EMBO/sd-character-level-ner huggingface dataset:
C, D Accumulation of neutral lipid in the cytoplasm under conditions of uncontrolled autophagy. (C) Fixed epidermis from larvae expressing the indicated constructs was stained with BodiPy 493/503 to mark lipid accumulations. (D) Quantification of the number of lipid puncta in an epidermal area of 10000 µm2 in the indicated conditions. A-D, n=7-20 larvae each genotype.
(B) Representative dot plots showing CD86 and CD40 expression by low‑density BMDCs stimulated with poly(I:C) for different periods of IFN‑α exposure.
(A) Immunolocalization of transfected Myc‐UBQLN (1 or 2) and GFP‐tagged microtubule‐associated protein 1 light chain 3 (GFP‐LC3) was analysed by microscopy. Inset: magnification of UBQLN1 vesicular staining (arrow), which, after acquisition of Z‐stacks, was found in all UBQLN structures analysed.
Plotting of the position of the first base (5' end) of Alu RNA fragments across the Alu consensus sequence produced by Alu RNA that has been processed in vitro for 90 minutes at 37C in the presence of HSF1 (upper panel) and compared with one of the in vivo samples of Figure 1 (lower panel). The x axis represents an Alu RNA metagene aligned at the start site of the Alu consensus sequence and the y axis shows the 5' end count for Alu RNA fragments aligning to any position downstream of position +1. The in vivo sample x axis depicts a 11nt sift compared to the in vitro one.
G HEK293T cells transfected with His-tagged ubiquitin and HA-tagged NEMO WT or NEMO lacking the CC1 domain together with Strep-tagged empty vector or MARCH2 were treated with MG132 (10 μM) for 6 h before harvest. Lysates were subjected to immunoprecipitation with an anti-HA antibody, followed by immunoblotting with an anti-His antibody. WCL were subjected to immunoblotting with anti-HA, anti-Strep, or anti-β-actin antibody.
Immunoelectron microscopy of macrophages 10 h after infection with HSV-1. (a-c) Accumulation of LC3 (a,b) and gB (c). (d) Fusion of four-layered membrane structures and lysosomes preloaded with bovine serum albumin-gold (BSA-gold; black dots). Original magnification, × 54,800 (a,b), × 69,000 (c) or × 38,000 (d). Images are representative of three (a-c) or two (d) independent experiments.
Neuroblastoma cells were treated with indicated concentrations of IBL-302 for 48 hours C. Brightfield photomicrographs of LU-NB-3 and SK-N-BE(2) c cells treated with 50 nM IBL-302. Scale bars represent 100 μm (LU-NB-3) or 200 μm (SK-N-BE(2) c). D. Quantification of neurite outgrowth presented as number of neurites/cell in LU-NB-3 PDX and SK-N-BE(2) c cells treated with IBL-302. For LU-NB-3 PDX cells, representative areas (n=4) were used and n=460 and n=216 cells/condition for CTRL and IBL-302, respectively, were counted. For SK-N-BE(2) c cells, representative areas (n=5 and n=7 for CTRL and IBL-302 respectively) were used and n=124 and n=260 cells/condition for CTRL and IBL-301, respectively, were counted. Statistical significance was determined by two-sided student's t-test. p=0.009 for LU-NB-3 and p=0.0003 for SK-N-BE(2) 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).
(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.
(B) Schematic diagram of proximity ligation assay (PLA), wherein primary antibodies against Hrs or Tau are detected by secondary antibodies conjugated to DNA. Ligation and amplification of DNA, resulting in green signal, only occurs when proteins are in close (20-50 nm) proximity
(G) Spine density in DiOlistically labeled prefrontal cortex (PFC) is similar in WT and crmp2-/- mice in both juvenile and adult mice. Scale bars: 5 μm. (H) Quantification of dendritic spine density of PFC pyramidal neurons, basal dendrites (WT, juvenile: 8.63±1.44 spines/10 μm, adults: 7.3±1.4; crmp2-/-, juvenile: 9.05±1.73, p=0.97, adults: 6.98±1.53, p>0.99), mean ± SD, 2-way ANOVA with Bonferroni's multiple comparisons test. P25: n=3, ≥50 dendrites; adults: n=3, ≥50 dendrites.
(a) Erythroblasts (EB; mitochondriahigh Syto16high), reticulocytes (RC; mitochondriahigh Syto16low) and EC (EC; Ter119+ PBC) were collected from the indicated embryos (E18.5), and the expression of each protein was examined by western blot. The asterisk indicates a nonspecific band. Uncropped images are shown in Supplementary Figs 11 and 12.
G) Larval NSCs of control (ctrl; yw) and pr-set7716/Df at 24h ALH were stained for Ebd1, Dpn and Mira. Yellow dotted boxes, zoomed-in area. White arrows point to NSCs. Data information: Scale bars, 15 μm (G).
Basal protein and mRNA expression in parental and VAR cells for the indicated proteins was measured using immunoblotting (upper) and reverse-transcriptase PCR (lower), with GAPDH expression used for normalization.
Two to three-month-old hemizygous M83 mice were injected into the striatum with sterile PBS or 10 μg of recombinant α-syn fibrils. The mice were treated with saracatinib at 10 mg/kg/day by oral gavage for 4 weeks. C. Immunohistochemistry of pSer129 α-syn was performed. Arrow indicates pSer129 α-syn positive inclusion. Scale bar indicates 50 μm. D. Scoring of pSer129 immunoreactivity in the cortex and the striatum of both regions. Values were derived from eight individual mice (n=8). *** P < 0.001, ** P < 0.01, * P < 0.05 compared to PBS group, unpaired t-test. Data are expressed as mean ± SEM.
(F) Fed Dcp-1Prev1/Dcp-1Prev1;UASp-GFP-mCherry-DrAtg8a/nosGAL4 flies showed diffuse GFP-mCherry-DrAtg8a staining in the germline. (G) Starved Dcp-1Prev1/Dcp-1Prev1;UASp-GFP-mCherry-DrAtg8a/nosGAL4 flies showed reduced autolysosomes in degenerating midstage egg chambers. (H and I) Fed flies overexpressing truncated Dcp-1 (tDcp-1) in the germline showed increased autophagosomes and autolysosomes in nondegenerating midstage egg chambers (H) and also contained degenerating midstage egg chambers that lose all GFP fluorescence and fluoresce red (I). Bars: (main images) 25 µm; (insets) 12.5 µm. Insets in A-I show diffuse cytoplasmic staining of Atg8a or autophagosomes and autolysosomes. (J and K) The percentages of autolysosomes (autolysosomes/total autophagic structures) were manually calculated in at least eight egg chambers for each genotype as indicated. Error bars represent the means ± SD. Statistical testing was performed using one-way ANOVA with a Dunnet post test (****, P < 0.0001) or a two-tailed Student's t test (**, P < 0.005).
(K) The PARylation of KLF4 was increased after exposure to DNA damage. KLF4 complex was purified using immunoprecipitation from MDA-MB-231 cells that were treated with 5 µM doxorubicin and were then collected at different times (1h, 2h, 4h and 8h). PARylation was detected by using antibody against PAR.
Three pairs of TGF-β-treated isogenic NSCLC cell lines (H1975, HCC4006, and HCC366) and two mesenchymal NSCLC cell lines (Calu6 and H1792) were incubated with 50nM volasertib for 24 hours, lysed, and subjected to reverse phase protein array analysis. Experiments were done in triplicate.
D qRT-PCR analysis of naïve related genes in pRH6, 2a2iL-, and TGFβ2iL-RH6. **P<0.01, ***P<0.001. One-way ANOVA. Data presented as mean ± SD (n=3).
Figure 2. Dynamic reorganization of chromosomes during replication. (C) p(s), i.e., average intra-chromosomal contact frequency p between two loci with respect to their genomic distance s along the chromosome (log-log scale) during replication (color code identical to FACS profiles and PCA).
D-F. Over-expression analysis of the activated form of β-catenin (βcat S33Y). (D, E) Expression plasmids of βcat S33Y were introduced to P5 mice EGL and immunostaining for PROX1 (magenta) was performed the following day. Nuclei of electroporated cells were detected with GFP (green). Images of H2B-GFP (D, Control) and βcat S33Y (E, S33Y). Arrows indicate the cells of interest where GFP signals are co-localized with PROX1. (F) Proportions of PROX1-positive cells in electroporated cells. Bars and error bars are representing as Mean± SEM. Student T-test between Control and S33Y was performed. ***p < 0.001. Number of cells: 89, 90 and 55 cells from N=3 mice (Control), 32, 111 and 56 cells from N=3 mice (S33Y).
(E) Representative images of DLD-1 CENP-B (+/+) or (-/-) cells in late M phase following one CENP-AOFF/ON cycle. EdU staining was used to confirm successful wash-out of Palbociclib and cell progressing through S-phase. Yellow dashed lines contour nucleus of cells in late M phase. Scale bar 5 µm. (F) Quantification of indicated events observed in late M phase cells in the indicated cell lines. Error bars show SEM from 4 independent experiments.
(A) L2 was tagged with HA at the C-terminus and expressed from the doxycycline-inducible promoter.
D. Immunofluorescence staining of Prom1 in mouse liver tranduced with adenoviral particles. Confocal microscope images of WT mouse liver infected with adenovirus harboring sh-Con or sh-Rdx linked with IRES-GFP. Scale bar, 20 μm.
G Representative images depicting CAG-GFP-IRES-Cre transduced adult-born neurons in control and β-catex3 mice at 17dpi. Scale bar= 20µm
RNA sequencing (RNA-seq) of week 15 COs and transcriptome analysis (WT: n=2, KO: n=4). qPCR analysis for selected Wnt target genes validating the results of the RNA- seq. Y-axis indicates relative expression fold change. Data are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=4 from 1 batch, KO: n=4 from 1 batch, W-AAV: n=4 from 1 batch). Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
C Phospho-kinase array was performed to find the mechanism by which ANGPTL4 increased C5a in human THP1-derived macrophage cells (n=3). Cells were treated with ANGPTL4 (100 ng/ml) for 6 h. After phospho-kinase array analysis, the expression of AMPKα, JNK, AKT, P70S6K, and EGFR was confirmed by western blotting.
2B4 NFAT-GFP reporter cells transduced with TREM2 or AD variant R47H were incubated with various concentrations of Aβ42 oligomers for 12 hours. GFP expression was detected at 1 µM Aβ42 oligomers with TREM2 transduced cells. No GFP expression was detected with Aβ42 oligomers from control transduced cells. Results were expressed as the percentage ± GFP cells averaged of ± standard error (n = 3, * p < 0.05 compared with mock, **** p< 0.0001 compared with mock, # p < 0.05 compared with TREM2, #### p < 0.0001 compared with TREM2, Two-way ANOVA, Bonferroni multiple comparisons, see Appendix table S3 for p value)
B, Serum P4 levels on days 4 and 6 of pregnancy were comparable between Rb1f/f and Rb1d/d mice. In Rb1d/d mice, pre-implantation P4 treatment on days 2 and 3 increased serum P4 levels on day 4 but did not affect those on day 6 (mean ± SEM, Student's t test; n=4 mice for each group).
Top: Relative mRNA expression of Ecad after treatment with 2.5μM ionomycin or 10ng/ml TGFβ for denoted time. Bottom: Western blot analysis of total E-cadherin and Vimentin protein for denoted time. Experiment was run in duplicate with three different murine PDA cell lines. Statistical analysis by ANOVA (***, p<0.001; non-significant (NS); ±SEM).
HSF1 expression was diminished in MCF10A cells for 48 hours using siRNA prior to treatment with either DMSO, EtOH, DMSO, 25uM 4HNE or 5uM RSL3. Isolated protein was assessed by immunoblotting for prominin2, phospho-HSF1 (Ser326), total HSF1, phospho-p38 (Thr180/Tyr182), total p38, and β-actin expression. Shown is one replicate of three independent experiments (n=3).
Figure 2. Dynamic reorganization of chromosomes during replication.(A) FACS profiles and representative DAPI-stained cells of G1 synchronized cells released in S phase.
(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).
(D) Confocal images of live GRAMD1 TKO HeLa cells that stably expressed either EGFP control or EGFP-GRAMD1b constructs as indicated. Cells were stained with recombinant mCherry-D4H proteins (10 mg/ml) (accessible PM cholesterol biosensor) for 15 min at room temperature before imaging. Insets show at higher magnification the regions indicated by white dashed boxes. Note that expression of EGFP-GRAMD1b-WT, but not EGFP-GRAMD1b-R189W, reduced binding of mCherry-D4H to the PM, in GRAMD1 TKO HeLa cells. Further reduction in mCherry-D4H binding to the PM was observed in GRAMD1 TKO HeLa cells that stably expressed EGFP-GRAMD1b-G187L compared to GRAMD1 TKO HeLa cells that stably expressed EGFP-GRAMD1b-WT. Scale bars, 10 µm. (E) Values of mCherry-D4H signals at the PM after background subtraction, as assessed by confocal microscopy and line scan analysis of GRAMD1 TKO HeLa cells that stably expressed either EGFP or indicated EGFP-GRAMD1b constructs as shown in (D) [mean ± SEM, n = 30 cells for all the conditions; data are pooled from three independent experiments; Dunnett's multiple comparisons test, **P < 0.0001].
C. BMDM cells, as described in panel (A), were additionally treated with DMSO (control), EIPA, or Akti-1/2(Akt1/2 inhibitor) as indicated, for 1 hour after CCL5 stimulation. Representative immunoblots from n=3 biologically independent experiments are shown. D. Relative pSer473-Akt levels were normalized to total Akt and calculated as fold of naïve cells (without CCL5 stimulation). Each dot represents one experiment, with error bars showing ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with *** representing P< 0.0005, ****P< 0.0001.
(I) Quantitative RT-PCR analysis of expression of MMP2, MMP9, IL6, and the JAK-STAT targets IFNAR1 and IFNAR2, in normal placentas and placentas from IUGR-complicated pregnancies. Results are from four human normal placentas and four placentas from IUGR-complicated pregnancies. Data information: Values are means + SEM of at least three experimental repeats. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) P<0.001.
FACS analysis of the concentration‐dependent effect of BMP4 on TRA‐1‐81+/cKIT+ PGCLCs on day 4. B25: 25 ng/ml BMP4; B50: 50 ng/ml BMP4; B100: 100 ng/ml BMP4.
MCFDCIS xenograft tumors have increased expression of AIB1-YAP, EMT, and 1q21.3 genes during the invasive transition. Schematic of experiment shown. Gene set enrichment analysis of RNA-seq data generated from MCFDCIS xenografts at indicated time points.
(C) The p53-activating drug Tenovin-1 (TEN1) protects spinal cord motor neurons from SOD1G85R-induced proteotoxicity. Rat spinal cord cultures were grown as described in Materials and Methods and treated with 0.8 μM TEN1 or vehicle (VEH) DMSO, followed by infection of HSV-SOD1G85R or the HSV-LacZ control after 24 h. At day 5 post-infection, cells were fixed and stained with a motor neuron-specific anti-neurofilament H (NF-H) antibody. Left: representative images of motor neurons in each condition. Scale bar = 50 μm. Right: quantification of motor neuron survival (one-way ANOVA with multiple comparisons and Tukey correction). Data represent means ± SEM.
Purified IL-2-stimulated NK cells (100 U/ml for 4 days, 106) were incubated with the SRC kinase inhibitor PP2 (25 μM), the vehicle DMSO, or medium only (Ø) for 30 min before being added to immobilized MICA-129Met-Fc, MICA-129Val-Fc, or OVA-Fc fusion proteins (10 μg/ml) for 10 min. The protein lysates of these cells were separated by SDS-PAGE, and the blot was probed subsequently with an anti-phospho-Tyr mAb and an anti-β-actin mAb as a loading control. The blot is representative for two independent experiments.
Proliferation curves of parental and AGO2KO HeLaS3 cells are plotted (n=6 experimental replicates).
Significantly reduced numbers of RA-SVs in IHCs of 1/2-DKO compared to Wt after stimulation, as quantified using random ultrathin sections of ribbon-occupied AZs from several IHCs per genotype. Mann-Whitney test; ***p < 0.001.
E. The relative abundances of abundant bacterial taxa (> 0.5% of the mean abundance).
(h) The bar graph shows the gene categories most strongly correlated with CRTAC1 belonging to the GO category of "transcription regulators". The dotted line marks a correlation coefficient of zero.
The effect of candidate genes on HIV latency was further verified in J-Lat 10.6 (B) models of HIV latency. GFP expression in J-Lat 10.6 cells was analyzed by flow cytometry
C,D Bar graphs showing ex vivo radiance ± SEM in different organs harvested from females bearing MM134 or SUM44 intraductal xenografts for the mice described in B. Statistical significance determined by Student's unpaired t-test, two-tailed. * p < 0.05, ** p < 0.01, n.s not significant.
(D) Maximum Ca2+ flux after stimulation under Ca2+ free conditions and after Ca2+ was added back. Two-tailed paired t test, ns = not significant, *P < 0.05.
D, E. Heatmaps showing the variations in RNAPII kinetic rates (Log2FC) on Myc-dependent (D) and Myc-independent (E) genes during the LPS time-course. The grey scale represents the starting level for each parameter in unstimulated cells. The first column on the left reports the changes in Myc share for the same genes. Genes were grouped using hierarchical clustering.
(A_B) Vero E6-TMPRSS2 (A) or Vero E6 (B) cells were inoculated with a total of 126 clinical NP swab samples representing a range of viral RNA levels, both total E RNA (Clinical Sample E CT detected by WHO-E primer/probe set) and subgenomic E RNA (CT detected by the Mills-SgE primer set developed in this study). Culture supernatants were harvested when cells displayed CPE or at day 7 if no CPE was observed earlier, clarified, and presence (blue square) or absence (yellow circle) of SARS-CoV-2 determined by immuno-focus assay.
(E) Dot plots show quantitation of intratumoral vessel density, mural cell coverage using Desmin and aSMA co-staining, and tumor necrosis (mean ± SD, n = 9 mice). All comparisons were rendered non-significant according to two-tailed Mann-Whitney U test.
E. Accumulation of macro-ER-phagy cargos in aberrant intracellular structures using live-cells microscopy. Shown from left to right: DIC, GFP, DsRed and Merge. Shown from top to bottom: Snq2, Snc1-PEM, Snq2+Snc1-PEM, and % cells with co-localization (relevant only for co-overexpression) (% cells with ER-phagy cargo accumulation in aberrant intracellular structures is shown in S5A Fig). F. Fluorescence level of intracellular structures (ratio over PM) in atg11∆ mutant cells. The bar graph shows Snq2-yEGFP (green) and Ds-Red-Snc1-PEM (red) fluorescence (20 cells were analyzed for each strain). When co-overexpressed in atg11∆ mutant cells, the fluorescence level of either protein accumulating in aberrant structures is ~5 fold higher than when overexpressed individually. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.
(F) Meta plots showing CHR11 and PIE1 occupancy on PIE1-dependent H2A.Z target genes. TSS, transcription start site; TTS, transcription termination site. The CHR11-Flag and PIE1-Flag ChIP-seq data are from two independent biological replicates.
E GSEA enrichment plots demonstrating downregulation of YAP, E2F1, EGFR and c-MYC pathway signatures after YAP/TAZ knockdown in resistant SKMEL28 and WM3248 cells.
(D) Splenocytes from Pacs1+/+ and Pacs1−/− mice were stained with cell surface antibodies to identify FOB cells and treated with 100 µM H2O2 for 35 minutes. Cells were then labelled with TMRE to monitor MMP by FACS analysis. Low TMRE fluorescence indicated susceptibility to H2O2 treatment. Data are presented as mean ± SD of 3-4 replicates, two-tailed unpaired t test, ***P < 0.001. Results are representative of three independent experiments performed on different Pacs1+/+ and Pacs1−/− pairs.
Rad53 phosphorylation was analyzed in WT and rtt105∆ cells by immunoblotting of protein extracts with anti-Rad53 antibodies. Ponceau S (Pon S) staining was applied as a loading control.
B,C- Pol II pause position remains unchanged upon CTD shortening. Histogram of estimated positions of paused Pol II in RPB1-52R and RPB1-25R cells at genes encoding major isoforms with constitutive first exon longer than 100 bp.
(D) Effect of CCB02 in the presence of control siRNA and CPAP siRNA in MCF10A (i) MDA-MB-231 cells (ii) CPAP depletion does not prevent centrosomes from clustering (second rows of i and ii). Note, in CPAP depleted cultures, centrosomes are marked with Cep152 (green) where CPAP (magenta) is detected faintly. In CPAP depleted cells, CCB02 does not prevent extra centrosomes from clustering (fourth rows of i and ii). Note CCB02 does prevent extra centrosomes from clustering in cultures treated with control siRNA (third rows of i and ii). Cells are stained for Cep152 (green), CPAP (magenta), microtubules (α-tubulin, red) and DNA (DAPI, blue). Scale bar, 2µm
Immunoprecipitation assays of biotin-labeled CHIRP probes incubated with liver CSC lysates.
B Estimated frequency of rearing activity as measure of exploratory drive in the open field (ANOVA: genotype F1, 24=39.40 p<0.0001, age x genotype F2,26=8.204 p=0.0017). Mutants showed little exploratory activity at 9- and 14-months.
D, E BMMΦ from wt and iNOS-/- mice were stimulated with LP and aminoguanidine (1mM) for 6 days and lysed with RIPA-buffer for western blot analysis of γH2Ax (D)
(D) Sequencing clusters obtained from HITS-CLIP experiment. Left are for let-7, and right for tRNAs.
(G). Confocal analysis of PAR and FLAG-tagged KLF4 showing that KLF4-Zinc 2-YYR/AAA mutation significantly reduces KLF4 PARylation in U2OS cells. Scale bars, 10 μm.
A) gfp::znfx-1; pgl-1::tagrfp animals were mutagenized with ethyl methanesulfonate (EMS). F2 progeny of mutagenized animals were fed bacteria expressing dpy-11 dsRNA. Mutant animals that failed to inherit dpy-11 RNAi (were non-Dumpy) were isolated. Lineages established from these animals were examined under a 63X oil objective on a Zeiss Axioplan fluorescence microscope for defects in number, size, morphology or subcellular distribution of Z granules using GFP::ZNFX-1 as a marker of Z granules.
Figure 2: FMN2 is highly expressed in mossy fibers and is required for synaptic depotentiation.C. Representative immunoblot images showing FMN2 levels in the hippocampus of wild type and Fmn2 -/- mice
(K) Translocation of the trans-Golgi protein to the vacuolar membrane induced by AmphoB treatment. GFP-Sft2-expressing atg5Δ cells (trans-Golgi marker protein) were incubated with or without AmphoB (2.5 µg/ml, 24 hr), and localization of GFP-Sft2 was assessed by confocal microscopy. Arrowhead indicates the GFP signal on the vacuolar membrane. Scale bars = 2 µm.
(C) DCs and LCs were infected with SARS-CoV-2 pseudovirus and infection was determined after 5 days by measuring luciferase reporter activity. As positive controls Huh7.5 cells were infected. Data information: Data show the mean values and error bars are the SEM. ordinary one-way ANOVA with Tukey's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (n=4 measured in triplicate), DCs: Dendritic cells, LCs: Langerhans cells, RLU: relative light units