293T cells were transfected with expression constructs encoding MYC-FOXP3, His-Ubiquitin, and either a wild type or catalytically inactive (C282S, "cs") version of USP44 tagged with FLAG. Transfected cells were treated with 20 μM MG132 for 4 hrs and subsequently harvested for cell lysis. Pull-down of His-labeled proteins using Ni-NTA beads allowed recovery of ubiquitinated FOXP3 species that were then visualized by immunobot probing with antibodies specific for MYC. B. Effect of USP44 knock down on FOXP3 ubiquitination. Two distinct shRNA lentiviral constructs each containing a G418 resistance cassette were delivered into HEK293T cells. After selected for 7d, cells were transfected with MYC-FOXP3 and His-Ubiquitin, then treated with 20 μM MG132 for 4 hrs before harvest and lysis. Ubiquitinated FOXP3 proteins were visualized as in A. C. Levels of polyubiquitinated FOXP3 in the presence or absence of USP44. As in Fig 2b, murine iTregs were generated from naïve CD4+ precursors isolated from wild type and mice globally deficient in USP44-/- mice (n=3/group/experiment). Ubiquitinated proteins were extracted from iTreg lysates with anti-ubiquitin antibodies (anti-Ubi) prior to resolution by SDS PAGE and immunoblot analysis, probing for FOXP3. D, E. HEK293T cells were transfected with plasmids encoding MYC-FOXP3, FLAG-USP44 (either wild type USP44, "wt" or the C282S mutant). These cells were subsequently treated with cycloheximide (CHX; 5μg/ml) for the indicated time points before harvested and cell lysis. FOXP3 levels and the relative turnover rate of this factor was determined by immunoblotting analysis with anti-MYC antibodies. F, USP44 knock down in human Tregs. Naïve CD4+ T cells (CD4+CD25-CD45RA+) were isolated from the peripheral blood of healthy donors by FACS, and iTreg were generated after 7 days of in vitro skewing conditions. As in Fig. 3b, shRNA constructs targeting USP44 were delivered by lentivirus. Here human iTregs received either shCK (control), or one of two different shUSP44 constructs. Endogenous FOXP3 and USP44 protein levels were visualized by immunoblot (F) G. USP44 knock down in human Tregs. Naïve CD4+ T cells (CD4+CD25-CD45RA+) were isolated from the peripheral blood of healthy donors by FACS, and iTreg were generated after 7 days of in vitro skewing conditions. As in Fig. 3b, shRNA constructs targeting USP44 were delivered by lentivirus. Here human iTregs received either shCK (control), or one of two different shUSP44 constructs. FOXP3 expression was assessed by intracellular staining followed by flow cytometry (G). H. Murine naïve CD4 T cells (CD62L+/CD25-/CD4+) from WT and USP44-/- mice (n=2-3/group/experiment) were FACS purified and activated in vitro for four days by CD3/CD28 cross-linking antibodies (1ug and 4ug/ml, respectively) in the presence of 100 U/ml IL-2 and the indicated dose of TGF-β. Down-regulation of FOXP3 was observed in USP44-/- mice by intracellular immunostaining and flow cytometry.
Figure 2A. The per million mapped reads (RPKM) values (extracted from GSE36552) showing the MOS expression level in human oocytes and early embryos (n= over 3 biological replicates). B. The immunofluorescence images show the pERK1/2 (red) dynamics in immature (GV, n=5), mature (MII, n=10), and fertilized oocytes from unidentified control patients (n=6), as well as in mature oocytes from patient 1 carrying homozygous MOSAsn95Lys variants. pERK1/2 activation was inhibited after U0126 treatment (n=6). FITC-α-tubulin (Green) and DAPI (blue) were used for co-staining. Scale bar=10 µm. C. Western blot analysis of the MEK1/2 and ERK1/2 activation after transfection of FLAG-tagged wild-type MOS and identified MOS variants in HEK 293 cells. D. Statistical analysis of the protein expression level of FLAG, pERK1/2 and pMEK1/2 (n=2 technical replicates). The protein intensities were quantified by Image J software. E. Diagram showing the experimental design, including procedure of MOS mRNAs injection and oocyte culture. GV oocytes were overexpressed via microinjection with wild-type MOS or MOS variant mRNAs combined with mCherry mRNAs, followed by culture for 4 h in medium containing 2.5 µM milrinone. F. Immunofluorescence of FLAG (green) and pERK1/2 (grey) in oocytes after injection of different MOS variant mRNAs combined with mCherry mRNAs. The signal of mCherry was directly captured after fixation, with DAPI staining for DNA visualization (blue). (n=over 30 oocytes in each group). Scale bar=10 µm.
Figure 8Diagram of the Sgs1 acidic patch (AP) truncations used in this study. Histogram showing abundance of Sgs1 peptides in Dpb11-HA co-immunoprecipitation mass spectrometry experiments from cells expressing either SGS1 acidic patch truncation mutants or wild type SGS1.. Each mutant was compared in a SILAC experiment against the wild-type control. Error bars represent SEM of 2 or more Sgs1 peptide-spectrum matches (PSMs). Serial dilution assay of sgs1Δ cell lines expressing either SGS1 or sgs1ΔACIDICPATCH. BIR assay in rad9Δ sgs1Δ cell lines expressing either SGS1 or sgs1ΔACIDICPATCH. Error bars represent SEM of 3 independent experiments. Error bars represent SEM of at least 3 replicate experiments. P value was calculated with a two-tailed, unpaired t-test. * = p≤0.05; ** = p≤0.01; *** = p ≤0.001.
Figure 2A: mRNA analysis of expression of endothelial (Tie2, CD31, Cdh5), epithelial (Cdh1), and early hematopoietic (Scl, CD41) marker genes in the indicated sorted cell populations. Error bars represent standard deviation (SD), n=3. Student's t-test. *p<0.05. B: Confirmation of the purity of sorted populations by qRT-PCR: c-Kit is highly expressed only in the c-Kit+ (D20LSK and D20LK) populations, and Sca1 is highly expressed only in the in Sca1+ (D20LSK and D20LS) populations. Error bars represent standard deviation (SD), n=3. Student's t-test. *p<0.05. C: qRT-PCR analysis of Gata1, Gata2, Lyz2, and Scl expression in the sorted cell populations on day 20. Error bars represent standard deviation (SD), n=3. Student's t-test. *p<0.05. D: FACS analysis of ESC-derived cells confirming that D20LS, D20LK, and D20LSK cell populations express the early hematopoietic marker CD41 and the pan-hematopoietic marker CD45.
Impaired autophagic substrate degradation in IBMPFD mouse muscle.Figure 7.(A) Representative images using in vivo bioluminescence of control or IBMPFD mutant RH-expressing mice (RH12) 2 d after electroporation (baseline) or after 24 h of nutrient deprivation (starvation) of polyQ80 (Q80)- or polyQ19-luciferase (Q19) in the right and left tibialis anterior, respectively. The ratio of polyQ80-luciferase/polyQ19-luciferase is indicated below each image set. Values are in ×104 photons per second per square centimeter per steradian. (B) Box and whisker plot of the change (Δ) in the ratio of polyQ80-luciferase/polyQ19-luciferase activity in the left and right tibialis anterior muscle of control, VCP-WT, or one of two VCP-RH (RH12 or RH9) transgenic mouse lines after 24 h of starvation. The graph is representative of three animals per group. The p-value for RH12 was 0.05 and 0.06 when compared with control animals or VCP-WT transgenic. The p-value for RH9 was 0.03 when compared with either control or VCP-WT transgenic. The p-value was 0.01 and 0.02 when combined RH12 and RH9 animals were compared with control or VCP-WT groups, respectively. There was no statistical difference between control or VCP-WT groups.
Mapping of SARS-CoV neutralizing antibody epitopes.Fig. 4 Mapping of SARS-CoV neutralizing antibody epitopes.The epitopes of SARS-CoV neutralizing antibodies m396 and 80R, which target the RBD, are labeled in the SARS-CoV sequence aligned with the sequence of 2019-nCoV RBD. Epitope residues of m396 are indicated by black dots; epitope residues of 80R are indicated by red dots.
Figure 5Overview of the known molecular characteristics of totipotent and pluripotent cells in vivo and in vitro, now including metabolic features.
Figure 5(A) Anti-CHIKV IgG titers from the vaccination phase were determined with a CHIKV virion-based ELISA. Sera from four animals were pooled and diluted 1:500 for the assay. Statistical analyses were performed using two-tailed paired T-test (*P =0.029 RH-CHIKV 6 dpv, *P =0.029 RH-CHIKV 90 dpv, *P =0.029 RHEV-CHIKV 6 dpv, *P =0.029 RHEV-CHIKV 90 dpv, *P =0.029 EV-CHIKV 90 dpv). (B) Half maximum inhibitory concentration (IC50) of pooled sera collected on 90 dpv were characterized in HEK293T cells infected with WT-CHIKV (MOI 5). Sera were serially diluted at 1:625000, 1:125000, 1:25000, 1:16000, 1:8000, 1:5000, 1:4000, 1:2000, 1:1000, 1:500, 1:250. Sera dilution to obtain IC50 are shown in parenthesis. (C) WT C57BL/6 mice were vaccinated subcutaneously with 1E6 PFU of WT-CHIKV or CHIKV nsP-mutants (RH, RHEV and EV) at the metatarsal region of the footpad. Challenge with 1E6 PFU of WT-CHIKV was performed via the same route at 90 dpv. Viremia were monitored over 2 weeks. Viremia is detected with CHIKV nsP1 probe via qRT-PCR. (E) severity of joint inflammation were monitored over 2 weeks.