EMBO/sd-character-level-ner huggingface dataset:
(A) EGFR phosphorylates STING in vitro. Purified STING-GFP was incubated with increasing amounts of EGFR for in vitro kinase assays; the products were analyzed on phos-tag gel by Western blot using GFP antibody (upper panel). The substrate was analyzed, before the assay incubations, by the same method (lower panel).
(B) Park2-/- BMDMs pretreated or not treated with IFN-γ were infected with GFP-Mtb for 24h and immunostained with UBQLN1 specific antibody; Arrows indicate Mtb co-localized with UBQLN1. ns- not significant.
Quantitative RT-PCR of IL-6mRNA normalized with S26mRNA of C57BL/6J and Cx3cr1GFP/GFP, and Cx3cr1GFP/GFP ApoE−/− peritoneal macrophages cultured for 24 h with CX3CL1 (n = 5 per group, one-way ANOVA/Bonferroni post hoc multi-comparison tests: C57BL/6J versus Cx3cr1GFP/GFP *P < 0.0001 and Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/−‡P < 0.0001. Mann-Whitney U-test: C57BL/6J versus Cx3cr1GFP/GFP *P = 0.0079 and Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/−‡P = 0.0079. The experiment was repeated twice with similar results.).
B, C. Illustration of the Incucyte readout. Infection of A549 or ACE2-expressing A549 cells with SARS-CoV-2 encoding a GFP expression cassette. Infection of cells was monitored microscopically as green fluorescence through live-cell imaging at 24 hours post infection (hpi) (B) or during the time-course of 48 h and shown as the mean of the GFP-positive area relative to the whole area covered by cells in the same well (C). Error bars represent means ± SD derived from biological triplicates (N = 3). Scale bar, 500 µm.
Neuro-2a cells expressing doxycycline-inducible shRNA knockdown of TDP-43 were transfected with the indicated TDP-43 constructs, and the alternative exons in Dnajc5 and Sort1genes were examined by PCR. Representative gel images were shown (top panel). TDP-43 protein levels were examined by immunoblotting (bottom panel).
E. Dot plots showing the expression levels of the top markers for myoblast clusters identified in the reference cell atlas across the cluster of myoblasts in 1151>Ama-RNAi and 7 myoblast clusters in control dataset.
(D) U2OS cells were transfected with control siRNA and cortactin siRNA. Autophagosome-lysosome fusion was analysed with or without starvation (6 h) using the mCherry‐GFP‐LC3 reporter as described in Figure 3A.
D Fraction of high exchanging subpopulation for peptides 159-168, 169-175, and 176-189 under the different indicated pre-incubation conditions: ‑chaperones, +Hsc70 (70), +DnaJB1 (B1), and +ATP incubated for 0 or 30 min at 25°C. Shown are means ± SD (n=3).
ATP levels in whole brain homogenate from 9 month old WT and MPSIIIA mice (n=6 mice per group). Data are expressed as mean ± STDEV and were tested by unpaired t-test. WT vs. MPSIIIA P<0.02
(C) Scatterplot with the transcription factors that are most likely to explain the dynamics shown in A. The x axis is the linear correlation between the best motif score and the log2 fold change EN over ENR under each significantly changing peak for a given dataset as shown in A. The y axis is the feature importance of the motif in a random forest model in percentage increase in mean squared error (MSE), where motifs that are important for the model to accurately predict the given fold changes get a high error increase. Different datasets are color coded. For motifs with more than one transcription factors associated to it, we show the transcription factor name with the most significant change in protein expression. The vertical line over each dot represents two times the standard deviation of the MSE
D. Cell lysates of LN229 cells with or without Wnt-3a treatment for 4 hours were subjected to IP using Axin antibody or control IgG, followed by IB with FoxM1 and Axin antibodies..
A series of major Rab family proteins were immunoprecipitated from HeLa cell extracts and then probed with an anti-BAG6 antibody. Co-precipitation of Rab8a (T22N) with BAG6 was used as a positive control for this experiment.
(C) Western blot of acid extracted histones and WCE from HeLa cells, treated with HATi (CTK7A, 100 μM and CPTH2, 50 μM) or HDACi (TSA, 1 μM) for 4h or mock treated as indicated
Schematic diagram of the identification of protein complexes associated with SRSF3 and hnRNP K. Nuclei were extracted from HEK 293 cells overexpressing FLAG-tagged SRSF3 (3XFLAG-SRSF3-IP) or FLAG-tagged hnRNP K (3XFLAG-HNRNPK-IP), followed by isolation of the high molecular weight (HMW) fraction (Damianov et al., 2016). After mild treatment with MNase to release proteins from chromatin, immunoprecipitation (IP) was performed using anti-FLAG antibody and mass spectrometry analysis was performed. Naïve cells not expressing a FLAG-tagged protein were used for control IP (Cont-IP).
Human pancreatic cancer cell line Colo357 was orthotopically implanted into NOD SCID mice. After tumor establishment, mice were randomized to receive rat IgG Mac84 (control), 2G8 or anti-mouse IL-6 antibody MP5-20F3 (each 30 mg/kg 2x/week, n = 6/group) for 3 weeks. For NK cell depletion, prior to therapies, mice received 50 μg of control rabbit IgG or anti-Asialo-GM1 three days in a row. For maintenance, 25 μg of control rabbit IgG or anti-Asialo-GM1 were given 2X/week throughout the whole study. Tumors were harvested for analysis and metastatic burden was determined by histologic evaluation of H&E-stained liver tissue. Ten sections of the anterior lobe of the liver (n = 6 per group) were scored for lesions.
Mice, implanted with control cells (Ctrl) or myoblasts expressing low (V Low) or medium (V Med) VEGF levels, were treated with the anti-NRP1A antibody blocking Sema3A/NRP1 binding (AbαNRP1A) or with control IgG2a. Analyses were performed after 1 week.E Immunohistochemistry for Sema3A protein on frozen muscle sections with H&amp;amp;E counterstaining. The intensity of Sema3A stain was quantified as reciprocal intensity on a scale out of 250. Scale bar = 100 μm. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group: No stain, n = 3; Ctrl IgG2a, n = 23; Ctrl AbαNRP1A, n = 12; V Low IgG2a, n = 53; V Low AbαNRP1A, n = 38; *P < 0.05 and ***P < 0.001 by Kruskal-Wallis analysis with Dunn's multiple comparisons test: V Low IgG2a versus No stain P = 0.0007; V Low IgG2a versus Ctrl IgG2a P < 0.0001; V Low AbαNRP1A versus Ctrl IgG2a P = 0.0142; V Low IgG2a versus V Low AbαNRP1A P = 0.0001.
N, Representative electron micrographs of IHCribbon synapses in OtofI515T/I515Tand Otof+/ (conventional embedding without immunogold) after pre-incubation for 10min at the indicated temperature followed by high K+ stimulation or 0 Ca2+ inhibition for 1min 45sec. Scale bars 100nm.
(A) qPCR levels of fibronectin (FN), fibronectin extracellular domain A (FN-EDA), and fibronectin extracellular domain B (FN-EDB) in PSCs, normalised to RPLP0 and relative to control
B Mass spectrometry analysis of CVF, CLys and CSN and identification of distinct and shared proteins. From the shared proteins in CLys and CSN, relative detection scores of GDH and IDH were presented.
F Representative Haematoxylin-Eosin (H&E) staining of mouse prostate tissue and organoid sections (scale bars = 50 μm).
Confocal micrographs of EphA2 (red) in HGSC cells cultured in 3D collagen for 7 d and treated for 72 h with 10 μM cisplatin (5 μM for OCKI_p01). The intensity of EphA2 is comparable only between mock and treatment conditions for each patient. Scale bars: 20 μm.
G. Thapsigargin, DMM, DMS and ENZA treatment for the indicated time points increased intracellular calcium flux in LNCaP cells as visualized with calcium sensitive Fluo4-AM probe under confocal microscope.
Representation of the mechanism of action of the Gata6-PGI2-IL-10 active inhibitory signal on IL-1β processing.
(B) Alignment of the BH3‐like domain of Beclin‐1 with established BH3 domains. L116A Beclin‐1 and F123A Beclin‐1 describe two Beclin‐1 mutant peptides.
Electron micrographs of MyoIAts control midguts, or expressing svb-RNAi and SvbACT. Brush border microvilli are pseudo-colored in green, and high magnification views are shown in inlets. Nuclei are pseudo-colored in purple, and visceral muscles located above the basement membrane are in pink. Arrowheads point to impaired cell contacts, the arrow points to a pyknotic nucleus.
C (Left) ANS fluorescence spectra of PDI WT and ­­S357E. Controls with ANS alone was shown. A.U., arbitrary units. (Right) Enhancement factor were quantified. Data information: All data were shown as mean ± SEM of three independent experiments. **p < 0.01, ***p < 0.001 (two-tailed Student's t-test).
A Images of B. subtilis Δbkd cells stained with membrane dye FM 5-95 and expressing GFP fusions of DNA-binding protein HBsu (top), cell division protein FtsZ (middle) or cell elongation protein MreB (bottom). Cells were grown with IB or depleted for precursors for 90 min (IBPF). For further examples and additional time points, see Appendix Fig S4A. B Images of E. coli fabA(Ts) cells stained with FM 5-95 for the outer membrane and with DNA-intercalating dye DAPI (top), or expressing GFP sandwich (SW) fusions to the cell division protein FtsZ (middle) and the cell elongation protein MreB (bottom), respectively. Depicted are cells grown at 30°C or with a temperature shift to 37°C for 120 min. For a more detailed view on the influence of low membrane fluidity on membrane dissociation of RNase E as well as on the cell division machinery, see Figs EV2, EV3, EV4, and S5. For further examples and additional time points, see Appendix Fig S4B. D
(a) Immunoblot analysis of ASC immunoprecipitates of differentiated THP-1 cells transfected for 2h with poly(dA:dT) (1.5 μg/ml), probed for ubiquitin (Ub), then stripped and reprobed for K63-linked ubiquitin (Ub-K63), then stripped and reprobed for ASC.
Heat map summarizing the SCNAs detected by sWGS of 28 genes of interest in tumor biopsies and CSF from patient G1 (4 tumor subparts and 1 CSF sample). Amplifications are shown in dark blue, deletions in orange, and copy number neutral regions are in light grey
(F) Survival curve for infected WT and Clec12a−/− mice (n = 9 mice per genotype).
G THP-1 Caspase 1 -/- cells were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. LDH release measured from supernatants of mock treated or infected cells at 12 hpi or 24hpi. n=3 for 12hpi, n=5 for 24hpi. Medians are indicated, statistical analysis was performed by one-way ANOVA.
(A) Four-weeks old transgenic wild-type Col-0 (WT) and atg7-2 mutant plants expressing GFP-ATG8a were treated with (45°C) or without (22°C) heat shock for 3 h and then placed at room temperature for 0.5 h. The leaves were visualized by fluorescence confocal microscopy of GFP signal. (B) Numbers of punctate GFP-ATG8a spots representing autophagosomes per 10,000 µm2 section. Means and SE were calculated from three experiments. According to Duncan's multiple range test (P = 0.05), means do not differ significantly if they are indicated with the same letter. Bar = 20 µm.
KIN10 reduces the stability of PIF4 through 26S proteasome-mediated degradation. Transfected mesophyll protoplasts were pre-treated with or without 20 µM MG132 for 4 hours and then incubated in the presence of 100 µM cycloheximide (CHX) for the indicated time. Total protein was extracted from the protoplasts, and analyzed by immunoblotting with anti-GFP or anti-Myc antibody. Quantification of relative PIF4-Myc levels is shown in (C). PIF4-Myc levels are presented as values relative to those at 0 h. In (C), different letters above each point indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05; n = 3). Error bars indicate s.d. (n = 3).
The number of KLS- (c-Kit+Lin-Sca-1-) is higher in mice treated with CoPP than in mice treated with G-CSF. Numbers of lineage committed progenitors: GMP (granulocyte-macrophage progenitors, KLS- CD48+CD150-CD34+) and MEP (megakaryocyte-erythroid progenitors, KLS- CD48+CD150+CD34-) were higher after CoPP administration than after G-CSF. G-CSF and CoPP similarly increase EP numbers (erythrocyte progenitors, KLS- CD48-CD150-CD34-).
(f) As in e, immobilized GST-ΔN-PINK1 (top panel), GST-Parkin (middle panel) and immobilized GST alone (bottom panel) were incubated with combinations of IVT Flag-Parkin, T7-ΔN-PINK1 and Fbxo7-HA as indicated. Input and bead-bound proteins were analyzed by immunoblotting with the indicated antibodies. All western blots were performed a minimum of three times. Full-length blots are presented in Supplementary Figure 9.
A549 cells were infected for 4 hours with WT or P. aeruginosa mutants STY, ΔS and ΔTY). Cell lysates were evaluated by immunoblotting.
(G) HeLa cells were treated with CCCP and cycloheximide as depicted, followed by immunoblotting with the indicated antibodies. LDH, lactate dehydrogenase. (F and G) Asterisks indicate a cross-reacting band.
B. Cells were stimulated with increasing (doubling) doses of Activin A (0.625-20 ng/mL) for 1 h. Data information: In all cases, whole cell extracts were Western blotted using the indicated antibodies. Representative experiments are shown. The levels of pSMAD1/5 relative to Actin were determined and normalized to the 20 ng/mL ligand dose in parental HEK293T cells Act, Activin A; Par, parental HEK293T cells, KO1, ACVR1 KO clone 1.
A. Depletion of Ccdc108 causes disorganized basal body distribution in MCCs. Representative 3D-SIM images (x-y) and 3D-reconstructions (x-z) of MCCs of embryos show a failure of apical trafficking of basal bodies in Ccdc108 depleted cells. Cell membranes (mGFP, blue), basal bodies (Cetn1, green) and distal appendages (Cep164, purple) were labeled with indicated antibodies. Select area of Cep164 channel was zoomed to show centriolar Cep164 accumulations. Orange arrowheads and numbers mark individual centriole. > 70 MCCs from 6 embryos for each condition were counted. Data information: Quantitative data were from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001 ; ** p<0.01; * p<0.05). Mean ± s.d. values are presented.
Western blotting for mTORC1 and mTORC2 activity in mESCs transduced with Rptor and Rictor shRNAs.
(c) PTPL1 stimulates the binding of FBXL2 to wild-type p85β, but not p85β(Y655A). HEK293T cells were transfected with GFP-tagged FBXL2 and either FLAG-tagged p85β or FLAG-tagged p85β(Y655A). Where indicated, HA-tagged PTPL1 or HA-tagged PTPL1(C/S) were also transfected. The experiment was performed as described in a.
(B to D) Analysis of protein abundance shift in cellular compartments in published datasets. Density plots for protein fold change distributions in different compartments for (B) lung vs. liver cells (Geiger et al, 2013), (C) hepatocytes vs. Kupffer cells (Azimifar et al, 2014) and (D) healthy human kidney vs. renal carcinoma cells (Guo et al, 2015). Distribution of protein fold changes for ten different compartments are shown as boxplot (inset); mean fold changes (log2) are indicated below the density plots; below the boxplot, asterisks mark the cellular compartments that show significantly different distribution of fold changes compared to the whole proteome (Mann-Whitney test p<0.01). Related to Figures EV1 and EV2 and Dataset EV1, EV2 and EV3
(f) ITM, ARM and ASM of 30-d-old flies expressing mushroom body-specific Odc-1 compared with their genetic controls (n = 8-9 independent experiments; F = 11.73 for ITM, F = 0.50 for ARM, F = 16.6 for ASM; one-way ANOVA with Bonferroni correction). **P 0.01, ***P 0.001; ns indicates not significant, P > 0.05. Data are presented as mean ± s.e.m.
(D) H&E staining showing representative histological features (overview and high magnification of microvascular proliferation) of Rheb1fl/fl and Cx3cr1-Rheb1Δ/Δ GL261 tumours. Scale bar is 125 μm (top, H&E) and 80 μm (all other images)
(A) Hepsin knockout mice harbor a 50bp frameshift deletion in the 4th exon of the Hpn gene (TM= Transmembrane domain, SRCR= Scavenger Receptor Cysteine Rich domain, SPD= Serine Protease Domain; Red bar indicates gRNA binding site). Immunoblot from whole mammary lysates against hepsin protein (Hpn+/+ =Wt, Hpn+/- = heterozygous deletion, Hpn-/- = homozygous deletion) (representative of 3 mice).
(e) Mouse fibroblasts stably expressing KFERQ-PS-CFP2 were plated in 384-well plates, maintained in the presence of serum supplemented media and leave untreated (white bar) or subjected to the indicated treatments (black bars). In both (d, e), after fixation, images were subjected to high content image analysis and the percent of responders was calculated in >200 cells per condition. Values are mean+s.e. of results in three different experiments. *P0.001 (range 0.0002-0.0007, t-test). Scale bars, 5 μm.
(E) Flow chart illustrates A151 administration and experimental design. Mice received daily i.p. injections of A151 (300μg) or an equal volume of PBS vehicle for 3 consecutive days.
A, Cell surface biotinylation assay performed in HEK293T cells transfected as indicated and treated with 10 μM MG132 to prevent proteasomal degradation. Consecutive immunoprecipitations were carried out as indicated in the scheme. The Avidin pulldown was carried under denaturing conditions (1st IP). The unrelated receptor CD147 was used as loading control for the avidin pull down, and CUL1 for the total input of cell lysate.
(C) Summary of the functional categories of mouse genes significantly enriched in response to PLX4720 and COMBO. GO analyses were performed individually on down- or up-regulated genes using DAVID tool (biological process). GO terms are ranked by p-value corrected by BH method, and the number of genes is indicated. For a complete list of significantly enriched GO groups see Appendix Tables SIII-SIV.
B Chromatin expansion in absence and presence of intercalating DNA dye (- and + DNA damage) and pretreatment of 1h with 1μM of Olaparib PARP inhibitor. Boxplots represent single cell measurement of chromatin expansion at 120 sec. post DNA damage from 3 biological replicates with n > 30 cells each. The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values (*, p < 0.05 using unpaired, two-tailed Student's T-test).
(A, B) Maximum projection of 2z slices of representative Hela cells stained for Anillin (A) and pMLC- S19 (B), showing reduction of these proteins in cells depleted of centralspindlin protein RACGAP1, but is unaltered upon Aurora B inhibition with ZM. Scale bar - 10 µm
A. All SGNs of Wrbfl/fl:CreA mice had spontaneous firing rates (SR) below 20 Hz (p>0.05, Kolmogorow-Smirnov test).
H. Residual mRNA levels of BZLF1 and BRLF1 after termination of transcription via ActD treatment in CNE2EBV cells transfected with YTHDF1-specific siRNAs or the siNC control following EBV reactivation. After 24 h of siRNA transfection, CNE2EBV cells were induced with NaB (2 mM) for 24 h, treated with ActD and analyzed by qRT-PCR. The relative mRNA level at 0 h after the ActD treatment was normalized to 1.
(J) Mouse dermal fibroblasts (MDF) or MDF-p53-/- fibroblasts were transfected with empty vector (EV) or p53-overexpressing vector (p53OE), then the cells were treated with vehicle (water) or doxorubicin (250 nM for 24 hours) and qRT-PCR was performed for Igfbp3 and Lif genes (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001. EV, empty vector
(B) Confocal images showing a TNT connecting two cells loaded with α-synucleinfibrilsATTO-550 immunostained with Lamp1 (in green). Labelling of TNTs is performed using a plasma membrane dye (WGAAlexa-488, in blue). The image represents a Z-projection of the several middle stacks where the TNT is located. The merged image with WGA-Alexa 633 (blue), the orthogonal view and insets showing 3D reconstructions of the TNT (with WGA in white) are in the right panel. Several α-synuclein fibrillar foci (in red) are present in the lumen of the TNT and embedded in Lamp1 positive vesicles (in green). Of note, only tubular structures physically connecting remote cells and not touching the substratum were identified as TNTs to distinguish them from filopodia. Scale bars represent 10 μm (confocal images) and 3 μm (insets). The merge of α-synucleinfibrils (red) and Lamp1 (green) channels and the respective single channels are showed on the left panel.
Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. mRNA expression of MyHC I, MyHC IIa, PGC-1α, myoglobin, TnnT1 MyHC IIb, MyHC IIx and TnnT3 in the gastrocnemius muscle (n=5-6).
Acute inhibition of Dyn2 reversibly disrupts autophagic lysosomal reformation (ALR) and lysosomal tubule scission. (A-D) Still frames from time-lapse movies of Hep3B cells expressing LAMP1-mCherry. Cells were starved for 2 h in HBSS and subsequently treated for 30 min with either DMSO (A and B) or 40 µM Dynasore (C and D), which induced extensive tubulation of LAMP1-positive compartments. Bars (A-D): 20 µM; (A′-B′) 2 µM; (C′-D′) 10 µM. (E-G) To demonstrate the reversibility of this tubulation, Dynasore-treated cells were washed extensively with drug-free media containing 10% FBS and monitored by time-lapse microscopy for 45 min. Frequently, after drug washout, LAMP1-positive tubules exhibited noticeable varicosities (E and F, arrows; bars, 10 µM) along their length. These sites are suggestive of areas of scission and resumed budding of nascent protolysosomes from the reformation tubules (G; bars, 10 µM). (H) Tubules from cells undergoing drug washout were quantified by tracing their lengths at the beginning and end of these movies. Still frames from a representative movie show tubule content at t = 10 and 45 min after drug washout. Bars, 20 µM. (I) Analysis of five independent movies showed an average decrease in total tubulation of ∼50% after drug washout. Data represent the average relative change in total tubule length between the first and last frames of the time-lapse movies. Error bars represent SE; *, P < 0.05.
(D) Representative images from live-cell movies of Nuclear-mCherry+ WT, Sas4cKO and Sas4cKO;Usp28cKO NPCs. White arrows showing the nuclei of daughter cells following cytokinesis. (E) Plot showing quantification of mitotic duration of WT, Sas4cKO and Sas4cKO;Usp28cKO NPCs. Triangles represent individual cells; triangles of the same color represent cells derived from the same embryo. Circles represent the average mitotic duration of cells from each embryo. WT n = 202 cells, N = 4 embryos; Sas4cKO n = 138 cells, N = 3 embryos; Sas4cKO;Usp28cKO n = 191 cells, N = 3 embryos; two-tailed Welch's t-test. WT and Sas4cKO data are from Figure 1K and shown alongside for comparison. (F) Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from Sas4cKO;Usp28cKO E14.5 embryos. Each bar represents one cell; its height represents the amount of time the mother cell spent in mitosis; bar color represents the fate of the progeny. Dashed line is set at 30 min. Sas4cKO;Usp28cKO n = 271 cells, N = 3 embryos. (G) Percentage of proliferating, arrested and apoptotic progeny within each group of the indicated mitotic duration, calculated from data shown in F; #, chi-square test with *, post-hoc analysis, comparisons are made to the 0-30min group.
(C) Scatter plot of 5hmC changes (Y-axis) and gene expression changes (X-axis) between CD4+ naïve T and WT Treg cells. DhmRs associated with a differentially expressed gene are denoted in color (blue: WT Treg cells; black: CD4+ naïve T cells).
Representative photomicrographs of cultured hippocampal neurons immunostained for TDP-43 (green), FLAG (red) and counterstained with DAPI (blue) in control conditions (untransduced) or infected with lentivirus expressing FLAG-tagged human TDP-43 12xQN construct.
Under the same growth conditions (permitted water, free food, and a 12 h/12 h dark/light cycle at 20±2°C), eight-week-old male C57BL/6 mice were continuously fed with methionine- and choline-supplemented (MCS) or methionine- and choline-deficient (MCD) diets for 6 weeks, and at the same time, gavaged with LicoB, MCC950 n=6 mice per group Real-time quantitative PCR was used to detect the mRNA levels of IL-1β (G), and IL-18 (H) in the mice livers, as described in (A) (n=6 mice per group).