EMBO/sd-character-level-ner huggingface dataset:
a) HeLa cells transfected with two rounds of control, clathrin heavy chain or AP2 siRNA for 4 days were collected for immunoprecipitation (IP) with anti-Atg16L1 antibody.figf8(a) HeLa cells transfected with two rounds of control, clathrin heavy chain or AP2 siRNA for 4 days were collected for immunoprecipitation (IP) with anti-Atg16L1 antibody. Western blot for total lysate (bottom) and IP (top) were performed with anti-Atg16L1 and anti-clathrin antibodies. Note the decrease in Atg16L1-clathrin interaction with AP2 knockdown. The graph shows quantification of the Atg16L1-clathrin or Atg16L1-AP2 with knockdown of control, clathrin heavy chain or AP2 from two independent experiments. (b) HeLa cells transfected for 24 h with Flag-tagged wild-type Atg16L1, GFP-PLC(PH) together with empty vector (control) or dominant-negative dynamin II mutant (K44A dynamin; DN-Dyn) were fixed and analysed by confocal microscopy. Note the co-localization of Atg16L1 with PLC(PH) at the plasma membrane; this is quantified in the graph at the right. Asterisk, P 0.01. n = 20 cells. Scale bars, 5 μm. DAPI, 4′,6-diamidino-2-phenylindole. (c) HeLa cells treated for 5 h with HBSS alone or with HBSS containing 80 μM dynasore were collected for immunoprecipitation (IP) with anti-Atg16L1 antibody. Western blot analyses for total lysate (TL) and IP were performed with anti-Atg16L1 and anti-clathrin antibodies. Note the strong Atg16L1-clathrin interaction with dynasore treatment. The graph shows quantification from two independent experiments. All error bars represent s.e.m. d, Representation of the contribution of the plasma membrane to autophagosome precursor formation. Cholera toxin is internalized by clathrin-dependent and clathrin-independent endocytosis. Clathrin knockdown significantly decreases cholera toxin uptake. Clathrin-coated vesicles (CCV) budding immediately from the plasma membrane (EEA1-negative) are precursors to early endosomes (EE) (EEA1-positive). Previous studies showed that delivery of fully formed autophagosomes to lysosomes requires fusion of such autophagosomes with early or late endosomes or multivesicular bodies (LE/MVB) to form amphisomes, which are Atg16L1-negative, LC3-positive and positive for endosomal markers (red arrows). We show here that inhibition of clathrin-dependent internalization inhibits the formation of early Atg16L1-positive precursors that mature to form phagophores and, later, autophagosomes (blue arrows). These Atg16L1 vesicles were positive for other early autophagosomal markers (Atg5 and Atg12) but not for early endosomal markers (EEA1). AP2 is a clathrin adaptor at the plasma membrane, whereas AP1 localizes to the trans-Golgi network and endosomes. Uncropped images of blots are shown in Supplementary Information, Fig. S9.
Dieldrin is a strong T cell activator causing transcription of several HERVs (A-E) Jurkat T cells were treated with either DMSO (vehicle) or with 100μM of either Dieldrin or PMA for the indicated times.Figure 2Jurkat T cells were treated with either DMSO (vehicle) or with 100μM of Dieldrin for the indicated times. Abundance of mRNA for the indicated genes or HERVs was assessed by RT-qPCR. Jurkat T cells were treated with either DMSO (vehicle) or with 100μM of PMA for the indicated times. Abundance of mRNA for the indicated genes or HERVs was assessed by RT-qPCR. Jurkat T cells were treated with either DMSO (vehicle) or with 100μM of either Dieldrin or PMA for the indicated times. Abundance of mRNA for the indicated genes by RT-qPCR. Jurkat cells were exposed to either DMSO, PMA in DMSO, or dieldrin in DMSO for 30 min in triplicates. RNA-seq was then performed on cDNA libraries prepared with poly(A) selection: principal component analysis on the 485 genes the most affected by DMSO, PMA, or Dieldrin. Venn diagrams reporting genes up- or down-regulated by dieldrin or PMA as compared to DMSO. Gene ontology analysis on genes up- and down-regulated by either dieldrin or PMA as estimated from RNA-seq data.
Figure 5Electron micrograph (EM) images from mid sagittal section of corpus callosum showing increased number of myelinated axons in AAV9-hSynI-mWwox-injected mice compared to Wwox-null mice (KO) at P17.   Graph showing average number of myelinated axons in corpus callosum from WT (n=3), KO (n=3) or KO mice injected with AAV9-hSynI-mWwox (n=3) per field of view (FOV).   g-ratio analysis showing reduced myelin thickness of axons in corpus callosum KO (n=3) compared to the KO+AAV-Wwox (n=3). Axon diameter and myelin thickness was calculated from the electron micrograph images of corpus callosum (total no of axons n=350 per each group).   EM images from mid sagittal section of corpus callosum showing similar number of myelinated axons in rescued mice (AAV9-hSynI-mWwox, n=3) compared to wild type (n=3) at the age of 6 months. Unmyelinated axons are noted with the red star symbols in EM images. Graph represents the number of unmyelinated axons (corpus callosum) per FOV counted from representative EM images shown in D. Graph represents the measured g-ratio of myelinated axons (n=300 per each group) in WT and rescued mice at 6 months (P value 0.011334). EM images showing increased number of myelinated axons in optic nerve in AAV9-hSynI-mWwox injected mice compared to KO mice at P17.   Graph showing the average number of myelinated axons (counted per FOV) in optic nerve from WT (n=3), KO (n=3) and KO+AAV-mWwox(n=3).   g-ratio's of myelinated axons from optic nerves showing improved myelin thickness (less g-ratio) after WWOX restoration (KO+AAV-mWwox) compared to KO alone (n=3 per genotype, total no of axons n=200 per each group).
Figure 1A. Glioma tumour micro arrays (grade II-IV gliomas) were stained to visualise MDGI (red). Samples from the epileptic brain served as a negative control. Scale bar 100 µm. B. Overall survival of grade II and III glioma patients (n = 76) was significantly better when none/low MDGI (blue line) was detected compared to patients with moderate/high MDGI (red line) expression (P = 0.007). The cumulative survival rates were estimated by using the Kaplan-Meier method. C. Analysis of the association of MDGI mRNA expression with glioma patient survival using the GlioVis data portal. In the TCGA GBMLGG dataset (n = 667), patients with low MDGI expression (green line; n = 333, events = 101, median = 67,5) show significantly better survival than patients with high MDGI (red line; n = 334, events = 138, median = 41.5) expression (P = 2e-04). HR = 0.62 (0.48 - 0.8). The cumulative survival rates were estimated by using the Kaplan-Meier method. D. In glioblastoma samples, highest MDGI expression was observed in the mesenchymal subtype (M) compared to the classical (Cl) or pro-neural (PN) ones. However, it did not reach the statistical significance. GlioVis data portal, TCGA GBM dataset, n = 156. Pairwise t-test with corrections for multiple testing, p-values with Bonferroni correction. E. MDGI expression was high in the leading edge of the tumour (LE) and in infiltrating tumour cells (IT) in the IVY_Gap dataset (n = 122) where the gene expression profile in different anatomical structures of glioblastomas was analysed. LE, leading edge; IT, infiltrating tumour cells; CT, cellular tumour (cells within the tumour mass); PC, pseudopalisading cells; MP, microvascular proliferation. Numbers in the graph depict the P values: Pairwise t-test with corrections for multiple testing, p-values with Bonferroni correction. F. Western blot analysis shows MDGI expression in human glioma cells including patient-derived spheroids (S24, ZH161, ZH305, BT3, BT11, BT12, BT13), patient-derived adherent cells (BT5 and BT5R), and long-term cell lines (U87MG, LN229, LN308). Numbers above the lanes show the relative expression of MDGI compared to the levels of β-tubulin that served as loading control in each cell line. Representative Western blot image of 3 separate experiments is shown. G. High MDGI and HIF-1α expression in the patient-derived glioblastoma spheroids decreased significantly when cells were cultured in medium containing 10 % of serum (FBS) for seven days. Numbers show the relative expression of MDGI and HIF-1α compared to the levels of β-tubulin. Expression in the serum-free medium was set as 1. Representative Western blot image of 2 separate experiments is shown. H. MDGI expression in adherent glioma cell lines and in glioma cell spheroids after 24h culture under hypoxia (1 % O2) or normoxia (21 % O2). Numbers show the relative MDGI expression compared to the levels of β-tubulin. Expression under normoxia was set as 1. Representative Western blot image of 3 separate experiments is shown.
Figure 2(A) Pictorial representation of stages (black font) of a typical life cycle of RNA viruses with host viral restriction factors (red font) induced in IRGM/Irgm1 depleted cells. Created using Biorender.com (B, C) Heatmaps representing the expression pattern of viral restriction factors from RNA sequencing data in (B) control and IRGM knockdown shRNA stable HT-29 cells (3 biological replicates) and (C) irgm1+/+ and irgm1-/- mice BMDMs (2 biological replicates). (D) RNA was isolated from irgm1+/+ and irgm1-/- BMDMs and subjected to qRT-PCR with indicated viral restriction factor genes (E) Western blot analysis with lysates of control and IRGM+/- HT-29 cells with indicated antibodies of viral restriction factors. (F) Western blot analysis with lysates of control or IRGM+/- HT-29 cells or Flag-IRGM complemented IRGM+/- HT-29 cells with indicated antibodies of viral restriction factors. (G) RNA isolated from control or Flag-IRGM transfected or Flag-IRGM S47N transfected THP-1 cells subjected to qRT-PCR with indicated genes. (H-J) RNA isolated from control and IRGM knockdown HT-29 cells and subjected to qRT-PCR with indicated genes of (H) Immunoproteasome complex (I) TAP complex (J) human leukocyte antigen (HLA) system. (K) Antigen uptake assay shown by representative flow cytometry analysis of control and IRGM siRNA transfected THP-1 cells treated with OVA conjugate AF488 (5 µg/ml, 30 min). The graph depicts the mean fluorescence intensity of control and IRGM knockdown THP-1 cells treated with OVA conjugate AF488 (L, M) Antigen processing assay shown by representative confocal images of control and IRGM siRNA transfected THP-1 cells treated with DQ-OVA (green) (10 µg/ml, 30 min). Scale Bar, 10 µm. (M) Graph depicts percentage of control and IRGM knockdown THP-1 cells with DQ-OVA puncta's (N, O) SIINFEKL based Antigen presentation assay shown by representative flow cytometry analysis of H-2Kb-SIINFEKL on the surface of Irgm1+/+ and irgm1-/- mouse BMDMs treated with OVA (2 mg/ml, 3 h). (O) The graph depicts the mean fluorescence intensity of H-2Kb- SIINFEKL on the surface of Irgm1+/+ and irgm1-/- mouse BMDMs treated with OVA (2 mg/ml, 3 h) (P) Representative confocal images of H-2Kb-SIINFEKL (red) on the surface of Irgm1+/+ and irgm1-/- mouse BMDMs treated with OVA (2 mg/ml, 3 h), Scale bar 5 µm or 8 µm (as indicated). (Q) Western blot analysis with lysates of control and IRGM siRNA knockdown HT-29 cells with indicated antibodies of stress granules signaling pathway. (R) Representative Immunofluorescence confocal images of irgm1+/+ and irgm1-/- mouse BMDMs immunostained with dsRNA (green) and TIA-1 (red) antibody. Scale bar 3 µm.
Female Coq9Q95X mice develop a mild myopathic phenotype with exercise intolerance.Figure 9A-C Voluntary wheel running test. Distance traveled on the wheel and average speed during the use of the wheel were decreased in female Coq9Q95X mice at 6 months of age (B, C). D Hanging wire test. Coq9Q95X female mice obtained less reaches score in the 'fall and reaches' method. E Open-field test. Coq9Q95X mice showed a reduction in the average distance traveled in Coq9Q95X female mice at 6 months of age. F Grip test: Muscle strength was not affected in Coq9Q95X mice at 6 months of age.
SUMO2 modification of FXR at K277 is important for increased FXR recruitment and decreased NF‐κB occupancy at inflammatory genes A FXR binding near inflammatory genes in mice fed a ND or HFD treated i.Figure 7A FXR binding near inflammatory genes in mice fed a ND or HFD treated i.p. with vehicle (−) or GW4064 (+) for 1 h. The sequence of an NF‐κB binding site present within the FXR binding peak is shown. B Luciferase reporter assays using direct FXR target inflammatory gene promoters (n = 3-6). C-E Gel mobility shift assays. Purified flag‐FXR was incubated with flag‐RXRα and the radiolabeled oligonucleotide probe containing the FXRE or NF‐κB binding site from the SHP or TNFsf4 promoter, respectively (C). Increasing amounts of flag‐FXR or SUMOylated flag‐FXR were incubated with purified RXRα (D) or whole‐cell extracts containing NF‐κB (E), and the complexes were detected by gel electrophoresis. F ChIP assay of hepatocytes from WT mice treated as indicated. Occupancy of endogenous p65, FXR, and RXR was detected by qPCR. G ChIP assay of hepatocytes from WT mice infected with Ad‐flag‐FXR‐WT and treated as indicated. H ChIP assay of hepatocytes from WT mice treated as indicated. I, J ChIP assays of hepatocytes from FXR‐KO mice infected with Ad‐FXR‐WT or Ad‐K277R and treated as indicated.
a, b) C38 cells were transfected with either 50 nM human CFTR siRNA or scrambled oligonucleotides (a, b) or cultured with CFTRinh-172 (b) in the presence or absence of cystamine (250 μM) or EUK-134 (10 mM).figf4(a, b) C38 cells were transfected with either 50 nM human CFTR siRNA or scrambled oligonucleotides (a, b) or cultured with CFTRinh-172 (b) in the presence or absence of cystamine (250 μM) or EUK-134 (10 mM). (a) Immunoblot of beclin 1, LC3 and p62 in C38 cells on starvation. Uncropped images of blots are shown in Supplementary Information, Fig. S10. (b) Percentage of cells containing more than five GFP-LC3 punctate dots per cell. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus scrambled oligonucleotides; circle, P 0.01 versus CFTR siRNA or CFTRinh-172; ANOVA. (c-g) A549 cells were transfected with 1 μg of pGFP--CFTRF508del or pGFP (empty vector), in the presence or absence of 10 mM EUK-134 or 250 μM cystamine. (c) Immunoblot of TG2 and PIASy. (c-g) A549 cells were transfected with 1 μg of pGFP--CFTRF508del or pGFP (empty vector), in the presence or absence of 10 mM EUK-134 or 250 μM cystamine.(d) FRET analysis of TG2-Alexa 546 fluorescence after SUMO-1 Cy5 photobleaching. (c-g) A549 cells were transfected with 1 μg of pGFP--CFTRF508del or pGFP (empty vector), in the presence or absence of 10 mM EUK-134 or 250 μM cystamine.(e) Immunoblot of beclin 1 and LC3 in A549 cells after starvation. Uncropped images of blots are shown in Supplementary Information, Fig. S10. (c-g) A549 cells were transfected with 1 μg of pGFP--CFTRF508del or pGFP (empty vector), in the presence or absence of 10 mM EUK-134 or 250 μM cystamine.(f) Percentage of cells containing more than five GFP-LC3 punctate dots per cell. Means ± s.d. for three independent experiments. Asterisk, P 0.001; two asterisks, P 0.01; ANOVA. (g) Immunostaining with anti-p62 (red) and anti-GFP (green) antibodies in starved A549 cells. Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm. Each experiment was repeated three times.
Figure 1(A) Pedigree of the patients P1 (II-1) and P2 (II-2) presenting with CRAC channelopathy. Filled circles (females) and squares (males) indicate homozygous patients; dotted symbols indicate heterozygous asymptomatic carriers; symbols with "?" indicate asymptomatic family members not available for DNA sequencing. (B) Sanger sequencing of genomic DNA isolated from PBMC of the mother (I-2), P1 and P2. (C) STIM1 and STIM2 mRNA expression in PBMC of P1, P2, mother and a healthy donor (HD) that were left unstimulated or stimulated with anti-CD3/CD28 for 24 hours. mRNA levels of STIM1/2 normalized to 18S. Graph shows the mean ± SEM of duplicates from one experiment. (D) STIM1 protein expression in CD4+ and CD8+ T cells from P1, P2, the mother and a HD analyzed by flow cytometry. Shaded histograms: polyclonal rabbit IgG control antibody; open histograms: polyclonal anti-STIM1 antibody. Bar graphs show the delta MFI calculated as MFISTIM1 - MFIIgG control that was normalized to HD T cells. Bar graphs are mean ± SEM of 2 independent repeat experiments. (E) STIM1 protein expression in expanded human T cells analyzed by immunoblotting. One representative Western blot of 3 is shown. Bar graphs are the mean ± SEM of STIM1 expression normalized to actin from 3 independent experiments. (F) Ca2+ influx in Fura-2 loaded PBMC of P1, P2, the mother and a HD. T cells were stimulated with 1 μM thapsigargin (TG) in the absence of extracellular Ca2+ followed by addition of 1 mM extracellular Ca2+. Bar graphs show the integrated Ca2+ influx response (AUC, area under the curve) from 400-800 sec and the peak Ca2+ influx normalized to baseline Ca2+ levels (F340/380) at 400 sec. Data represent the mean ± SEM from 2 experiments. Statistical analysis by unpaired Student's t-test. **p<0.01, ***p<0.001.
Figure 5A,B Representative images (A) and quantitative analysis (B) showing aberrant accumulation of mitophagosomes labeled by LC3 and Cyto c antibodies in the hippocampal mossy fibers of snapin-cKO mouse brains. Note that white arrow-labeled mitophagosomes are not readily observed in WT mouse brains. The average number of mitophagosomes per section (320 μm × 320 μm) was quantified. Representative blots showing aberrant mitophagic retention at synaptic terminals in snapin-mutant mouse brains. The levels of Rheb along with LC3-II, p62, and Rab7 are significantly elevated in the synaptosomal fractions (Syn) purified from snapin-deficient mouse brains, relative to those of WT littermate controls (red box). Equal amounts (10 μg) of Syn and post-nuclear supernatants (PNSquantitative analysis The levels of Rheb along with LC3-II, p62, and Rab7 are significantly elevated in the synaptosomal fractions (Syn) purified from snapin-deficient mouse brains, relative to those of WT littermate controls WT and snapin-mutant mouse brains were sequentially immunoblotted with antibodies against TOM20 on the same membrane after stripping between each antibody application. The purity of synaptosomal fractions was confirmed by relative enrichment of SYP and PSD95 and less abundance of GAPDH. Data were quantified from five independent repeats. E,F Representative blots (E) and quantitative analysis (F) showing mitophagic accumulation in the brains of snapin conditional KO (cKO) mice by crossing with Thy1-cre Tg mice. Note increased levels of Rheb along with LC3-II, p62, and Rab7 in mitochondrial (Mito) fractions isolated from snapin-deficient mouse brains, relative to those of WT littermate controls (red box). Data were quantified from five independent repeats. PNS: post-nuclear supernatants. G,H Representative TEM images (G) and quantitative analysis (H) showing abnormal presynaptic retention of mitophagosome-like structures in the hippocampal regions of snapin-deficient mouse brains. Mitophagosomes, indicated by black arrows, are not readily detected in WT mouse brains. Data were quantified from the total number of the imaging fields (n) indicated in parentheses (H). I,J Representative images (I) and quantitative analysis (J) showing that deletion of snapin in mouse brains induces synaptic damage. Note that the density of presynaptic terminals in the hippocampal mossy fibers revealed by SYP immunostaining is significantly reduced in snapin-mutant mice crossed with CamKII-cre Tg mice at the age of 8 months, relative to that of WT littermate controls.
Figure 2(A) Schematic diagram of CRISPR/dCas9-3×sfGFP targeting loci on mouse X chromosome. sgRNA mX26 and mX8 target microsatellite repeats on Dxz4 and Xist loci, respectively. (B) Experimental design: female ESCs are engineered to stably express two sgRNAs, dCas9-3xsfGFP, and a nuclear localization signal (NLS)-fused H3K27me3-mintbody (mCherry). Upon ESC differentiation by LIF withdrawal H3K27me3 accumulation occurs at the inactivating X (Xi) but not at the active X (Xa). Green foci allow marking of two X-linked loci (Dxz4 and Xist). (C) Live imaging of female mESCs as in (B) in undifferentiated conditions (+LIF) or upon 3 days of LIF withdrawal. Double arrowheads mark Xa and single arrowhead marks Xi. Shown are maximum intensity projections of 11 z-plane confocal sections. Scale bar = 10 μm. (D) ESCs as in (B) were engineered to express H3K27me3-mintbody (SNAP/JF646) and H4K20me1-mintbody (mCherry). Cells were cultured in the absence of LIF for 2 days. Time-lapse images for 12 z-plane confocal stack were acquired every 1 h. Maximum intensity projection images are shown with elapse time (hh:mm). Arrowheads mark Xi. Scale bar = 10 μm
Figure 3 A. NS-EM micrograph of the T4SS3-10+D4 complex. A few representative particles of the T4SS3-10+D4 complex are circled in yellow. Scale bar 50nm. B. Representative class averages of T4SS3-10+D4 complex obtained after initial alignment and classification. C. The upper row represents typical class averages of the T4SS3-10+D4 IMC complex (i.e. with the outer membrane core complex (OMC) masked out). The second row shows projections of the T4SS3-10+D4 IMC complex in the same directions determined for the classes above. The third row displays projections of the T4SS3-10 IMC complex (EMD-2567) in the same directions. The bottom row shows the differences between the projections above (plus or minus TrwB/VirD4) corresponding to positions of the TrwB/VirD4 protein. D. FSC of the electron density map of the T4SS3-10+D4 complex. The 0.5 line crosses the FSC at a resolution of 28 Å.
deficient HSPCs accumulate DNA-damage during compensatory proliferation triggering cell death.Figure 6Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of fetal liver cryosections of the indicated genotypes at E13.5 and quantification. Scale bar 100x = 200 µM, 400x = 50 µM. The percentage of TUNEL+ area/field was assessed across three random fields from two control (Chk1fl/+) and two knock-out embryos (Chk1fl/- Vav-Cre) using ImageJ, as reported by us before (60). Bars represent means ± S.D. Total fetal liver cell suspensions of Chk1fl/+ (+/+), Chk1fl/+ Vav-Cre (+/-) and Chk1fl/- Vav-Cre (-/-) E13.5 embryos were processed for Western blot analysis using the indicated antibodies. Each lane represents samples isolated from an individual littermate embryo. DAPI-negative viable LK- and LSK-cells of the indicated genotypes (littermates) were sorted from E13.5 fetal livers and immediately fixed in 70% EtOH. Fixed cell suspensions were stained for ɣH2A.X and DAPI intracellularly. Shown here are representative dot-plots and quantification of N=4 embroys per genotype; Bars represent means ± S.E.M. Total E13.5 fetal liver cells were processed for intracellular Ki67 staining in combination with cell surface antibody staining to discriminate LK (top panel) from LSK (lower panel) cells. Shown here are representative dot-plots of Ki67 and DAPI staining in LK and LSK cells, quantified in (E). Bars represent means ± S.E.M. N=4 for Chk1fl/+ and N=5 for Chk1fl/- Vav-Cre.