EMBO/sd-character-level-ner huggingface dataset:
(G) Time course of normalized EGFP signal, as assessed by TIRF microscopy, from GRAMD1 TKO (TKO) HeLa cells expressing either wild-type EGFP-GRAM1b (WT) or mutant EGFP-GRAM1b (G187L) as indicated. Cells were cultured in the medium supplemented with 10% lipoprotein deficient serum (LPDS) and mevastatin (50 µM) for 16 hours before imaging. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 36 cells (WT), n = 37 cells (G187L), data are pooled from two independent experiments for each condition; two-tailed unpaired Student's t-test, **P = 0.0003]. Note the rapid PM recruitment of G187L mutant compared to WT.
(E) Control white RNAi muscles starved and treated with CQ accumulate GFP-Atg8a punctae, but very few colocalize with ubiquitin (Ubi). (F) Knockdown of Rpn1 in starved + CQ muscle strongly inhibits the formation of GFP-Atg8a punctae. The remainder colocalize with ubiquitin. (G) Quantification of the mean GFP-Atg8a punctae area and Mander's overlap coefficient for GFP-Atg8a and Ubi for genotypes shown in E and F. Error bars indicate SEM for n = 8 ventral longitudinal muscles from individual animals, with p-values for Rpn1 RNAi relative to white RNAi control (***p<.001).
C) Germlines dissected from animals expressing the indicated proteins were fixed and stained with α-FLAG and α-HA antibodies, followed by secondary antibodies conjugated with Alexa 488 and CF430, respectively. Pachytene germ-cell nuclei were imaged with a Leica TCS SP8 microscope. Scale bar, 2μm.
In situ hybridization of HAND2-AS1 in HCC tumor tissues. HAND2-AS1 staining is shown in tumor tissues (left panel). Quantitation of HAND2-AS1+ cells in liver tissues was examined in 10 high-power fields (HPF) of sections from ten different HCC patients (right panel). Results are shown as means ± SEM. Scale bar, 100 μm.
C, Potential processes that contribute to embryo invasion through uterine RB-induced CCA and necroptosis of luminal epithelial cells.
Nrp-1 expression in pre-retinal tufts (OIR model, P17). Abundant expression of Nrp1 in endothelial cells. Pericytes at vascular tufts were immunostained with anti-NG2. Scale bar, 20 μm.
A) 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).
C. Co-immunoprecipitation assay. The solubilized lysates from 2×1010 E. coli K-12 W3110 cells co-producing the indicated FLAG-tagged TssMP variants (exported in the periplasm) and VSV-G-tagged MltE protein (Total, T) were subjected to immune precipitation on anti-FLAG-coupled agarose beads. The immunoprecipitated material (IP) was subjected to 12.5%-acrylamide SDS-PAGE and immunodetected with anti-FLAG (upper panel, TssM domains) and anti-VSV-G (lower panel, MltE) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.
Reporter activity of the sensor across (D) multiple FBP levels Reporter activity is given by the YFP/mCherry ratio, calculated through the quantification of YFP and mCherry fluorescence along culture time using flow cytometry. Both YFP and mCherry fluorescence levels were first corrected for background using the same strains harboring the YCplac33 plasmid (Appendix Table S8). The control is the wildtype and TM6 strains expressing only the reporter plasmid without CggR. Error bars represent the standard deviation of at least three replicate experiments.
(P) ChIP analysis of KLF4 binding to the BRCA1 promoter in MDA-MB-231 at -.3K positions relative to the TSS in untreated and Olaparib (10µM for 8h) or 5Gy radiation treat cells. No significant difference of KLF4 binds to BRCA1 promoter between untreated and Olaparib or radiation treat cells. n=3, p=0.80, one-way ANOVA assay.
(A, B) Co-localization analysis of neosynthesized alcAp-UapA-GFP with early (SedV) or late (PHosbp) markers, respectively, tagged with mCherry or mRFP. Images show single hyphal cells. For conditions of transcriptional derepression see Materials and methods. Quantification of co-localization was performed by calculating Pearson's Correlation Coefficient (PCC). One sample t-test was performed to test the significance of differences in PCCs. Biological/technical replicates:2/9 for alcAp-UapA-GFP mCherry-SedV and 2/8 for alcAp-UapA-GFP mRFP-PHosbp. For the definition of the two categories of replicates see Materials and methods. Results of quantification, shown on the middle, suggest that there is no significant overlapping fluorescent signal of UapA with SedV (PCC=0.25±0.09, P<0.0001) or with PHOSBP (PCC=0.34±0.06, P<0.0001), as PCC values close to 0.2-0.3 are also commonly obtained when distinct compartments (e.g. ER and early Golgi or early and late Golgi) are followed with different fluorophores. Scale bars: 2 μm.
B Western blot analysis of ALKBH5 and TRAF4 in control, WT- and MUT-ALKBH5 overexpressing cells and quantification of protein levels normalized to β-actin expression.
E COBRA-LINE-1 of global DNA methylation levels in CCRF-CEM cells upon ATO or DAC treatment in the indicated conditions. The signal density of unmethylated and methylated DNA were quantified by ImageJ.
(A) RT-qPCR quantification of relative levels of pri-miR-21, pre-miR-21, miR-21-5p and miR-21-3p levels in healthy human skin and human squamous cell carcinomas (SCC). pri-miR-21 Healthy skin vs SCC P = 0.043; pre-miR-21 Healthy skin vs SCC P = 3E-05; miR-21-5p Healthy skin vs SCC P = 7E-05; miR-21-3p Healthy skin vs SCC P = 1.4E-06. ≥ 5 independent biopsies per condition.
In organello transcription analyses using heart mitochondria isolated from 8-week-old control and Tefm knockout mice (n= 5 mice for each group). The steady-state levels of 12S rRNA and tRNAV were used as endogenous controls, due to their unchanged levels. VDAC was used as loading control for the equal input of mitochondrial extracts. This experiment has performed five times with either two or three replicates.
A) Metaphase II oocytes from mice carrying a conditional, oocyte-specific invalidation of separase (Separaseflox/flox Zp3 Cre+, or Sep-/-) and oocytes from litter mates (Separaseflox/flox, or Sep+/+) were obtained after superovulation. In vivo matured control (Sep+/+) and Sep-/- oocytes are found in oviducts at comparable numbers, enclosed by cumulus cells. Example of cumulus enclosed oocytes from the oviduct is shown below. In (A), n indicates the number of mice and a mean number of oocytes per mouse of each genotype, error bars are + s.e.m.
(N) Quantification of autophagy phenotypes from panels D-M. SEM is indicated, with n = 8 ventral longitudinal muscles from individual animals and **p<.01; ***p<.001.
(C) M and Z α1-antitrypsin were subjected to equilibrium unfolding into different concentrations of guanidine hydrochloride (gdnhcl) in the presence and absence of GSK716, with bis-ANS dye added to report the presence of the unfolded intermediate. The normalised fluorescence intensity data were fitted with an equation describing a three-state unfolding curve. Values shown are the mean of three independent experiments and the error bars represent ±SEM.
WT primary hepatocytes were infected with an adenovirus harboring FERM-Myc (Ad-FERM-Myc) or LacZ (Ad-LacZ) for 48 hours. the nuclear localization of p-CREB after glucagon stimulation for 10 minutes (E) was determined by immunofluorescence staining. Scale bar, 20 μm.
(B) IF of DAXX and ATRX in zygotes at different pronuclear (PN) stages (PN0, n=8; PN2, n=6; PN5, n= 33). Data information: All panels: images of paternal and maternal pronuclei at given stage/condition are from same zygote.
(A) Line plots depicting fluctuations in gene expression levels from vsn normalized probe intensities across human T cell development for both αβ- and γδ-lineages (n=2 biological replicates and error bars indicate SEM).
Quantification of the SA-β-gal staining of HeLa cells stably transfected with Ctrl or FIS1 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days.
C Schematic overview of live microscopy using mCherry-tagged PCNA to monitor cell cycle progression. At the onset of S phase, nuclear dots are formed, which disappear when S phase is completed. PCNA leaks into the cytosol when cells become apoptotic.
(B) Analysis of autophagy over time. Cells were grown at 24°C and shifted to starvation medium at 24°C or 37°C for the indicated time periods. The cells were harvested and proteins were extracted from cells by TCA precipitation. The extracted proteins were analyzed by SDS-PAGE and Western blotting against GFP.
HAND2-AS1 overexpression enhances the capacity of oncosphere formation. Scale bar, 100 μm. oe: overexpression. Right panel: Data are shown as means ± SD (n = 3 per group).
D. Brightfield photomicrographs of LU-NB-3 and SK-N-BE(2) c cells treated with 0.36μM IBL-202 or 0.05 μM IBL-301. Scale bars represent 100 μm (LU-NB-3) or 200 μm (SK-N-BE(2) c). Arrows indicate neurite outgrowths and asterisks indicate where inserts are magnified. IBL-301 treated cells were stained for Tuj1. DAPI was used to visualize nuclei. E. Quantification of neurite outgrowth presented as number of neurites/cell in LU-NB-3 PDX and SK-N-BE(2) cells treated with IBL-301. For LU-NB-3 PDX cells, representative areas (n=2) were used and n=344 and n=240 cells/condition for CTRL and IBL-301, respectively, were counted. For SK-N-BE(2) c cells, representative areas (n=2 and n=3 for CTRL and IBL-301 respectively) were used and n=141 and n=130 cells/condition for CTRL and IBL-301, respectively, were counted. Statistical significance was determined by two-sided student's t-tes. P=0.003 for LU-NB-3 and p=0.08 for SK-N-BE(2) c.
E Representative fluorescent scan of an SDS-PAGE gel of APC/C-dependent polyubiquitination of Ub-CyclinB* by a panel of UBE2SHTH charge-swap variants. Quantification in Fig EV5C.
B Representation of the ubiquitin (UB) first neighbors subnetwork within the CBL and CBLB CN. Edge width is coded according to Pearson correlation coefficient (R<0.8 or R>0.8). Red edges depict direct interactions reported in the BioGRID database. Ubiquitin (UB) is highlighted in yellow.
Three representative protomers within a VCP hexamer are shown in the model. The D2 in the central protomer (N) is highlighted. The conformational changes observed in the four nucleotide states apo, ATP, ATP‡ and ADP over a cycle of ATP hydrolysis are schematically represented. The largest conformational changes occur between the preactivated states i and ii (apo and ATP) and the activated states iii and iv (ATP‡ and ADP). (i) In the apo state, the D2 domain (dark yellow) and D1-D2 linker (orange) are in a disordered state. (ii) Upon ATP binding, only small conformational changes occur. (iii) ATP interactions with D2 elements induce major conformational changes that result in an overall more ordered D2 and D1-D2 linker (orange) in the activated ATP state (ATP‡; red), with formation of the allosteric inhibitor binding site (violet). (iv) ATP hydrolysis leading to the ADP state induces a repositioning of the arginine finger (indicated by the dotted arrow) that in turn assists γ-phosphate release in the N + 1 protomer. Binding of allosteric inhibitors, which most likely occurs through interactions with the ISS motif, hampers the movement of the arginine finger toward the γ-phosphate, thus preventing ATP hydrolysis propagation and freezing D2 in the ADP-bound state.
(K) m6A RIP assay HPRT1 serves as negative control. *** P<0.001 as compared between indicated groups (mean ± SD, n = 3 biological replicates, Student's t‐test).
B Yeast strains of the indicated genotypes and carrying a YCplac33-ULP2 cover plasmid were streaked on SD+5-FOA to evict YCplac33-ULP2; cells were grown at 30°C for 3 days.
D, Quantification of the subcellular localization of the respective Fluc-EGFP protein in the presence of the indicated mHTT constructs. N=3 independent experiments. Three-way ANOVA with Tukey's multiple comparisons test. ANOVA: Localization, *p=0.019; HTT, n.s.; Fluc, n.s.; Localization x HTT, **p=0.0012; Localization x Fluc, ****p<0.0001; HTT x Fluc, n.s.; Localization x HTT x Fluc, ***p=0.0009.
A-B, In vitro analyses using primary mouse uterine epithelial cells demonstrated that TNFα induces the expression of annexin V, a marker of PS presentation at the outer membrane of Rb1f/f epithelial cells but does not in Rb1d/d ones, and P4 restores TNFα-primed annexin V expression in Rb1d/d uterine epithelial cells (mean ± SEM, Student's t test). Using three lines of primary mouse epithelial cells obtained from both Rb1f/fand Rb1d/d mice, the in vivo experiments were performed three times. Each of the annexin V-positive ratios was demonstrated. Scale bar = 100μm; green signal, annexin V.
Volcano plot highlighting differentially expressed genes (DEGs, false-discovery rate [FDR] <0.01; red upregulated and blue downregulated) at each study time point versus pre-vaccination b) Same as (a) but 24 hours post-vaccination (5553 DEGs, n=31)
In organello replication assays were performed on heart mitochondria isolated from 8-week-old control and Tefm knockout mice (n= 5 mice for each group). VDAC was used as loading control.
(g) DLD1 cells were stimulated with LY294002 or PKB inhibitor VIII for 24 h. Cell lysates were analysed for protein levels of LC3 and actin. Shown are representative blots (n=4). Uncropped images of blots are shown in Supplementary Figs S7 and S8.
J LAPTM4B N‐terminus binds multiple phosphoinositides including PtdIns(4,5) P2 in liposome‐binding assay.
(A) Subset of cell line-specific drivers that were identified as rCCS genes in this study. Highlighted entries indicate that the gene was identified as a rCCS gene in that modality.
(D) The ab initio shape envelopes of the xrRNA1-2s of DENV2 (left), ZIKV (middle) and WNV (right). The atomic models from rigid-body modeling by Xplor-NIH are superimposed onto the respective envelopes. The back-calculated scattering profiles of the atomic models were fitted to the respective experimental scattering profiles in (A).
(B) Autophagosome spatial distribution was quantified by measuring the distance of each LC3 vesicle from the nucleus (in pixels) and by analysing the frequency distribution.
usage.Determination of virus infectivity in HeLa cells with or without the expression of ACE2. h, human; b, Rhinolophus sinicus bat; c, civet; s, swine (pig); m, mouse. ACE2 protein (green), viral protein (red) and nuclei (blue) was shown. Scale bar=10 um.
G NEMO ubiquitination assay. HEK293T cells were transfected with different doses of Strep-tagged MARCH2 in the presence of a HA-tagged NEMO plasmid. Lysates were immunoprecipitated with anti-HA antibody followed by immunoblotting with anti-K48 antibody.
(F) Exponentially growing cells were treated for 1 hour with Torin1, which was added at increasing concentrations, 7 and 21 µM, to rich medium. Dimethylsulphoxide (DMSO) was used as the vehicle and added as a negative control.
(A) Immunofluorescence analysis of the old APPPS1 slice in co-culture using antibody 6E10 to detect amyloid plaques (green) and Thiazine Red (TR, red) to visualize fibrillar amyloid cores. At 7 DIV, the majority of amyloid plaques contain 6E10 and TR positive dense core surrounded by 6E10 positive halo of diffuse Aβ. In contrast, at 14 DIV, the majority of amyloid plaques are core-only plaques co-stained with 6E10 and TR without detectable 6E10 positive halo. Lower panels are higher magnification images of boxed regions. Scale bars: 50 µm.
(A) Representative western blots showing the level of SQR, TST, ETHE1 and SUOX proteins in two controls (C1, C2) and two patients fibroblasts (P3, P4). (B-E) Relative levels of proteins normalized to Vinculin.
Representative immunoblot of isolated mitochondria from the indicated groups treated with / without trypsin and probed for HSP60, ATP5a and TOM20. Bar graphs represent mean ± SEM of the ratio of densitometric levels of the indicated proteins (with : without trypsin) from at least 3 independent experiments
(D) Quantification of mitosis (phospho-histone H3 (PH3)+ cells) in low demand midguts at the indicated time points. **, P = 0.0015; n.s. not significant from one-way ANOVA test. Data information: In the boxplots, the box extends from the 25th to 75th percentiles and whiskers show all points, min to max. The line in the middle of the box is plotted at the median
human cells were infected with VSV-particles pseudotyped with the lineage B chimeric spike panel. Pseudotypes were either left untreated or incubated with trypsin before addition to the cells. Luciferase was measured and normalized to particles produced without pseudotype. Shown are the data for 3 replicates.
B, C Responsiveness of Unc93b1-/--3ddiTHP1 cells treated with T2.5 and challenged with heat inactivated (hi) bacteria (triangle, decreasing doses; n=3).
A. Facilitation of Fn biofilm formation by amyloid-like FadA. An aliquot of 10 μg each of the detergent-resistant pellets prepared from of Fn 12230 (wild type), US1 (∆fadA) and lam, or PBS, was added to the 96-well plates and incubated at 370C for 5 hours. Fresh culture of Fn 12230 was diluted to OD600 of 0.2 and an aliquot of 200 μl was added to the wells, followed by incubation at 37 0C for 48 or 72 hours under anaerobic conditions. Following washes, the biofilms were incubated with 100 μl 0.1% crystal violet for 15 min. After washing, 100 μl 95% ethanol was added to each well and the optical density at OD550 was measured in a microplate reader. The results shown are the average of four experiments each performed in duplicate. The error bars indicate SD. A representative image following 72 hours of incubation is shown in the top panel. ** p<0.01. ***p<0.001 (t-test).
Schematic representation of Aβ42 oligomers (right panel) and APOE4 (left panel) binding with soluble TREM2-Fc at various concentrations. Curves were fit by setting buffer control (no ligand) to y=0
Gel analysis of HAND2-AS1 RNA binding BMPR1A promoter in liver CSCs. LacZ and PVT1 served as negative controls.
ChIP assays comparing control IgG versus FOSL2 antibodies demonstrate specific recovery of the GUARDIN promoter by RT-PCR assay. UPe and UPk served as a positive and negative controls, respectively [54].
Correlation between protein and mRNA expression for differentially expressed proteins. Proteins that were down-regulated (< twofold change) in Dnmt2-deficient cells are indicated in red, and up-regulated proteins (> twofold change) are shown in green. The Pearson correlation coefficient is −0.03.
Ferrozine-based colorimetric measurement of intracellular iron in wild-type (wt) L6 and RHEB-Q64L L6 cells after iron treatment (50 µM or 250 µM) for 24 h.
(e,f) HeLa cells were cultured in complete or starvation medium for 30 min or in the presence of 100 nM of rapamycin for 3 h and then protein extracts were immunoprecipitated using anti-ULK1 antibody or with IgG as a negative control. Purified complexes and total extracts were analysed by western blotting as in a. Quantifications are shown on the right. In both panels, n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.003 and P = 0.0015, respectively.
(F) Model for the role of MCL‐1 in regulating both autophagy and apoptosis. See text for details.
(B) Schematic drawing of DiI injection and axon tracing. Axons that initially enter pyramidal tract fail to prune in crmp2-/- mice (red line).
Silencing of Nrp-1 by lentiviral particles expressing shRNA. After culturing 72 h in selection medium, RNA was harvested for qPCR analysis. NS shRNA vs. Nrp-1 shRNA: #1, *P = 0.017 and #2, *P = 0.02; n = 3; mean ± s.e.m. Cell lysates were subjected to SDS-PAGE and probed with anti-Nrp-1.
D, Heat map of untargeted metabolomics results showing the clustering of the normal - wild type (dark blue), normal - uninduced (light blue), tumor (yellow) and residual (green) populations and the most altered metabolic pathways (n=4). Hierarchical clustering was based on the complete linkage method using the Euclidean distance metric. Significance is defined as a false discovery rate < 0.05 versus the normal population, calculated using unpaired two-sided t-tests and adjusted for multiple hypothesis testing according to Storey's and Tibshirani's method PPP, pentose phosphate pathway; TCA, tricarboxylic acid cycle; OXPHOS, oxidative phosphorylation; AA, amino acids. Data information: Number of replicates corresponds to individual mice used to derive organoids.
(b) Quantification of the average number of rhabdomeres per ommatidium in Huntington's disease flies with and without pan-neural mGpx1 overexpression at days 1 (n = 14 (Htt93Q); n = 9 (Htt93Q + mGpx1)), 7 (n = 7 (Htt93Q); n = 9 (Htt93Q + mGpx1)) and 10 (n = 8 (Htt93Q); n = 9 (Htt93Q + mGpx1)) after eclosion. Significant effects of genotype (F1,50 = 82.3, P = 3.82 × 10−12) and age (F2,50 = 94.3, P = 1.06 × 10−17) were observed, as well as a significant interaction between genotype and age (F2,50 = 7.76, P = 0.00116), indicating that mGpx1 reduces the rate of neurodegeneration in Htt93Q flies. Post-hoc Newman-Keuls comparisons showed significant rescue of neurodegeneration in mGpx1-expressing flies at days 1 (*P 0.05), 7 (***P 0.001) and 10 (***P 0.001). Browne-Forsythe analyses for equality of variance revealed a significant inequality of variance (P = 0.02) due to a single group (Htt93Q + mGpx1, day 1). The nonparametric Mann-Whitney test confirmed a difference at day 1 between the two groups (P = 0.03), and between Htt93Q + mGpx1 flies at days 1 and 7 (P 0.0001).
A Schematic of the two-state model of transcription coupled to translation of a protein (TNF) that positively feeds back on its own transcription rate.
(j) Diffusion map of human AT2 cells colored by cell type identity and inferred pseudotime.
Model schematizing the proposed view of SMC5/6 action in genome integrity and DNA repair. SMC5/6 acts differently from non-homologous end joining and downstream of RAD51 filament formation and of homologous recombination branches mediated by DDX11, RAD17 and FANCD2, RAD17.
F Immunoblots of FACS-sorted RPE cells with stable expression of the FUCCI system. Cells were sorted based on expression of truncated versions of and Azami Green-tagged Geminin (amino acids 1-130) and Kusabira Orange-tagged CDT1 (amino acids 30-120), respectively. Blots are representative examples of four independent replicates derived from two different stable RPE-FUCCI clones.
E, HBMEC were treated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii (T) and then processed for electron microscopy at 5 h post-challenge. Images at the bottom represent magnification of the areas within the boxes. Arrow indicates the PVM; arrowhead indicates the double membrane structure around the vacuole.
Growth assay of wildtype and Art2P748A,P749A,Y750A (Art2PPY*) strains containing GID2 or GID5 deletions on SD-Lys (-) and SD-Lys containing 1.0 μg/mL thialysine (+). Spots represent 1:2.6 serial dilutions.
E, F, Overexpression of wt OTUB1 (E) or the catalytically dead mutant OTUB1 C91S mutant (F) promotes serum-induced MAPK activation in cell expressing wt NRAS. Whole cell lysates were analyzed by immunoblotting using the indicated antibodies.
Western blot analysis for CSTB in the CM from hCOs after 4 days in culture (C). The CM was collected from hCOs at different time points in culture (d19, d38, d60, d80). Protein extract (PE) from 60 days hCOs was used as a positive control. GAPDH immunostaining was used as a negative control on hCOs CM. Ponceau red staining is shown to indicate the presence of proteins at comparable levels in the CM samples
HDFs (C) were cultured in media containing increasing concentrations of r.h. TGF-β1 at 0, 0.1, 0.3, 1, 3 and 10 ng/ml for 24 h, before protein lysates or total RNAs were harvested. Western blot analysis of Smad7 was performed with protein lysates and semi-quantitatively analyzed with densitometry. β-actin served as loading control. Data is given as mean ± SEM of three independent blots. *