EMBO/sd-character-level-ner huggingface dataset:
D. SIM1 directly interacts with AGO2. Bacterial lysates expressing MBP control or MBP-AGO2 and GST or GST-SIM1 were mixed and GST pull down assay was performed. The presence of proteins in the pull down samples was analyzed by WB using specific antibodies. The extent of GST pull down was monitored by coomassie staining of the membrane (coomassie).
Genome browser plots showing RNA-seq reads, H3K27me3 and H2AK119ub nChIP reads around the Abcb7 gene for Xist FL (left) and Xist ΔB+C (right) at day 2 of differentiation in both DOX and noDOX conditions.
Mice were injected with control (Ctrl) or STING-KO MC38 cells, and subjected to treatments, with each dose consisting of 25 mg/kg, 50 mg/kg or 75 mg/kg 5-FU, or PBS. B. Tumor volumes were quantified at the indicated days post cancer cell injection. N=5.
F) Inhibition responses were smaller and less sustained for mixed stimuli (50% 405nm, 50% 525nm) compared to 405nm only (N = 14 biological replicates).
Activation of WOX5 by DEX treatment of 35S::GVG-WOX5 in Col and hag1-6. Root explants of 35S::GVG-WOX5 and 35S::GVG-WOX5 hag1-6 were transferred onto the indicated combinations of CIM and SIM supplemented with or without 20 μM DEX. DEX was treated for 3-d-pulse period on CIM or/and SIM (see Materials and Methods for details and Appendix Fig S11A for schematics of the experimental procedure). Morphology of shoots regenerated from 35S::GVG-WOX5 and 35S::GVG-WOX5 hag1-6 root explants (A). Explants with shoots were scored at 3 w after transfer onto SIM with or without DEX (B).
(D) F-Actin in control and PPM1G depleted cells was stained using rhodamine labelled Phalloidin and the cytoskeleton arrangement was detected by using confocal microscope. Scale bars, 20 μm.
FACS quantification of CD45+ cells from ear skin of control, DKO* and DKO*K15 mice at different time points during psoriasis-like development. n > 5 per group. Statistical significance *p<0.05, **p<0.01, ***p<0.001 (t-student two tailed-test relative to controls).
Illustrated schematic representation of our single-molecule assay. Cas9 assembled with tracrRNA and Cy5-labelled crRNA is immobilized on the surface via biotin-streptavidin links (left). After addition of Cy3-labelled DNA substrate interaction between Cas9 and DNA brings the dyes together resulting in FRET signal (right). EmCCD images show Cas9:RNA immobilized on the surface with green and red laser illumination (left). No fluorescence is seen when illuminated with green laser. Immobilized Cas9 can be seen when illuminated with red laser. After addition of Cy3-labeled DNA spots appear on the EmCCD in the green channel when illuminated with green laser (right). Size of EmCCD images is 25µm x 25µm.
B (left) Immunoblotting of LDHA in melanoma cell lines transfected with non-targeting siRNA (NT-siRNA) or LDHA-specific siRNA for 72 hr. (right) Proliferation of melanoma cell lines transfected with NT-siRNA or LDHA-specific siRNA for the indicated timepoints. Data information: Statistical analysis was performed by two-way Anova for time-dependent proliferation changes and by one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. In (B), statistical comparison for the treatments that showed a significant change.
Ratio of membrane to cytosol intensity of tPH-CynA-KikGR in wildtype Dictyostelium AX3 cells before and after LY2924002 treatment, T-test was carried out by GraphPad Prism, **P ≤ 0.01 versus -LY2924002 group (mean±SEM, n = 7).
A-D Percentage of EdU incorporation in T47D (A, C) or BT20 (B, D) depleted of EGFR or not, transfected with wt or T693A, and stimulated or not with FGF10 (A, B) or with TGFα (C, D) and pre-treated (C, D) or not (A, B) with FGF10 for 40 min. Values represent the median ± SD of N= 4. P value =<0.05 *, <0.01 **, <0.001*** (one-way ANOVA with Tukey test).
(C,D) The threshold for action potentials increased over maturation in WT but not in Cox6a2-/- PV+ interneurons. As a result, the threshold for action potentials was significantly lower in P34-45 Cox6a2-/- PV+ interneurons when compared to their WT counterparts. D: two-tailed unpaired Student's t-tests.
C, D The analysis of male-female mating behaviors showed that the frequency (C) and duration (D) of mating to females of the miR-200b/a+REST DKD mice were significantly improved compared with those of the miR-200b/a KD mice (NC: n=10 mice, miR-200b/a KD: n=9 mice, miR-200b/a+REST DKD: n=13 mice; data represent the mean±SEM; C: F=6.162, D: F=6.360, *p<0.05, **p<0.01; one-way ANOVA and Bonferroni pairwise comparisons).
(a) LC3-II levels, which closely correlate with autophagic activity, were higher in mutant lines (expressing Gln74 huntingtin; Q74; lanes 3 and 4) than in wild-type lines (expressing Gln23 huntingtin; Q23; lanes 1 and 2). Actin was used as a loading control. Data are shown for two independent clonal lines expressing mutant or wild-type huntingtin. Quantification of the band intensity is shown in the right panel.
D: Surface representation of the MSP domain of MOSPD2 in complex with the conventional FFAT of ORP1. Data information: Phosphorous, nitrogen, and oxygen atoms are colored in orange, blue, and red, respectively. Carbon atoms are shown in slate blue, grey and rose in the MSP domain, the classic FFAT motif, and the Phospho-FFAT motif, respectively. the protein surface is colored according to the YRB scheme, showing hydrophobic, negatively and positively charged atoms in yellow, red and blue, respectively; the other atoms are in white
Mass spectrometry-based analysis of formaldehyde-induced SUMOylation changes. His10-SUMO2 conjugates from HeLa/His10-SUMO2 cells subjected or not to formaldehyde were purified on Ni-NTA under stringent conditions and analyzed by mass spectrometry. All proteins displaying significant upregulation of SUMOylation in response to formaldehyde treatment were subjected to network analysis using the STRING database, at the default interaction confidence setting of 0.4. Proteins not connected to the network were omitted.
(B) IFN-γ release by NK cells cultured in the presence of supernatants recovered from untreated DCs (untreated) or DCs treated O/N with LPS (LPS). Where indicated IL-18 was added to the cultures.
E, F Representative micrographs of LGALS1-stained (green) BT12 control (E) and MDGI-silenced cells (F) six days after silencing. Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.
B. RT-qPCR quantification of viral RNA (SARS-CoV-2 N gene relative to RPLP0) upon infection of A549-ACE2 cells with indicated SARS-CoV-2 strains at 24 hpi. Cells were pretreated with BB94 (10 µM), DPC-333 (10 µM) or DMSO for 6 h. Data are normalized to DMSO (N = 3-7). A two-sided independent Student's t-test with Benjamini-Hochberg FDR correction was performed. Data information: Data are represented as means ± 95% CI from at least three , B) biological replicates *p < 0.05, **p < 0.01, ***p < 0.001.
FACS analysis of IFNγ (left panel) and IL17A (right panel) expression in tumor extracted T cells after αGr1 + CXCR2i (n=7) or IgG + DMSO (n=7) treatments.
f, Slightly less quenching was observed for the F408Dgr;-TRPML1Va-expressing cells.
B) Short BDNF stimulation changes the levels of only a few microRNAs, such as miR-16-5p and miR-551b-5p. Volcano plot of small RNA sequencing data from BDNF- or control-treated cortical neurons (6DIV) was based on differential expression analysis using the edgeR package. Individual points represent the average fold change obtained in three independent experiments for each miRNA, and plotted against obtained p-values. MiRNAs above the red dashed line are significant (p<0.05, t-test type 2, n=3).
H, I Number of GFP::MTM‐3(C459S)‐positive puncta and co‐localization of GFP::MTM‐3(C459S)‐ and LGG‐1‐positive puncta in various strains [mtm‐3(tm4475), atg‐2(bp576), lgg‐1(bp500), vps‐34(h797)]. At least 10 (I) and 15 (H) embryos were scored per strain. Data are shown as mean ± SD. Data from different mutant backgrounds were compared with wild type. **P 0.0001; N.S.: not statistically different.
a, Heat map of HCIPs (represented by APSMs) for HA-Flag-PARKIN and mutants in response to depolarization in 293T cells, with or without Btz or BafA. Proteins indicated had weighted and normalized D-scores ≥ 1.0, Z-score ≥ 5, and APSMs ≥ 2 unless otherwise noted (see Methods). b, Summary of PARKIN-interacting proteins in 293T and HeLa and integration with diGly sites.
Mean number of TAF α-actinin positive CMs from C57BL/6 mice. Data are mean ± SEM of n=4-7 mice per age group.
Postulated organization of the amphipathic H3 helix in the post-fusion trimer. Left panel: proposed arrangement of the H3 helix in our post-fusion trimer model, lying flat on the fused membrane (in light orange) and projecting the TM segments (not drawn) away from the trimer axis. Second panel: potential conformation of the sE448r trimer, with the H3 helices trimerizing via their hydrophobic surfaces. Third panel: the sE-linker construct. As the linker is not long enough (only 8 residues), the H3 helix cannot trimerize as in the full-stem sE448r trimer. It would be exposed on the sides of the trimer, potentially leading to aggregation. Right-hand panel: the sE-linker* did not lead to aggregation, and at high concentrations it crystallized in a form that had the tip disordered. Trimerization of the three H3 helices in the trimer may have caused this effect, as they must displace the trimer tips in order to oligomerize. This is a further measure of the highly dynamic breathing behavior of the E trimers, as the sequence required to complete the stem zipper was absent from this construct.
Distribution of global transcript and protein abundance in all tissues. It is apparent that the dynamic range of protein expression (iBAQ scale) exceeds that of mRNA expression (FPKM scale; see Appendix Fig S10 for the corresponding plot for RNA and protein copy numbers).
F. Cells in (E) were pulse-labeled with EdU, stained with DAPI and analyzed for DAPI and EdU signal intensity using QIBC.
K. The predicted JunD binding sites within Pbk promoters of mouse, human, and rat genomes via software PROMO3.0.
(C) Representative microscopic appearances of the corneal epithelial cells of the chimeric piglet staining by H&E. Scale bars, 100 μm
C. Cells treated as in (B) were pre-extracted, fixed and stained with PCNA or MCM2 antibody, and subsequently analyzed by QIBC (n>2000 cells per condition).
Percentage of EdU+ epidermal cells (CD45-CD49f+) after blocking with anti-TSLP in DKO* and DKO*15 mice
(D) Top: Representative images of in situ PLA performed between S9.6 (DNA-RNA hybrids) and anti-γH2AX antibodies in BRCA2 deficient DLD1 cells (BRCA2-/-) bearing BRCA2 (WT) or BRCA2-T207A (T207A). When indicated, cells were transfected/treated with RNase H1 (RH) or treated with Cordycepin (Cordy) for 2h at 37°C before fixation. Single antibody controls in non-irradiated BRCA2 WT cells are shown. Scale bar indicates 10 µm. Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. Bottom: Quantification of the number of PLA spots per nucleus. At least 600 cells were counted per condition from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.
8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on HFD-feeding for 12 weeks. n=5 per group. (J) Protein levels of KLF10 in the livers (left) and its expression was normalized to β-actin (right). Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test
C Quantification of H3P immunostaining in intact animals following control or Lrig-1 knockdown (n = 9-10 technical replicates).
(E) NIH-3T3 cells were stably transduced with retroviral vectors expressing M45 or GFP, and treated for the indicated times with PI-mix, 10 mM NH4Cl, or 5 µM lactacystin, respectively. Levels of the indicated proteins were analyzed by immunoblotting.
(F) Western blotting analysis confirming high-throughput results for selected ATM substrates.
NIH-3T3 cells were treated with rotenone, antimycin, myxothiazol or vehicle control for 1h, and proline abundance was measured by GC-MS. Values are normalized to control-treated cells.
(D) Immunofluorescence (IF) assay was performed to determine the subcellular location of GDH1 with or without aa starvation. GDH1 localization was indicated by GDH1 antibody and MitoTracker (left). Signal intensities and distances were quantified (right). Scale bars: 20 μm.
(e) Left: Overlay of 2D-[15N, 1H]-TROSY spectra of BakΔTM in [Ni2+]-caged liposomes (magenta) and Bid-BH3 activated BakΔTM in nanodiscs (green). Right: Plot of the relative NMR intensity of Bid-BH3 activated BakΔTM in nanodiscs. Peaks that are also visible in liposomes are indicated by horizontal lines in magenta at the bottom, while the chemical shift-derived secondary structure elements are indicated at the top.
(e) Proteins were extracted from 16 paired samples from patients for western blotting with anti-FoxO1 or with anti-p62 antibody. Protein bands were scanned with a phosphorimager and relative band intensity was normalized to the β-actin band. FoxO1 of normal tissue, 0.37; FoxO1 of tumour tissue, 0.10; P = 0.001, two-sided t-test. p62 of normal tissue, 0.32; p62 of tumour tissue, 1.00; P = 0.009, two-sided t-test. Data in e are means ± s.d. (n = 16).
A) M. florum doubling time at different incubation temperatures measured by colorimetric assays. The bars represent the mean and standard deviation values obtained from three technical replicates. The asterisk indicates the absence of significant growth, preventing the calculation of a doubling time.
a) Photomicrographs represent 2D optical sections from a Z stack acquisition of whole mounted skin displaying IFE harbouring multiple clones of different colours at 3, 5, 12 and 24 weeks (bar represents 50 μm). In each image a clone is circled in white, illustrating the progression of clone size at different times. b) Histogram charts showing the relative frequency of clones according to their size between 3 and 24 weeks in mice induced at P21 and P42. The frequency of larger clones increases over time. Chi square test p<0.0001 for P21 group and p=0.0056 for P42 group. The number of clones evaluated at each time point is indicated above each histogram series. c) and d) Correction for contiguous clones harbouring a same colour.
B. Chromatin accessibility and p300 binding at differential ATAC-seq peaks visualized using K-means clustering.
F. As in B and C but with transfection order reversed (green =Cry1 expression in ProkR2+ cells only, n=6) (p=0.006; baseline vs. ProkR2.Cre *p=0.03, baseline vs. ProkR2.Cre and Prok2.Cre **p=0.009, ProkR2.Cre vs. ProkR2.Cre and Prok2.Cre ns. p=0.1).
(E) Purified GST or GST-fused PAQR3 (1-71) (GST fused NH2-terminal 71aa of PAQR3) on glutathione agarose beads was mixed with purified His tagged proteins as indicated. After incubation at 4 °C for 3 h, the samples were washed extensively and subjected to IB.
(B) HPLC-QgQ-MS/MS quantification of the m6A/A in mRNA of siCtrl or siFto 3T3-L1 cells. FTO knockdown efficiency was measured by Western blot. Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test.∗∗p < 0.01, ∗∗∗p < 0.001.
The activation of the Wnt/β-catenin signaling at the matured osteoblasts transcriptionally induces CXXC5, and its binding to Dvl is followed by suppression of the Wnt/β-catenin signaling (left panel, suppression of the signaling marked by thin arrows) (Kim et al, 2015). Blockade of the Dvl-CXXC5 interaction with KY-02061 or KY-02327 releases the negative feedback regulation, resulting in high activation of the Wnt/β-catenin signaling (marked by thick arrows) and subsequent induction of osteogenic genes stimulating new bone formation and high bone mineral density.
Cideb interacts with the 416-800 portion of SCAP. Flag-tagged Cideb was co-expressed with the indicated HA-tagged SCAP truncation mutants in 293T cells and immuno-precipitated with an anti-Flag antibody. Levels of the co-immunoprecipitated protein were detected by an anti-HA antibody following SDS-PAGE.
The mFc-tagged ACE2s from human, mouse, rat and European hedgehog were captured by anti-mIgG Fc antibodies immobilized on the CM5 chip, and sequentially tested the binding with serially diluted SARS-CoV-2 RBD and its mutants (K417R, Q498H and K417R-Q498H).
(C) Distribution of the levels of all proteins identified after isolation of the RNA-bound proteome in U2OS cells treated with protamine (30 μM) or (PR)20 (20 μM) for 180´. RNA-binding proteins (RBPs) were identified as those significantly enriched after the isolation of mRNA-binding factors. Note that treatment with either peptide leads to a selective reduction in the levels of RBPs bound to mRNA that is not observed for the rest of the factors (Not RBPs) (n=2). P values were calculated with two-sided two sample t-tests assuming equal variances. Center lines indicate median values; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquantile range from the 25th and 75th percentiles and outliers are represented by dots.
(A) TEM images of a single ommatidium from wild type photoreceptors of flies reared in dark (i) day 1 (D1) and (iii) day 14 (D14), scale bar: 1µm (red asterisk indicates R7 photoreceptor). Magnified image showing a single photoreceptor from the same ommatidium (ii) day 1 (iv) day 14. Scale bar: 200 nm. Origin boxes for magnification shown. Arrow in (Aiv) indicates the SMC forming an MCS with the microvillar PM. M- Microvillar plasma membrane, S- Sub microvillar cisternae, MCS- Membrane Contact Site, P- Pigment granules. (B) Quantification of the number of MCS per wild type photoreceptor of day 1 and day 14 old flies reared in dark (from figure 6A), Y-axis indicates the number of MCS/photoreceptor. n=30 photoreceptors from 3 separate flies (R1-R6) for (i). n=9 photoreceptors from 3 separate flies (R7) in (ii).
(G) The sequence from RING1-IBR was aligned by overlaying the HOIL-1 AlphaFold prediction (residues 282-404, AlphaFold Uniprot ID: Q9BYM8) with residues 699-827 in the crystal structure of HOIP-RBR (PDB: 5EDV). The location of the allosteric ubiquitin from that HOIP structure is also shown. HOIL-1 residues I358, F381 and E383 are highlighted in red.
(e) Rheb inhibits glucose starvation-induced Ulk1 activation. HA-Ulk1 and Myc-Rheb was transfected into HEK293 cells, which were incubated with glucose-free (-Glu), amino-acid-free (-A.A) medium, or 50 nM rapamycin (Rapa) for 4 h before lysis. HA-Ulk1 was immunoprecipitated and kinase assays were performed. Ulk1 activity was measured by 32P-autoradiogram and the protein level of HA-Ulk1 and GST-Atg13 used in the assay was determined by western blot and by Coomassie staining, respectively. Uncropped images of blots are shown in Supplementary Fig. S5.
HeLa cells treated with vehicle or 1 μM rapamycin for 4 h were stained for phosphorylated (S473) Akt (p-Akt) or HSP60 (mitochondrial marker). Merged images are indicated (merge). Scale bar 10 μm
293T cells were transfected with PGC1α wild type or QRE sites mutant 3'UTR fragments. RNA immunoprecipitation (RIP) to identify these QRE motif sites region bound to endogenous QKI by using anti-QKI antibodies. The results showed that endogenous QKI interacted with full-length wild type QRE motif regions within 3'UTRs of PGC1α
(D) Top table: absolute intracellular concentration of nucleotide monophosphates in carbon starvation. Bottom plots: Phosphatase activity of Phm8 and Sdt1 as a function of CMP concentration. The x axis represents CMP concentration and the y axis represents specific phosphatase activity (μmol of phosphate produced per minute per mg of enzyme, mean±range of N=2 replicates).
(C) The CSD domain of HP1c and the BTD domain of Su(H) are responsible for the interaction by yeast two-hybrid analysis.
(C) WT or RagA/B knockout (KO) HEK293A cells were co-transfected with HA-tagged ArfGAP1, or HA-tagged RFP1 (control) with Myc-tagged S6K1. Cells were starved of amino acids (-AA) for 2 hours and then 4 mM of glutamine (+Gln) or asparagine (+Asn) were added for 2 hours. Cell lysates were assed for mTORC1 activity as in by the phosphorylation of S6K1 at Thr 389 (pS6K1) and probed for HA, Myc, S6K1, RagA, RagB, and Vinculin.
D. Representative electron micrographs of cells subjected to AICD untreated (upper panel) or treated with 100nM rapamycin (lower panel). 24h after AICD induction cells were fixed and processed for electron microscopy, Scale bar, 2μm.
(b) A549 cells were infected with adenovirus harbouring shRNA for Rubicon or control (luciferase). The cells were cultured in N or S medium in the presence or absence of E64d (50 μg ml−1) and pepstatin A (50 μg ml−1) for 1-4 h. Cell lysates were subjected to western blotting with the antibodies indicated.
(D) Cells expressing either vector or SFB-B56δ were fractionated into nuclear and cytoplasmic fractions and levels of indicated proteins were determined using western blotting. Tubulin and Histone H3 were used to mark cytoplasmic and nuclear fractions respectively.
Kinetics of in vivo depletion of indicated bone marrow cells in WT mice (n=3) after γ-irradiation (9 Gy).
(E) Ratios of living tumour cells after co-culture with B220+CD11c+NK1.1+cells that had been primed with lung- or liver-CM. Shown are averages (N = 6/group) with SEM and one-way ANOVA
C. Time-lapse of TagRFP-ER dynamics in hippocampal neurons (DIV16-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A, SCRN1 shRNA or VAPA/B shRNAs. Intact ER structures (dark arrowheads), intact and dynamic ER structures (light arrowheads) and impaired an non-dynamic ER structures (arrows) are indicated. Scale bars: 5 µm (full size) and 2 µm (zoom).
(E) Quantification of autophagosomes and autophagolysosomes. The error bar represents the standard error.
(K) Fractional shortening measured before, 2 weeks and 4 weeks after AAC. n=6:6. Two-way ANOVA followed by Bonferoni post-tests was used for statistical analysis.
Figure 4 - GO term enrichment analysis for unstable (A) and stable (B) proteins. Horizontal bar chart representations are given of significantly enriched GO terms in the human proteome (FDR q-value ≤ 0.05).
D CTRL, Bax/Bak-deficient or Bcl-XL-over-expressing HeLa cells were infected with Salmonella Typhimurium (MOI=50). Extracellular bacteria were killed using non cell-permeable antibiotic. At the indicated time points cells were lysed, and aliquots were plated on agar plates. Recovered colony forming units were calculated. Data are means/SEM of four independent experiments. Significant genotype effects were found between mutant and control cells (Bax/Bak p<0.0001; Bcl-XL p 0.0001).
(B) The frequency of L-ESLAM HSCs within the divided fraction in 5-FU recipients or undivided fraction in untreated recipients. Numbers within each histogram represent the CFSE+ cell number detected.
Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. Percentage of secondary recipient mice with GFP chimerism in BM KLS (G) and KLS-(H) cells higher that 1% is similar in G-CSF and CoPP groups.
Analysis of covalent adduct formation between CtUba4 WT/cysteine mutants and CtUrm1-COSH in the presence of oxidizing agents. HP: Hydrogen peroxide; D: Diamide.
N A tethering assay using the β-globin-6xMS2 reporter and MS2-tagged proteins was performed in HEK293T cells. The transfection mixtures contained plasmids expressing wild-type DCP2 (-) or the catalytic DCP2* mutant (+). The panel shows a northern blot of representative RNA samples analyzed as described in (panel M).