EMBO/sd-character-level-ner huggingface dataset:
Fig.Figure 3(A) Superimposition of camrelizumab/PD-1 with PD-1/PD-L1 complex structure (PDB code: 4ZQK). PD-1 is shown as surface format in light blue, while PD-L1 is shown as deep teal cartoon and camrelizumab-scFv is depicted as cartoon. VL of camrelizumab-scFv is colored in yellow and VH in deep salmon. (B) Competitive binding surfaces of camrelizumab with PD-L1 on PD-1. The residues contact with PD-L1 alone are colored in deep teal while the residues bind to camrelizumab alone are colored in deep salmon. The residues contacted by both camrelizumab and PD-L1 are colored by magenta. (C) The binding surface of PD-L1 and FDA approved MAbs on PD-1 is presented in different colors. The binding area of PD-L1 and the MAbs targeting PD-1, camrelizumab, toripalimab, nivolumab, pembrolizumab, GY-5 and GY-14 are colored in deep teal, deep salmon, magenta, green, orange, light pink and yellow, respectively.
Figure 5A. A set of different constructs of PTEN, PTENα, PTENβ, and PTENε with a C-terminal GFP tag. The AUG start codon of canonical PTEN was mutated to AUA in PTENα, PTENβ, and PTENε constructs to abolish the expression of canonical PTEN and the corresponding start site of these PTEN isoforms was mutated into AUG to enhance their expression. A set of different constructs of PTEN, PTENα, PTENβ, and PTENε with a C-terminal GFP tag. Subcellular localization of C-terminal were introduced into Hela PTEN-/- cells. Thirty-six hours after transfection, cells were stained with DAPI, followed by imaging with confocal microscopy. C. The transfected Hela PTEN-/- cells were stained with an anti-β-catenin antibody and DAPI before being imaged with confocal microscopy. D. HEK293 cells were transfected with C-terminal GFP tagged PTEN and PTENε and subjected to cell fractionation, followed by western blotting with GFP, E-cadherin, and β-tubulin antibodies. C: cytoplasm. M: cell plasma membrane. E. Prediction of N-terminal structure of PTENε by Swiss Model (left panel); a different set of C-terminal GFP-tagged PTENε plasmids with or without fragment deletion in PTENε N-terminal sequence were constructed. To determine the sequences that are critical for the membrane localization of PTENε. Hela were transfected with constructs Thirty-six hours after transfection, transfected Hela cells were stained with anti-β-catenin antibody and DAPI, followed by imaging with confocal microscopy. The scale bars represent 5 μm (F). The transfected HEK293 cells were conducted with cell fractionation before western blotting with GFP, E-cadherin, and β-tubulin antibodies (G). C: cytoplasm. M: cell plasma membrane.
1,25D3 inhibition of HIV and M.Figure 4MDM were transduced with non-specific scrambled shRNA (shNS) or Beclin-1 shRNA (shBCLN1) and selected using puromycin resistance. Five days later, cells were incubated with 100 pmol/L 1,25D3 or vehicle control for 4 h before infection with HIV and/or M. tuberculosis (TB) for 3 h. Cells were then washed and incubated with 100 pmol/L 1,25D3 or vehicle control for 7 days. (A) Immunoblot analysis performed using antibodies raised to Beclin-1 or β-actin after initial pathogen exposure (Day 0) or after 7 days. (B) ELISA performed for HIV p24 antigen release over 7 d. (C) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and day 7. D) Intracellular mycobacteria harvested and assayed for viability based on time to positivity (75 growth units) at day 0 and day 7 using the MGIT 960. All bar and line graphs are reported as mean ± s.e.m. of three independent experiments performed in triplicate. § P<0.05; * P<0.001.
Figure 6(A) Enrichment of the ZEB1, AP-1 and TEAD4 DNA-binding motifs identified by HOMER known motif analysis on 200 bp regions centred on ZEB1 peak summits associated with repressed or activated genomic regions identified by ATAC-seq. (B) Comparison of ZEB1 ChIP-seq peaks with ZEB1-dependent changes in chromatin activation status identified by ATAC-seq on control and ZEB1 knock-down MDA-MB-231 cells. Percentage of ZEB1-only (n=5,963) and ZEB1/YAP/JUN peaks (n=1,993) falling into ZEB1 repressed or activated genomic regions are shown. (C) Genome browser tracks of ZEB1, YAP and JUN ChIP-seq signal intensity in wild type cells overlaid to ATAC-seq signal intensity in wild type, control (shCtrl) and ZEB1 (shZEB1) knock-down MDA-MB-231 cells. One known ZEB1 repressed target (CDH1) and one activated target (ANKRD1) are shown.
Figure 4(A) Confocal fluorescence microscopy-based filamentation assay at 561 nm shows mChRZZSF (4 µM), but not mChRZZ (4 µM) or mChSF (4 µM) as controls, forms filaments at 30ºC. Scale bar: 5 µm. Two technical replicates were performed. (B) Coomassie and ProQ Diamond-stained gels on mChRZZSF (4 µM) treated with the indicated kinases (1 µM) for 15 hours at 20ºC. Rev = reversine, used at 10 µM. (C) A filamentation assay as in A demonstrates sufficiency of MPS1 phosphorylation for filamentation. Experiments for panels B and C were technically replicated three times. (D) Levels of Zwilch at kinetochores of HeLa cells that had been previously synchronized in G2 phase with 9 μM RO3306 for 16 h and then released into mitosis. Subsequently, cells were immediately treated with 500 nM reversine, 3.3 µM nocodazole, and 10 µM MG132 for 1 hour and imaged while in mitosis. CREST serum was used to visualize kinetochores and DAPI to stain DNA. Scale bar: 10 µm. Three biological replicates were performed. (E) Cells treated as for panel D were treated for visualization of Spindly. Scale bar: 10 µm. (F) Scatter dot plots representing normalized total area of the Zwilch and Spindly signals, normalized to the reversine-untreated control, in the indicated number of cells from the experiment shown in panels C-D. Red lines indicate mean and standard deviation. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000. (G) Representative images showing the effects of a knockdown of the endogenous Spindly in HeLa cells. RNAi treatment was performed for 48 h with 50 nM siRNA. Before fixation, cells were synchronized in G2 phase with 9 μM RO3306 for 16 h and then released into mitosis. Subsequently, cells were immediately treated with 3.3 μM nocodazole for an additional hour. CREST serum was used to visualize kinetochores and DAPI to stain DNA. Scale bar: 10 µm. Three biological replicates were performed. (H) Scatter dot plots representing normalized intensity ratios of Spindly over CREST for individual kinetochores of cells from the experiment shown in panel G. Red lines indicate median with interquartile range. (I) Levels of CENP-E and Zwilch were assessed in control cells and in cells treated as in panel G to knockdown Spindly. Three biological replicates were performed. (J) Scatter dot plots representing normalized total area of the CENP-E and Zwilch signals, normalized to the RNAi negative control, in the indicated number of cells from the experiment shown in panel I. Red lines indicate mean and standard deviation. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000.
Fig.Figure 6 （A）Schematic illustration showing overlapping target miRNAs of circLMP2A predicted by miRanda and TargetScan. (B) The relative levels of 7 miRNA candidates were detected by real-time PCR in SNU719 and SNU-4th cells. (C-E) Real-time PCR analysis of 7 miRNA expression in SNU-4th cells transfected with sh-circLMP2A-2 or sh-control (C), and SNU719 (D) and YCCEL1 cells (E) transfected with circLMP2A or vector. (F-H) circ LMP2Ain specific probe and detected by real-time PCR (F) and RT-PCR (H). The relative levels of 5 miRNA candidates were detected by real-time PCR (G) and RT-PCR (H). GAPDH was used as a negative control. (I) RNA ISH showing the co-localization between circLMP2A and miR-3908 in SNU-4th cells. circLMP2A probes were labelled with Cy3, and miR-3908 probes were labelled with FITC. Nuclei were stained with DAPI. (J) Wild type and mutant circLMP2A and miR-3908 sequences are presented. Red font indicates mutant bases. (K) Dual luciferase reporter assay for luciferase activity of the indicated plasmids (circLMP2A Wt, circLMP2A mut1, circLMP2A mut2, circLMP2Amut3 luciferase reporter) in HEK-293T cells transfected with mimic-NC or miR-3908.
a–l, SEM images of fly eyes expressing the indicated transgenes.figf4a-l, SEM images of fly eyes expressing the indicated transgenes. The rough eye phenotypes caused by proteasome mutation (a) or by expression of polyQ-expanded AR (b) were both suppressed by rearing flies on the TOR inhibitor rapamycin (c, d). e, f, Rapamycin failed to suppress degeneration in an autophagy-deficient background created by knockdown of atg12, confirming that rescue by rapamycin is autophagy-dependent. g, h, Rapamycin also failed to suppress degeneration when HDAC6 levels were knocked down, demonstrating that suppression via the TOR pathway is HDAC6-dependent. k, j, HDAC6 failed to suppress degeneration in an autophagy-deficient background, confirming that rescue by HDAC6 is dependent on autophagy (compare k, l to i, j). 200 to >1,000 fly eyes of each genotype were examined. Quantitative analyses of eye phenotypes are presented in Supplementary Fig. S2.
Figure 1A. Cytosolicnon-heme iron levels in wild-type mice subjected to sham or I/R procedure two days after surgery. Two-tailed unpaired T-test was performed. N=4 mice for each group.B. Mitochondrialnon-heme iron levels in wild-type mice subjected to sham or I/R procedure two days after surgery. * P=0.024 with two-tailed unpaired T-test. N=4 mice for each group. C. Mitochondrial labile iron in H9c2 cells with or without H2O2 treatment measured using RPA fluorescence. * P<0.0001 with two-tailed unpaired T-test. N=8 independent samples for PBS group and N=10 independent samples for H2O2 group.D. Cytosoliclabile iron in H9c2 cells with or without H2O2 treatment measured using calcein fluorescence. * P=0.0042 with two-tailed unpaired T-test. N=11 independent samples in each group. E. Mitochondrialiron in humancardiac tissue sample from non-failing hearts and from hearts with ischemic cardiomyopathy (ISCM). * P=0.041 with two-tailed unpaired T-test. N=4 independent samples in each group.F. Cytosoliciron in humancardiac tissue sample from non-failing hearts and from hearts with ischemic cardiomyopathy (ISCM). Two-tailed unpaired T-test was performed. N=4 independent samples in each group. All data are expressed as mean ± SEM.
Impact of FLIP deletion on DISC assembly and caspase activation.Figure 5(A) Western blot analysis of FLIP, caspase 8 and FADD recruitment to the TRAIL-R2 DISC in parental (WT) and CFLAR deficient (KO) HAP-1 cells treated with AMG655 conjugated beads for either 45 minutes (45'), 90 minutes (90'), 180 minutes (180'), 3 hours (3h), 6 hours (6h) or 9 hours (9h). (B) Caspase activity assays of the unbound soluble fraction (C) Caspase-8 (i) and -3/7 (ii) activity assays and high content microscopy assessment of cell death (iii) in Parental (PAR) and FLIP knockout (CFLAR CRISPR) cells treated with escalating doses of isoleucine zipper TRAIL (IZ-TRAIL) for 6 hours. (D) Caspase-8 (i) and -3/7 (ii) activity assays in Parental (PAR) and FLIP knockout (CFLAR CRISPR) cells treated with escalating doses of isoleucine zipper TRAIL (IZ-TRAIL) for 24 hours. (iii) Flow cytometry assessment of cell death induction in Parental (PAR) and FLIP knockout (CFLAR CRISPR) cells treated with IZ-TRAIL for 24h. (E) Western Blot analysis of Parental (WT) and FLIP knockout (KO) HAP-1 cells treated with 10ng/mL IZ-TRAIL for 0, 3, 6 or 24 hours. (F) Cell viability assay of Parental (PAR) and FLIP knockout (CFLAR CRISPR cells) treated with an escalating dose (0-1000ng/mL) of IZ-TRAIL for 72 hours.
Figure 9A, B. Two C9ALS/FTD iNeuron cultures (183 and 201) and their matching controls (170 and 155) were treated with vehicle (Ctrl) or bafilomycin A1 (100 nM; 6 h - BafA1) and processed for immunoblot detection of LC3. LC3-II levels were normalized to α-Tubulin (Mean ± SEM; one-way ANOVA with Fisher's LSD test; ns, not significant; * P ≤ 0.05; ** P ≤ 0.01; **** P ≤ 0.0001; Ctrl 209/Pat 201, n=4; Ctrl 155/Pat 183, n=6).
Figure 7Representative images (left) and quantification (right) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination. Scale bars, 100μm. Data are representative of n=6 biological replicates. Representative images (left) and quantification (right) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination. Scale bars, 100μm. Data are representative of n=6 biological replicates. Representative images (left) and quantification (right) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination. Scale bars, 100μm. Data are representative of n=6 biological replicates. Representative images (left) and quantification (right) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination. Scale bars, 100μm. Data are representative of n=6 biological replicates. Representative images (left) and quantification (right) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination. Scale bars, 100μm. Data are representative of n=6 biological replicates. Representative images (left) and quantification (right) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination. Scale bars, 100μm. Data are representative of n=6 biological replicates. Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of n=3 (RTKi in G (DMSO in G Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of (DMSO in and ERKi in H) Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of (DMSO in (PI3Ki in Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of (DMSO in (PI3Ki in Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of (DMSO Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of Docetaxel (DMSO
Fig.Figure 1A. Binding of eight commercial antibodies to recombinant human LAG323-450 and murine LAG324-442 via indirect ELISA. Seven out of eight antibodies bound either human or mouse LAG3, while one antibody (LSB15026) recognized both species. B. Specific detection of murine but not human LAG3 using 4-10-C9 anti-LAG3 antibody is confirmed with Western Blotting. C. No detection of human LAG3 in neuronal or glial cell lines of human origin. The band for LAG3 was detected in activated T cells. D. No band for human LAG3 could be detected with Western Blot in lysates of fully differentiated human NSC-derived neural cultures. E. Violin plot showing the RNA expression levels of human LAG3 in human NSC-derived neural cultures. Identities annotate different clusters: Neuronal clusters are comprised of the following markers: GAD2, GABRG1, NTRK2, NEFM, SNCG, SLC17A6, SCN2A, DDIT3/HRK. Mixed glial clusters are defind by the following markers: GFAP, S100B, STMN2, NRN1, GPM6B, COL1A1, with astrocyte-specific clusters characterised by GFAP, S100B, GPM6B, COL1A1. LAG3 cannot be evidenced in any of the clusters beyond few random events. Data shown from 5'476 unique analysed cells from one out of two independent biological replicates. F. Dopaminergic neuronal cultures from control lines and glucocerebrosidase (GBA) N370S PD patients were immunoblotted for the presence of LAG3. No band for LAG3 could be observed in neurons. G. Using high power, high resolution laser scanning confocal microscopy, no human LAG3 signal could be detected in human neurons (Auto-hLAG3 transduced, DOX OFF) by two different anti-human LAG3 antibodies (17B4 and D2G40; left panel and zoomed-in insets) whereas LAG3 was clearly detected in human neurons induced to express hLAG3 (DOX ON; right panel and zoomed-in insets). Scale bars 25 µm. H. Human brain homogenates from autopsy material were immunoblotted for the presence of LAG3. No band for LAG3 could be evidenced in any of the brain homogenates. Control samples (tonsils and activated human T cells) show expected bands. I. Nuclei were isolated from 21 human dorsolateral prefrontal cortices from 16 donors, isolated, and were subjected to snRNAseq. Expression levels were quantified and are shown as violin plots. The LAG3 transcripts are non-detectable in all 34 distinct cell types, including multiple excitatory and inhibitory neurons, oligodendrocytes (ODC), oligodendrocyte precursor cells (OPCs) microglia (MGL), astrocytes (AST), or endothelial cells (EC). Cluster markers as detailed in Saez-Atienzar et al. 2021.
Chk1 loss generates replication-associated obstacles that impair nascent DNA elongation.Figure 2U2OS cells were labeled with CldU for 10 min, followed by increasing IdU labelling times and subjected to DNA stretching. (A) Labelling scheme and representative DNA fibers. Scale bars: 5 μm. U2OS cells were labeled with CldU for 10 min, followed by increasing IdU labelling times and subjected to DNA stretching. IdU track length after 10-80 min. Theoretical IdU track lengths are shown in red; the theoretical 10-min points were considered equal to the experimental values obtained for siLuc or siChk1, and the following theoretical points were calculated proportionally. Data were calculated as the mean (+SD) of mean IdU track lengths (B) n=4 for IdU times 20-60 min or n=3 for IdU times 10 and 80 min; for each experiment and condition 60-100 fibers were scored. U2OS cells were labeled with CldU for 10 min, followed by increasing IdU labelling times and subjected to DNA stretching. Fork speed for each IdU labelling time. Data were calculated as the mean (+SD) of mean fork speeds (C). n=4 for IdU times 20-60 min or n=3 for IdU times 10 and 80 min; for each experiment and condition 60-100 fibers were scored. DNA combing from HCT116 cells labeled with CldU and IdU for 20 min. The upper left panel shows the labelling scheme. The upper right panel shows representative sister replication forks that emanate from the same origin (ORI=origin of replication); a pair of sister forks is considered asymmetric if their lengths (CldU+IdU) differ in more than 33%. The percentage of asymmetric forks (lower left panel; a Western Blot of Chk1 and Ku80, loading control, performed in parallel to the combing experiments, is shown) and the lengths of each pair of forks (lower right panel; points outside of the dotted lines correspond to asymmetric forks) are shown. Data (siLuc, n=131; siChk1, n=117) come from 2 independent experiments; the numbers indicate the mean (+SD) percentage of asymmetric forks.
Figure 3(A) Crude membrane fractions from APP-DKO cells expressing C99WT or C99MUT were treated with 0.2% Triton-X-100, loaded onto continuous density sucrose gradients and centrifuged for 16h. Fractions from these gradients were analyzed by western blot to determine the migration of the indicated proteins [two parallel gels (bold vertical line)]. (B-G) Graphs represent the relative abundance of the indicated proteins in each fraction of the gradient, as measured by densitometry analysis of western blot signals (ImageJ). Note the reduced degree of co-migration of C99MUT with MAM markers (Acsl4), compared to C99WT.
Figure 5(A-B) Contour diagram of dissociated sensory neurons from Sst-Cre::ReteGFP/+ (A) and Avil-Cre::ReteGFP/+neurons (B), plotted according to their native eGFP expression and IB4 binding. Only one subset of Ret-eGFP cells can be defined within the Sst-Cre::ReteGFP/+ dissociated DRGneurons. This displays the same range of eGFPfluorescence as the Ret-eGFPLo:IB4Neg population and doesn't bind to IB4. (C) Quantitative RT-PCR analysis of selected transcripts in the Sst-Cre::ReteGFP (darker colors) and Ret-eGFPHi:IB4Neg (lighter colors) subsets displayed as differential expression compared to the Ret-eGFPHi:IB4Neg subset. Almost all the genes that are up-regulated in the Ret-eGFPLo:IB4Neg compared to the Ret-eGFPHi:IB4Neg subset are also enriched in the Sst-Cre::ReteGFP population (n=3)
Figure 1a, TEM analysis of P7 wt retinae (N = 4 eyes) showing a (photoreceptor- Multivesicular Body) PR-MVB in close proximity to (Muller glia) MG (red dashed box). PR-photoreceptor cell, MG (blue dashed lines) -muller glia cell, MVB (yellow and yellow arrow)- multivesicular body, m (green)-mitochondria, cc (pink)-connecting cilium, RPE (purple and purple dashed line) -retina pigment epithelium, RPE melanin-black arrows. Scale bar = 2µm left, 1µm right; b, Representative SEM microphotograph of cultured P8 Nrl.Gfp+/+ photoreceptors; arrows indicate membrane budding of processes or EVs; dashed circles denote EVs; Scale bar = 3µm; c, Representative TEM microphotograph of 100K EV pellet derived from Nrl.Gfp+/+ photoreceptor cultures (N = 10 independent preparations); Scale bar = 100nm; d & e, Representative Dynamic Light Scatter (DLS) plot of 100K EV sample (N = 13 samples), showing (d) average intensity and (e) volume against diameter (13 DLS measurements per sample); f, Representative Dot Blot of 100K EV pellet (each dot represents 3 pooled EV isolations, derived from 60*106 cells; N = 8 experiments) vs cell lysate (CL) from P8 Nrl.Gfp+/+ photoreceptors. EV markers = LAMP1, CD8, CD9; Golgi marker = GM130; phototransduction markers = Recoverin, Rhodopsin, Rod α-Transducin; g, RTqPCR analysis of 100K P8 photoreceptor pellets for Gnat1, Rec, Rho, Crx, Cre and Gfp, relative to β-actin, comparing EVs (N = 8) against the appropriate (Nrl.Gfp+/+ or Nrl.Cre+/-) photoreceptor cell lysate (N = 3). Gnat1, Cre and Gfp mRNA were present in all relevant samples; h, schematic representation of Cre-LoxP system to assess EV function in co-culture of Nrl.Cre+/- photoreceptors (PRs) with mTmGfloxed reporter retinal cells in non-proximity (transwell) versus physical proximity (contact); i, representative flow cytometry analysis of Nrl.Cre+/- & mTmGfloxed co-cultures. Samples were analyzed for expression of myrGFP (recombination) and CD73 (photoreceptor identity) versus CD73-ve fraction (other retinal cells). N > 3 independent cultures for each condition with technical triplicate for each sample; One way ANOVA, non-parametric, Kruskal-Wallis with Dunns' multiple comparisons test j, representative maximum intensity projection (MIP) confocal images of mTmGfloxed reporter retinal cells, (left) treated with AAV-Nrl.Cre virus (positive control), (middle) co-culture with Nrl.Cre+/- photoreceptors separated by transwell (non-proximity) or (right) in contact; Scale bar = 20µm; Red = myrRFP-expressing mTmGfloxed reporter, no recombination; green = myrGFP-expressing mTmGfloxed reporter, cells recombined upon acquiring Cre; blue = nuclei. N = 7 cultures.