EMBO/sd-character-level-ner huggingface dataset:
C) Data confirming that paclitaxel treatment significantly increases (adjusted p value = 0.0001[***]) the number of pH3+ cells in the hair matrix This effect was not observed when paclitaxel treated hair follicles were pre- and co-incubated with palbociclib. Analysis performed using N of 6-9 HFs per condition from 3 patients Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.
(H) Average number, normalized on cell size, quantified by image (as in e) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of LAMP-1+ endosomes. Results are the average ± SEM of two independent experiments for a total of 436 late endosomes in 13 shCTL/GFP cells (34 ± 6 endosomes per cell), 1452 late endosomes in 12 shSTIM1/GFP cells (121 ± 13 endosomes per cell), 615 late endosomes in 12 shSTIM1/GFP-STIM1 WT cells (51 ± 5 endosomes per cell), 1278 late endosomes in 12 shSTIM1/GFP-STIM1 NN cells (106 ± 15 endosomes per cell) and 506 late endosomes in 12 shSTIM1/GFP-STIM1 AA cells (42 ± 7 endosomes per cell) and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for shCTL/GFP and shSTIM1/GFP p≤ 0,001 ***, for shCTL/GFP and shSTIM1/GFP-STIM1 WT p≤ 0,001 ***, for shSTIM1/GFP-STIM WT and shSTIM1/GFP-STIM1 NN p≤ 0,05 *, for shSTIM1/GFP and shSTIM1/GFP-STIM1 NN p≤ 0,01 ** and for shSTIM1/GFP-STIM WT and shSTIM1/GFP-STIM1 AA p> 0.05 not significant (ns).
Percentage of labeled threonine and deoxyribose from protein and DNA hydrolysate shows de novo protein and DNA synthesis in non-dividing cells. After 6 hours of pulsing uniformly labeled 13C-glucose, cultures were lysed, and their macromolecules were washed free of latent metabolites and hydrolyzed to monomers. Labeling data are presented as the mean ± standard error of independent biological replicates (n = 3, all pairwise P < 0.02 as determined by one sided Student"s t-
Protein extracts from control or Tgif1KO mice (right) were immunoprecipitated using normal IgG, anti-TGIF1 or anti-Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 To control for loading, total cell lysates were analyzed by immunoblotting using antibodies to Twist1, TGIF1 or β-Actin.
(e) Pearson correlation coefficient observed in segmented E10.5 homozygous Venus::HES5 spinal cord ex-vivo slices showing correlation between mean nuclear Venus::HES5 intensity in any cell compared to up to 8 nearest neighbours (see Materials and Methods); dots indicate average per slice; bars indicate mean and standard deviation of 5 slices from 3 experiments (dataset is different from (d).
C-E. Western blot analysis for CCNA, CCNB, Cdk1 and GAPDH (loading control) protein expression in lysates from control (si-GFP) and YAP-depleted (si-YAP; si-YAP_1) (D) MDA-MB-231 and CAL27 cell lines. Representative images are shown.
(C) Ribbon diagram of the 2.56-Å resolution structure of pentameric PfFNT viewed from the parasite's cytoplasm. The five protomers of pentameric PfFNT are colored yellow, cyan, orange, slate and purple. (D) Ribbon diagram of pentameric PfFNT viewed from the parasite's membrane plane. The five protomers of pentameric PfFNT are colored yellow, cyan, orange, slate and purple. (E) Ribbon diagram of pentameric PfFNT viewed from the extracellular side of the parasite. The five protomers of pentameric PfFNT are colored yellow, cyan, orange, slate and purple. (F) Structure of a protomer of PfFNT. This ribbon diagram is colored using a rainbow gradient from the N-terminus (blue) to the C-terminus (red).
F Ratio of toxicity and viability in HCT116 spheroids treated as indicated for 48h (NEN 1.2 µM, Domperidone (Domp), Imipramine (Imi), Desipramine (Desi) and Amitriptyline (Ami), each 30 µM; Clomipramine (Clomi) 20 µM, ISRIB 1 µM).
Number of unique phosphorylation sites defined at different site localisation confidence values: ptmRS ≥ 0.99 (green); ptmRS ≥ 0.90 (yellow); ptmRS ≥ 0.75 (red). Light green, yellow or red indicate those sites of pLys or pArg mapped to the extreme peptide C-terminal residue at each ptmRS score cut-off.
M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). (E) Western Blot analysis of phenotype-switch markers on cell lysates from M238P cells transfected with the indicated siRNAs.
(F) Correct MHE innervation is required for hatching of Drosophila embryos from the eggshell (Friedrich et al., 2016). Hatching rate was calculated based on the number of L1 larvae observed after 24h in genetic crosses, depleted of Dfd (UAS-DfdRNAi) in neurons (elav-GAL4; n=217) compared to crosses with control animals (mock = elav>RFP; n=156). P-values were calculated by a two-sided Fisher test, ***: p < 0.005.
(B) Western blot analysis of PKM2, PKM1, NRF1, TMEM33, RNF5 and SCAP levels in MEF (PKM2fl/fl, Cre-ERT2) cells treated with 4-OHT in a time course experiment.
D) Neighboring cells can form new attachments underneath (basal to) a mitotic cell that has moved apically. Scale bars = 5 μm. Thin yellow lines in the XZ reconstructions indicate the z-position of the two XY focal planes
Representative pictures of dissected abdominal region of KC and KTC mice. Yellow arrows indicate metastatic nodules in liver. Yellow dotted lines show the pancreas.
A. Permeability assay using vibratome section showing the cadaverine 555 dye (red), CD31 (green) and CD13 (purple) in the brain vasculature of Glut1iECKO mice with (bottom) or without (top) lactate administration. Note the extravascular accumulation of cadaverine dye in control Glut1iECKO mice, but not in lactate supplemented Glut1iECKO mice.
E. Analysis of GO terms. Significance cut-off was set as α = 0.01 with at least 3 proteins identified in the respective group. GO term enrichment factor expresses the relative over-representation of the GO term within the group of proteins containing a phosphorylation site that is more abundant in WT compared with KD compared with the group of all proteins.
(P-R) Representative photomicrographs of GFAP immunostaining of the CA1 region (P, Q) and quantitation of GFAP immunoreactivity (R).
(K) Validation of CRISPR KO clones was assessed by PCR amplification using primers flanking the targeted AhR exon. Subsequent sequencing revealed indels at the expected locations.
C BMDMs from WT or TLR9−/−mice were infected with swollen conidia (MOI = 1), and after overnight stimulation, TNF-α was measured in the SN by ELISA. TLR9−/− BMDMs had significantly lower TNF-α levels.
C Binding of nanobodies to the quadruple RBD mutant was monitored by BLI. Immobilized RBD was incubated with 100 nM of nanobodies for 450 s, followed by assay buffer for 900 s.
(B) An in vitro cleavage assay was performed through mixing GSDMD and its mutants with caspase-1 p20/p10 complex, followed by immunoblotting with antibodies against the indicated protein tags. β-actin served as a loading control. FLAG-tagged GSDMD and its mutants were immunoprecipitated from HEK293T cells using anti-FLAG coupled magnetic beads. HA-tagged p20 and p10 were immunoprecipitated from HEK293T cells using anti-HA coupled agarose beads. Data information: Experiments were repeated for three times with similar results.
D Distribution of the IDO1.WT protein expressed by P1.HTR transfected cells in isopycnic sucrose gradients. Fractions were immunoblotted using antibodies specific for IDO1 and intracellular organelles or structures as indicated.
Quantification of tumor size in tibiae at 5-6 months of age (n=22 wt, 13 ColAREG)
G Parameters related to heart function generated by Echocardiography (ECHO) and Electrocardiography (ECG), n = 15 f, 15 m Uqcrh-/- and 15 f, 15 m WT mice, 6 weeks old.
(C, D) Targeted bisulfite-sequencing heat map showing non-conversion rates of cytosines in mitochondrial tRNA Met-CAU (C) and mitochondrial 18S rRNA (D). Each row represents one genetic strain analysed and each column represents one cytosine. Data information: In (C, D), the average of two experiments is plotted, n = 2 independent biological replicates, 10 clones sequenced per strain, per replicate. Similar effects were observed in all replicates analysed.
(B) Sagittal brain stem sections from gba+/+, gba+/−, and gba−/− mice were stained with α-synuclein antibodies. Scale bar, 1,000 μm.
(F) neurite length measurement and representative tracings (G) of primary cortical neurons of WT and Mfn2 KO mice co-transfected at DIV4 with GFP and the indicated plasmids. After 72 h, the neurons were fixed, immunofluorescence with anti-GFP antibodies was performed and neurite length was measured (n= 30 neurons from 3 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05 versus WT; #p<0.05 versus Mfn2 KO neurons, one-way ANOVA followed by Tukey's post-hoc test. Scale bar: 500 μm. E
D LAMP1 puncta in GCN5 KO HEK293 cells overexpressing Myc-tagged GCN5 or GCN5-E575Q (Scale bars, 10 µm).
(I) Expression of the selected glycolytic genes was quantified by RT-qPCR. Hsp90ab1 was used as the normalization control.
Venn diagram of intersection of down- and up-regulated protein-coding genes in Epi-HOT, -HOTΔP and -HOTΔL cells compared to Epi-CTR. HPL-neg and HPL-pos are gene sets down- and upregulated only by the full-length HOTAIR (Dataset EV2).
I RNA level of FOXO3 in YTHDF1-knockdown Bel-7402 cells by RT-PCR. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.
(B) A cartoon detailing how the heat shock controlled dual-color system driven by GreenEye-nanos-Gal4 labels preexisting (old) histones with eGFP (green fluorescent protein) and newly synthesized (new) histones with mCherry (red fluorescent protein), respectively.
(E) Representative images showing the phospho-eEF2 immunolabeling (Pseudo-colored) and newly synthesized proteins as FUNCAT signal (Pseudo-colored) in cortical neurons on the basal condition and on NMDA treatment for 5 minutes in the presence or absence of Compound C. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.
Heat map showing relative protein fold changes for members of selected complexes affected by aging. Names of significantly affected members in the 39 wph vs. 12 wph comparison (adj. p < 0.05 and absolute log2 fold change > 0.5) are highlighted in bold with a star. Related to Figure EV3 and Dataset EV5 and EV6.
(D) Expression of FLAG-FBW7α enhanced HA-SOX9-WT protein turnover, which can be attenuated with addition of 10 µM MG-132 (MG) in HEK293 cells. By contrast, forced expression of FLAG-FBW7α did not enhance HA-SOX9-T236A and -T236A/T240A protein turnover over the course of 4 hours protein chase. Protein loading for each sample was indicated by the level of β-actin protein. The set of blots shown is representative from 4 independent experiments.
(C) Samples were subsequently analyzed by 2D-BN/SDS-PAGE and immunoblotting with the indicated antibodies. The identities of MRC complexes and SCs are as in Figure 1.
Figure 6. Model for Mmi1/exosome YTH-mediated control of cell differentiation and heterochromatin gene silencing mediated by its targeting of nascent lncRNA and the induction of their transcription termination. The YTH domain of Mmi1 co-transcriptionally binds to specific lncRNAs expressed from either euchromatin or heterochromatin regions. We propose that Mmi1 binding to a euchromatic nascent lncRNA (in green) induces the recruitment of the exosome and together they degrade the lncRNA and promote robust termination of the lncRNA transcription, which otherwise will inhibit the expression of the downstream protein-coding gene (in blue). In the case of nam1-byr2 locus, the efficient transcription termination prevents the occurrence of read-through transcription from nam1 gene, which represses byr2 MAPKKK gene, a critical regulator of the entry into sexual differentiation. In parallel, Mmi1 co-transcriptional binding to the heterochromatic pericentromeric lncRNAs also recruits the exosome and contributes to heterochromatin gene silencing by degrading the nascent lncRNAs as well as by inducing precocious termination of their transcription. STOP sign, site of Mmi1-dependent lncRNA transcription termination.
(c) The competitive p62 activity against Keap1 was measured by luciferase assay. The expression plasmids for Nrf2, Keap1 and p62 wild-type (w.t.) or its mutants were transfected into Hepa1 cells along with pNqo1-ARE reporter plasmid and pRL-TK as an internal control. At 36 h after transfection, the luciferase activity was measured in accordance with the instructions provided by the manufacturer. Assays were performed twice in triplicate. Data are means and s.d. for six determinations.
Representative ProQ Diamond-stained 2D gel images of isolated proteins from testes in WT (left) and KO (right) mice. Testes lysates were separated by 2DE, and phosphoproteins were stained with ProQ diamond. Images in the boxes are enlarged in insets shown below. Circles with numbers indicate spots with significantly changed volume relative to those on WT gels.
(C) Schematics of normal BET1 exon 3-4 splicing (top) and the various aberrant splice products associated with the paternally inherited p.(Asp68His) variant in P1. Splice variant one utilizes normal splicing generating a full-length BET1 protein with the Asp68His substitution. Splice variant two skips exon 4 to non-coding transcript (NR_133908) exon 5, resulting in a shift of the reading frame and a premature termination codon after 18 amino acids. Splice variant three utilizes a cryptic splice acceptor within exon 4, deleting the first 34 nucleotides of exon 4, and producing again a frameshift and a premature termination. The maternally inherited frameshift variant c.134delC introduces a premature stop codon in exon 2 and is predicted to induce nonsense mediated decay (NMD).
(F) SMAD3-signature in pre-treatment biopsies from responders and non-responders to anti-PD-1 treatment (from the Hugo's cutaneous melanoma cohort, n=15 and 13 respectively) (Hugo et al, 2016).
WT and Mfn2 KO cells were transfected with plasmids encoding: G) GFP and ChiMERA or control (CT) plasmid. The effect of caffeine-induced (20 mM) ER Ca2+ release on mitochondrial Ca2+ was measured (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM. H) GFP and ChiMERA or control (CT) plasmids. The effect of ATP-induced (100 μM) ER Ca2+ release on mitochondrial Ca2+ was measured (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM. I Data information: *p<0.05, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.
(G-I) NIH3T3 cells stably expressing CFP‐Gal3 and YFP‐LC3, and either empty vector (control) (G, H) or mStrawberry‐Atg4BC74A (I) were treated with 1000 μM LLOMe for 2 h, fixed, and observed by confocal microscopy. The electron micrographs were taken in the same sample field as the transmission electron microscope. Green: LC3; blue: Gal3 and DAPI; black arrow: single membrane; white arrow: autophagosome; white arrowhead: ER membrane.
(F) Venn diagram displaying an overlap of Ig domain genes expressed in MNs and genes that are bound by Dfd based on the Dfd ChIP-Seq performed in a whole embryo (Sorge et al, 2012). p-value was calculated using a hypergeometric test using as a reference all protein coding genes n=13920.
(A) IFNγ primed WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were treated with LPS (5 µg/ml) for 4 h. Following treatment, cells were stained with anti-ASC antibody and Hoechst (DNA/nuclei). Representative images of ASC specks (white arrows point at specks) are shown and number of ASC specks per nuclei quantified. Scale bars: 20 µm.
A Gene Set Enrichment Analysis (GSEA) output images of cellular response to stress pathways displaying a correlation of differentially regulated genes in METTL3-knockdown HepG2, SMMC-7721 and Bel-7402 cells with the C5. biological process symbol set. (p < 0.001).
d, Vacuole morphology of cells expressing Atg18Sloop-GFP, imaged before and after osmotic shock as in Fig 1a. Scale bar: 5 µm.
(D) Representative Western Blot analysis of increasing amount of GST-STIM1 (cytoplasmic domain) pulled down by Cap-Gly-CC1-p150Glued-V5-coated beads, in absence or presence of FLAG-EB1 C-terminal. Colors label the protein as in (c). Negative control was performed using equal amount of empty GST protein, together with Cap-Gly-CC1-p150Glued.
(G) Confocal microscopy images of nuclei assembled for 120 min in mock depleted, Nup50 depleted (∆Nup50), and Nup50 depleted Xenopus egg extracts supplemented with recombinant Xenopus wild-type Nup50 or RCC1 binding mutants. Nuclei were fixed in 4 % PFA and 0.5 % glutaraldehyde, stained for NPCs (mAB414) and the chromatin (DAPI). Scale bar: 10 µm. Quantitation shows the average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of three independent experiments. Individual data points are indicated (H) Confocal microscopy images of nuclei assembled for 120 min in mock depleted, Nup50 depleted (∆Nup50), and Nup50 depleted Xenopus egg extracts supplemented with RCC1 excess as indicated. Nuclei were fixed in 4 % PFA and 0.5 % glutaraldehyde, stained for NPCs (mAB414) and the chromatin (DAPI). Scale bar: 10 µm. Quantitation shows the average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of three independent experiments. Individual data points are indicated.
F. Heatmap showing the expression dynamics of MOS-regulated decay genes in GV and MII oocytes from control or MOSAsn95Lys patient.
e) Southern Blot analysis to detect integration events in LV- and NILV-S/MAR-transduced single cell clones with DNA probed against the viral genome. A 6.8 kb positive control from a linearized plasmid (lane 3) was included for indication of the size of DNA circles formed by non-integrated NILV-S/MAR vector.
D-F Plots of reciprocals of EC50 vs. the effective in-situ affinities (AcKa) (D), the effective in-situ on-rate (AcKon) (E) and in-situ off-rate (koff) (F).
(I) Left: representative images of TA muscles isolated from Ctrl or YY1iKO mice 60 days post-CTX injury. Right: Masson's Trichrome staining of the above muscles to visualize the degree of fibrosis. Scale bar = 100 µm.
A. G-overhang assay for clone 35 upon POT1 removal. The bars show relative amounts of single stranded telomeric DNA ± SD. Experiments were performed in triplicate.
H&E (P2, left), ITGA6 (P2, middle) and LOR staining (P4, right) of Col17a1-/- mice (top) and littermate controls (bottom) . The regenerated epidermis is indicated by arrowheads. Scale bar: 200 μm.
The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). D: Cells were stimulated with 20 ng/mL 555-EGF for 15 or 30 min before immunofluorescence staining against EGFR. Scale bar: 10 μm.
E, F: Jurkat cells were transfected with expression vectors encoding GFP, GFP-FIP3-WT or GFP-FIP3-I738E. Endogenous Rac1 was detected by immunofluorescence.
(e) Confirmation of the CRISPR/Cas9 mediated KO of Samhd1 in CH12F3-2A cells. Beta-actin was used as a loading control.
C) Sidedness is affected by planar division orientation in an in silico simulated epithelium. One round of doubling extending over a six-hour period was simulated in a stretched tissue under different conditions for division orientation. The three time points shown reflect cell geometry in the first two hours (early), second two hours (mid), and last two hours (late)
(a, b) Doxycycline-inducible RAB2A-expression MCF10.DCIS.com (a) and MCF10A (b) cells were cultured in the absence (CTR) or presence (RAB2A) of doxycycline, and immunostained with anti-RAB2A (blue), anti-E-cadherin (green), and anti-TGN46 (red) antibodies. Merged images with DAPI (magenta) are shown on the right. Bars, 10 μm.
D283 cell lines were treated with PBS (vehicle, black) or with ActivinB (green). Growth curve experiments showing cell proliferation upon ActivinB treatment. The p-values were determined by unpaired t-test and two-way ANOVA for F. *p < 0.05, **p < 0.01, ***p < 0.001.
b, A representative male control (Atg5flox/+; nestin-Cre) and an Atg5flox/flox; nestin-Cre (Atg5flox/flox) littermate at three weeks of age.
(B) WT (n = 9), Irgm2-/- (n = 8), Casp11-/- (n = 7) and Irgm2-/-Casp11-/- (n = 10) mice were injected i.p. with LPS (8 mg/kg). Serum
(A) Venn diagram showing the total and overlapping numbers of genera enriched in the fer-4 mutant and under Pi starvation. Data information: All experiments were repeated three times with similar results.
D DJA6 can bind Fe2+ in chloroplast. Arabidopsis mesophyll protoplasts were transformed with plasmids that express the indicated gene constructs, respectively. The untransformed protoplasts and protoplasts transformed with non-fused EBFP proteins were used as controls. The protoplasts were further stained by FeRhoNoxTM-1 (red) to visualize the cellular distribution of Fe2+. Green, EBFP fluorescence signals; magenta, chloroplast autofluorescence signals; yellow, regions where the EBFP and FeRhoNoxTM-1 fluorescence signals merged. The speckles which DJA6-EBFP co-localizes with FeRhoNoxTM-1 are indicated by white arrows. Scale bars = 5 μm.
(G) Line graph showing the average normalized synaptic ATP level at various time points after DHPG treatment until 5 minutes of recovery in the presence or absence of anisomycin. Data presented as mean +/- SEM, *p<0.05, **p<0.01, ****p<0.0001 for comparison with 0 minute. ##p<0.01 and ####p<0.0001 for comparison between DHPG + anisomycin. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. n≥ 5 animals per group.
(J) ChIPseq was performed using chromatins from WT ASCs and the genomic distribution of 2031 YY1 binding peaks is shown.
F Immunohistochemical staining of UQCRC2 was performed on liver, heart and kidney derived from wild-type and Uqcrh-/- mice at 9 weeks of age. Staining of VDAC1/porin was also carried out G Graphs indicate score values of immunohistochemical staining intensity of both VDAC1/porin and UQCRC2. Data are given as mean ± SEM. * p < 0.05; n = 3, Student's t-test (unpaired samples).
Colocalization of AcGFP-BmAgo3 and mCherry-BmDcp2 in BmN4 cells. Line scans (white line in the enlarged region) show that Siwi-D670A mutant but not BmAgo3-D697A mutant overlaps with BmDcp2 foci. Line scans of fluorescence intensity were normalized to the highest value and depicted at the right panel. Scale bar, 8 μm.
N. Quantification of luciferase activity of midguts with indicated genotypes. Error bars show the SD of six independent experiments. Data information: Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.
(a-c) UVRAG interaction with beclin 1 and PtdIns(3) P, but not with RINT-1, is required for autophagy-induced Atg9 dispersal. (a) HeLa cells were stably transfected with control shRNA (first row) or UVRAG shRNA (second-sixth rows). The UVRAG-depleted cells were then transfected with empty vector (second row), Flag-UVRAG (third row), UVRAGΔ270−442 mutant (fourth row), UVRAGK78A/R82A mutant (fifth row) or UVRAGΔCCD mutant (sixth row), and incubated in complete medium (untreated, UT, first panel), starvation medium (HBSS 2 h, third panel), or treated with rapamycin (50 nM 2 h, second panel) or SMER28 (50 μM, 2 h, fourth panel). The distribution pattern of endogenous Atg9 (green) in the UVRAG (wild-type or mutant)-transduced cells (red) was analysed by confocal microscopy (a). Asterisks denote condensed (non-dispersed) Atg9 in UVRAG-depleted HeLa cells and arrows denote the cells complemented with wild-type or mutant UVRAG.