EMBO/sd-character-level-ner huggingface dataset:
a) Top: schematic representation of IRGM.figf5(a) Top: schematic representation of IRGM. The G1-G5 GTPase motifs and their sequences are shown. The change corresponding to the S47N mutation is indicated with an arrow. Bottom: splice variants of IRGM and Ras (p21). (b) HeLa cells were transfected with plasmids encoding fluorescent protein-tagged IRGMb, IRGMd WT (wild-type IRGMd) or IRGMS47N mutant (control cells; vector expressing GFP only), and after 48 h were labelled with MTR and imaged by live-cell confocal microscopy. Scale bars, 5 μm. (c) Fluorescence intensity analysis of green and red channels along a line drawn through two adjacent cells, one GFP-IRGMd positive and one GFP-IRGMd negative, as indicated in the inset. (d) Quantification of GFP+ MTR+ cells from an experiment performed as in b. (e) Nitrocellulose filters spotted with the indicated lipids were incubated with GST or GST-IRGM. Binding was detected by immunoblotting. (f) Agarose beads (control beads) and cardiolipin-bound agarose beads (CL beads) were incubated with GST or GST-IRGMd. Proteins were eluted from the beads and immunoblotted. Two lanes on the left indicate immunoblotting of samples used to incubate beads. (g) Nitrocellulose filters spotted with the indicated lipids (defined in e) were incubated with wild-type IRGMd, IRGMdS47N, and the IRGM isoform IRGMb at the indicated concentrations. GST control is shown in Supplementary Information, Figure S1d. Membranes in e-g were probed with anti-GST antibody.
Slug inhibition reduces vascular remodeling and proliferation of vascular wall cells.Figure 8(A) Representative images and quantification of Slug-positive macrophages (slug, red; CD68 green). (B) Relative expression of CD68 mRNA normalized to GAPDH. (C) Representative images and quantification of Slug-positive macrophages between non-fibrotic and fibrotic areas of the lung. Relative expression of PIP mRNA (D) normalized to GAPDH. Relative expression of PIP protein (E) normalized to GAPDH. (F) Representative images and quantification of PIP immunohistochemistry. (G) Proposed mechanism. In pulmonary fibrosis, vascular wall thickening is mainly seen in fibrotic areas of the lung; while in PH secondary to PF, vascular remodeling is seen both in fibrotic and non-fibrotic areas of the lung. The extension of vascular wall thickening to the non-fibrotic areas of the lung in PF-PH is concomitant with an increased expression of Slug and PIP in the lungs in both fibrotic and non-fibrotic areas. At the cellular level, Slug up-regulation in macrophages in PF-PH leads to increased PIP expression in the extra-cellular space, which in turn triggers SMC proliferation leading to vascular wall thickening observed in fibrotic and non-fibrotic areas of the lung. (ECM: Extracellular Matrix; SMC / EC : smooth muscle cells and endothelial cells). The number of samples per group for each experiment is included within each bar graph.
Figure 6(A) Larval NSCs of control (ctrl; yw) and cdk1B47 at 24h ALH were labelled for Earthbound1 (Ebd1), Deadpan (Dpn), and Miranda (Mira). (B) Quantifications of the corrected total cell fluorescence (CCF) of Ebd1 in genotypes in (A). CCF = Integrated Density - (Area of selected cell × Mean fluorescence of background readings). CCFs were 846.9±394.2 for control (ctrl; yw; n=167, 14 BL) and 354.3±245.3 for cdk1B47 (n=211, 13 BL). (C, D) Larval NSCs of control (UAS-dicer2) and UAS-cdk1-Myc induced with insc-Gal4 at 6h ALH were labelled for Ebd1, Dpn, and Mira. (D) Quantifications of the CCF of Ebd1 in genotypes in (C). CCFs were 111.3±80.2 for control (n=157, 12 BL) and 346.6±221.1 for cdk1-Myc (n=181, 12 BL). Data information: Data are presented in (B, D, as mean ± SD. n numbers in (B, D, were total NSCs counted with total brain lobes measured, while each NSC was considered as one biological replicate. White arrows point to NSCs (A, C, Yellow dotted boxes indicate the region of zoomed-in images (A, C, Statistical analyses were done comparing between two different genotypes using Mann-Whitney test (B, D) Scale bars, 15 μm (A, C, (E) qPCR analysis of cdk1 in larval brains of control (ctrl; yw) and ebd1240 at 24h ALH. n=3 as biological replicates. Statistical analyses were done comparing control (GAPDH1, GAPDH2, Tbp1 and Rpl32) and ebd1240, using two-tailed Student's t-test. The mRNA levels of control were normalized to 1. Data information: Statistical analyses were done comparing between two different genotypes using two-tailed Student's t-test (E). Larval NSCs of control (ctrl; yw), ebd1240 and pr-set7716/Df(3R) ED5644 (716/Df) at 24h ALH were stained for Senseless (Sens), Dpn and Mira. (F) Data information: White arrows point to NSCs F). Yellow dotted boxes indicate the region of zoomed-in images F). Scale bars, 15 μm F). G) Larval NSCs of control (ctrl; yw), Quantifications of the CCF in genotypes in (G). CCFs were 1349.7±792.5 for control (n=166, 13 BL), 505.8±395.9 for ebd1240 (n=206, 14 BL) and 859.7±619.0 for 716/Df (n=174, 13 BL). Data information: Data are presented in G) as mean ± SD. n numbers in G) were total NSCs counted with total brain lobes measured, while each NSC was considered as one biological replicate. Statistical analyses were done comparing among different genotypes using ordinary one-way ANOVA (G). *P < 0.05; ***P < 0.001. (H) A working model. Pr-Set7 promotes NSC reactivation by positively regulating Cdk1 and Wingless pathway transcriptional co-activator Ebd1 in NSCs. Cdk1 upregulates Ebd1 in NSCs, while Ebd1 may maintain an equilibrium of Cdk1 level during reactivation. Data information:
a, b) dRagC is not required for Rheb-induced cell growth.figf6(a, b) dRagC is not required for Rheb-induced cell growth. (a) The area of Rheb-overexpressing cells in control or dRagC mutant (dRagC−/−) backgrounds under fed conditions, relative to that of neighbouring control cells which were assigned a value of 1. Overexpression of Rheb led to a significant increase in cell area in both control and dRagC mutant backgrounds. Data are mean ± s.d., *P = 0.034 (n = 5), **P = 6.1 × 10−3 (n = 5), Student's two-tailed t-test (n represents number of experimental samples). (b) A representative example of a clone of Rheb-overexpressing cells in a dRagC−/− animal. Rheb transgene-expressing cells are marked by co-expression of GFP. Cell boundaries are labelled by phalloidin staining in red; nuclei are labelled by DAPI in blue. Scale bar represents 50 μm. (c, d) Expression of dRagAQ61L fails to rescue the growth impairment of Rheb mutant cells. (c) Relative area of clonally-induced Rheb26.2 homozygous mutant cells in a control background and in animals expressing dRagAQ61L throughout the fat body. Clonally induced Rheb26.2 homozygous mutant cells were significantly smaller than neighbouring control cells both in wild-type and in dRagAQ61L expressing backgrounds. Data are mean ±s.d., *P = 2.91 × 10−4 (n = 7), **P = 2.59 × 10−8 (n = 5), Student's two-tailed t-test (fed conditions where n represents number of experimental samples). (d) A representative example of Rheb homozygous mutant cells (marked by lack of GFP, arrows) in fat body ubiquitously expressing UAS-dRagAQ61L. GFP-positive control cells in this experiment are a mixture of Rheb+/− and Rheb+/+. Scale bar represents 50 μm.
Schematic model of the disruption of T cell help by HIV-1 NEF.Figure 7(A) Undisturbed physiological reactions in the absence of Nef. (B) Effects in the context of HIV-1 infection due to expression of NEF.
Figure 6 Mice were superinfected as described in Fig 3A (A) Frequency of IFNγ+ and IL-17+ immune cells following intracellular cytokine staining and flow cytometry Data information: Data are representative of at least two independent experiments with 3 mice per group. All data are represented as mean ± S.E.M. Statistical significanc was determined by unpaired student's T-test  Mice were superinfected as described in Fig 3A (B) Representative flow cytometry plots of total and IL-17+ lymphocyte subsets on day 3 post-secondary bacterial infected lungs following intracellular cytokine staining. Numbers in plots indicate the frequency of each IL-17+ cell subset over total numbers of respective cell-types Data information: Data are representative of at least two independent experiments with 3 mice per group. All data are represented as mean ± S.E.M  Mice were superinfected as described in Fig 3A (C) Frequency over total IL-17+ cells and absolute numbers of each IL-17 producing lymphocyte subset on days 0, 1 and 3 post-secondary bacterial superinfected lungs as assessed by flow cytometry of intracellular cytokine stained cells Data information: Data are representative of at least two independent experiments with 3 mice per group. All data are represented as mean ± S.E.M. Statistical significanc i (C) was determined by unpaired student's T-test
Figure 1T cells were collected from age- and gender-matched WT, EGR1KO and EGR4KO mice. (A) CD4+ T cells were isolated from the spleen by negative selection before incubating with anti-CD3/CD28 antibodies for the indicated time periods. Levels of expression of EGR1, EGR2, EGR3 and EGR4 were measured by qPCR. T cells were collected from age- and gender-matched WT, EGR1KO and EGR4KO mice. (B) WBC were collected from WT, EGR1KO and EGR4KO mice before staining and FACS analysis to determine composition (gating strategies in Appendix Fig S1). Subclasses of CD8+, CD4+ and Treg populations were determined as shown.
Apoptosis and autophagy are activated in a developmentally regulated manner in MCL‐1‐null animals.Figure 7(A) Brains from E12.5 Foxg1 Cre MCL‐1 animals were analysed by western blot for the presence of the caspase‐cleaved fragment of the caspase‐3 substrate p130CAS and the autophagic markers LC3 and p62. (B) Autophagy in Foxg1 Cre MCL‐1Δ/Δ mice. Brain sections from E15.5 animals with the indicated genotypes were stained for LC3 (green). Confocal images were taken using × 20 (bottom panel) or × 63 (top and middle panels) objectives. Scale bars=50 μm. (C) Colocalization of LC3 vesicles with the lysosomal marker LAMP1 was analysed by immunohistochemistry in E15.5 wild‐type embryos. Data are expressed as the percentage of LC3 vesicles that are LAMP1‐positive±s.d. About 100 LC3 vesicles were counted per section, with four sections per embryo. VZ, ventricular zone. (D) Primary cortical neurons and neuronal progenitor cells were analysed for the presence of the indicated apoptosis (BIM, Puma) and autophagy (ATG7, ATG5‐ATG12) related proteins as well as Tuj‐1 (neuronal marker), Nestin (neural progenitor marker) and HSC70 (loading control). (E) Primary neurospheres were treated for 8 h with EBSS and analysed by western blot for the presence of MCL‐1 and actin as a loading control. (F) Primary neurons and neurospheres were treated as in E and analysed by western blot for p130CAS, LC3 and actin as a loading control. Alternatively, they were analysed by immunofluorescence for c release (G) in the presence of 50 μM zVAD‐FMK to prevent apoptotic cells from detaching from the coverslip. Data are expressed as the average of 3-7 embryos±s.d. *P0.001.
– Lysosomal function inhibition affecting TLR8 activity is anti-inflammatory upon both, Gram-positive and Gram-negative bacterial infection ex vivo.Figure 4A Cytokine release of 3 days differentiated THP-1 cells of indicated genotypes upon TLR2 blockade (T2.5) and challenge with TLR ligands, heat inactivated (hi) or viable bacteria. B PBMC activity upon endosome function inhibition (chloroquine) and TLR2 blockade (T2.5) followed by TLR ligand challenge or bacterial infection. C Activity of whole blood culture upon endosome function inhibition (chloroquine) and challenge with TLR ligands, heat inactivated (hi), or viable bacteria.
Figure 5SH-SY5Y cells in the presence of 5 μM saracatinib, 10 μM SKI-1 were incubated with 1 μM α-syn fibrils for 30 min and these cells were further incubated with 50 nM BODIPY FL C5-LacCer and 2.5 μg/ml rhodamine-conjugated transferrin for 20 min. The cells were fixed and observed under confocal microscopy. The intensity was analyzed. Values were derived from five independent experiments (n=5). *** P< 0.001 compared to control, one-way ANOVA. B. NT, c-src KD #1 or c-src KD #2 SH-SY5Y cells were incubated with 1 μM α-syn fibrils for 30 min and these cells were further incubated with 50 nM BODIPY FL C5-LacCer and 2.5 μg/ml rhodamine-conjugated transferrin for 20 min. The cells were fixed and observed under confocal microscopy. The intensity was analyzed. Values were derived from five independent experiments (n=5). *** P< 0.001 compared to control, one-way ANOVA.
Figure 9Schematic representation of the proposed temporal regulation of BCL11B expression during critical stages of human T cell development as explained in more detail in the discussion of the manuscript.
Figure 5Immunofluorescence showing the Ago3 signals (green) in Siwi-depleted BmN4 cells (Siwi KD) and Siwi KD cells where Flag-Siwi (red) was expressed. DAPI shows the nuclei (blue). Scale bar: 10 μm. The panels on the right-hand side show high magnification images of the part indicated by the white dashed line box in the images on the left-hand side. This arrangement applies also for panel C. Bar chart indicates dot sizes of > 100 Ago3 particles in each case (Large: IntDen value > 105; Small: IntDen value < 105). A significant difference was found plus/minus Flag-Siwi expression by the Fisher's exact test (P < 0.05). Western blots show that the degree of Ago3 phosphorylation was reduced upon Siwi KA mutant as well as Siwi WT and DDH mutant expression. Immunofluorescence showing the Ago3 signals (green) in Siwi-depleted BmN4 cells (Siwi KD) and Siwi KD cells where Flag-Siwi KA mutant (red) was expressed. DAPI shows the nuclei (blue). Scale bar: 10 μm. Bar chart indicates dot sizes of > 100 Ago3 particles in each case (Large: IntDen value > 105; Small: IntDen value < 105). A significant difference was found plus/minus Flag-Siwi expression by the Fisher's exact test (P < 0.05).
Atg34 phosphorylation on homologous sites is required for Ams1 transport to the vacuoleComparison of Atg19 and Atg34 domain structures and C‐terminal amino acid sequences.Figure 3A Comparison of Atg19 and Atg34 domain structures and C‐terminal amino acid sequences. Green: Atg11 binding region. Serine residues S390, S391, and S396, which are phosphorylated in Atg19, and the conserved sites in Atg34 are shown enlarged. B atg34Δ yeast cells containing HTB‐Atg34 were grown to mid‐log phase and treated with rapamycin. Atg34 was affinity purified and subjected to mass spectrometric phosphorylation mapping. Phosphorylation sites: enlarged; Atg11 binding region: green; lysine substitution: gray. C Ams1‐GFP atg19Δatg34Δ cells containing protein A (Stag)‐tagged Atg34 as indicated were starved for 4 h. Processing of endogenous Ams1‐GFP was analyzed by Western blotting and quantified by calculating the ratio of free GFP versus uncleaved Ams1‐GFP normalized to the wild‐type. Expression of Atg34 proteins was assessed by anti‐protein A Western blotting. D Alignment of the Atg1 consensus sites in the Atg11 binding region of Atg19 and Atg34 with the known Atg1 phosphorylation sites of Atg9 and Atg2. E GST, GST‐Atg19, and GST‐Atg34 were purified from Escherichia coli and in vitro phosphorylated with immunoprecipitated Atg1‐TAP bound to IgG magnetic beads. F In vitro phosphorylation of recombinant Atg19, Atg19‐3A, and Atg19‐3D using wild‐type and kinase‐dead Atg1‐D211A as described in (E).
Figure 3(A) RIP assay in P493-6 cells treated with Tet for 0h or 72h. HPRT1 serves as negative control. *** P<0.001 relative to indicating groups (mean ± SD, n = 3 biological replicates, Student's t‐test). (B, Western blot analysis for protein levels in P493-6 cells that expressed NTC or YTHDF3 shRNAs and were then treated with Tet or not (B) HPRT1 and β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments. C) Western blot analysis for protein levels in Raji cells that expressed NTC or YTHDF3 shRNAs and knocked down MYC or not (C). HPRT1 and β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments. (D) Polysomes profiling assay in P493-6 cells that expressed NTC or YTHDF3 shRNA. (E) RT-qPCR assay for mRNA levels of MRGs (SPI1 and PHF12) in different fractions in (D). HPRT1 serves as negative control. Data were presented as mean (±SD), n = 3 biological replicates. ** P<0.01 and *** P<0.001 as compared to NTC group (Student's t‐test). (F) Click-iT AHA (L-azidohomoalaine) experiments were performed using IgG, anti-SPI1 or anti-PHF12 antibody. P493-6 cells expressing NTC or YTHDF3 shRNAs were incubated for 1 hour in medium containing 100 ug/mL AHA. The translated proteins were detected by Western blot. Arrow indicates translated MRGs. (G Western blot analysis for protein levels in P493-6 cells (G) β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments. H) Western blot analysis for protein levels in Raji cells (H) that expressed NTC, or ALKBH5 shRNA, or YTHDF3 shRNA. β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments. (I) m6A RIP assay HPRT1 serves as negative control. *** P<0.001 as compared between indicated groups, ns, not significant (mean ± SD, n = 3 biological replicates, Student's t‐test).
associates to telomeres in a TERRA-dependent manner A) Telomere interactors were identified and plotted as a volcano plot as in A, B and using tlc1 protein extracts.Figure 2A) Telomere interactors were identified and plotted as a volcano plot as in Figure 1 A, B and using tlc1 protein extracts. Interactors were identified using untreated protein extracts from tlc1 cells (left panel) or digested with RNase A and RNase H (right panel). B) Npl3 associates to TERRA. Cross-linked samples from the indicated strains were subjected to Npl3-TAP RIP. Enrichment of the indicated RNAs was determined by quantitative PCR on pulled-down reverse-transcribed RNA. Data represents mean % input +/- SEM n=3 (unpaired t-test two tailed *p<0.05, **p<0.01). Npl3 associates strongly to telomeres in the absence of Rat1. C) The Rat1-AID tagged protein is strongly degraded following 1h of 1mM IAA treatment. Protein levels are determined using anti-FLAG (Rat1-AID) and anti-actin antibodies. Abbreviations: IAA: indole-3-acetic acid, AID: Auxin inducible degron. Npl3 associates strongly to telomeres in the absence of Rat1. D) TERRA levels increase in cells expressing the Rat1-AID variant after 1h treatment with 1mM IAA. TERRA levels were determined by quantitative PCR on reverse-transcribed RNAs after phenol-chloroform extraction. RNA levels are normalized to 7S ncRNA levels. Data represents fold increase to corresponding -IAA samples n=2. Abbreviations: IAA: indole-3-acetic acid, AID: Auxin inducible degron. Npl3 associates strongly to telomeres in the absence of Rat1. E) Npl3-TAP associates strongly to telomeres after 1mM IAA treatment in cells with Rat1-AID. Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM n=3 (paired t-test one tailed *p<0.05, **p<0.01). Abbreviations: IAA: indole-3-acetic acid, AID: Auxin inducible degron. F-G) Npl3 associates to telomeres in an R-loop dependent manner. E) RNH1 was overexpressed from a galactose-inducible promoter. Protein levels are determined using anti-HA and anti-actin antibodies. F) Npl3-TAP associates to telomeres in an R-loop-dependent manner. Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM n=3 (paired t-test one tailed *p<0.05, **p<0.01). Abbreviations: eV: empty vector control, oE: overexpression H) Npl3 associates with TERRA at telomeres, likely through association with the nascent transcript. When Rat1 is absent, TERRA is stabilized and Npl3 would persist at telomeres bound to TERRA. RNaseH1 resolves telomeric RNA-DNA hybrids, which may release nascent TERRA with Npl3 bound.
Figure.Figure 1A. Results of CRISPR screening analyzed by the MAGeCK algorithm. B. Wild type (WT) and NCOA4 KO MEFs were pretreated with 25 μg/ml ferric ammonium citrate (FAC) for 16 h and then chased with 20 μg/ml cycloheximide (CHX) for the indicated times. Soluble ferritin expression levels were determined by immunoblotting with the indicated antibodies. C. WT MEFs untreated or treated with 10 μg/ml FAC for 12 h were lysed with SDS-containing sample buffer or Triton buffer, and analyzed by immunoblotting with the indicated antibodies. D. Schematic of fractionation procedures. E. Subcellular fractions of MEFs treated with 20 μM Dfo for 12 h, 10 μg/ml FAC for 12 h, or 100 μM Deferoxamine (Dfo) for 6 h after treatment with 10 μg/ml FAC for 12 h were prepared by differential centrifugation as described in (D). Each fraction was subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. F. WT MEFs were untreated, treated with 10 μg/ml FAC for 12h or treated with 10 μg/ml FAC, 10 μM E64d and 10 μg/ml pepstatin A (pep A) for 12h. The cells were immunostained with anti-NCOA4 and anti-LAMP1. Scale bar, 10 μm. Quantitative data are shown as the means ± SD of three biological replicates. At least 30 cells were quantified in each replicate. P=0.033 (Welch Two Sample t-test). G. NCOA4 puncta were photobleached in MEFs stably expressing NCOA4-GFP after treatment with 10 μg/ml FAC for 12 h, and then fluorescent recovery was monitored. p62 puncta were photobleached in MEFs stably expressing GFP-human p62, and then fluorescent recovery was monitored. Representative images are shown. Time 0 indicates the start of recovery after photobleaching. Scale bars, 2 μm. H. Quantitative data of fluorescence recovery in (G) shown as means ± SD. 26 dots (NCOA4) or 25 dots (p62) were quantified from three biological replicates. I. Time-lapse imaging of MEFs stably expressing NCOA4-GFP. Representative fusion and fission images are shown. Red arrowheads indicate the fission event. Scale bars, 5 μm.
Cyclin D1b expression promotes double-strand breaks and PARP-1 activity.Figure 6Ccnd1+/+ and Ccnd1KI/KI cells were transfected with a pool of siRNA directed against the N-terminus of the murine cyclin D1 transcript or scramble control for 48 h (as in Figure5). Cells were then fixed and stained for markers of double-strand breaks (p-H2AX and 53BP1, 400× objective). Total number of foci for each cell was quantified and represented as the % of cells with > 10 foci/cell (p-H2AX) or % of cells with > 2 foci/cell (53BP1). Error bars represent ± SEM. Ccnd1+/+ and Ccnd1KI/KIMAF lines were grown in serum-proficient media for 24 h and probed for expression of auto-modified PARP1 via immunoblot. Gapdh and cyclin D1b serve as controls. Cells were plated as in (B) and treated with 0.5 μM etoposide for 3 h. Cells were harvested and then probed for markers of DNA damage via immunoblot. Lamin B serves as a control. Indicated MAF lines were treated as in (A) and harvested 48 h post-transfection. Cells were analyzed for total PAR levels via immunoblot. cyclin D1 levels serve as siRNA validation controls. cyclin D1b expression was induced in the prostate cancer cell line LNCaP (previously described (Augello et al, 2014)) and stained for p-H2AX and 53BP1 foci as in (A) via immunofluorescence (400× objective). Total number of foci/cell is reported for LNCaP vector control and cyclin D1b-expressing isogenic pairs in biological triplicate. Isogenic pairs from (E) were grown in serum-proficient media for 24 h, harvested, and probed for total PAR levels via immunoblot.