EMBO/sd-character-level-ner huggingface dataset:
Figure 2(A, B) Site-specific methylation analysis by whole-transcriptome bisulfite sequencing. Scatter plots show individual cytosines and their respective non-conversion rates in rRNAs (A) and tRNAs (B) of wild type and noNSUN strains; pie chart showing most frequently methylated tRNA isoacceptors. Data information: In (A, B n = 3 independent biological replicates. (C) Heatmap showing non-conversion rates of tRNA positions methylated in stoichiometry higher than 50% and of tRNA positions predicted to be targets of DNMT2 and NSUN6. Data information: In n = 3 independent biological replicates. (D) Site-specific methylation analysis by whole-transcriptome bisulfite sequencing. Scatter plot shows density of cytosines and their respective non-conversion rates in mRNAs of wild type and noNSUN strains. Data information: In n = 3 independent biological replicates. (E) Heatmap showing non-conversion rates of small non-coding RNA positions methylated in stoichiometry higher than 50%. Data information: In E), n = 3 independent biological replicates.
a) UVRAG is released from the ER membrane in cells overexpressing MTMR3.figf3a) UVRAG is released from the ER membrane in cells overexpressing MTMR3. HeLa cells transfected with MTMR3-mStrawberry or the catalytically inactive mutant MTMR3C413S were stained with anti-UVRAG antibody and processed for confocal microscopy. Asterisks indicate MTMR3-transfected cells (left). The percentage of transfected cells with diffused staining of UVRAG was quantified (middle). Data are mean±s.d.; n = 240 cells collected from four independent experiments; **P0.01. Western blot analysis shows the levels of endogenous UVRAG in the presence or absence of MTMR3 (right). (b) Wortmannin treatment releases UVRAG from the ER. HeLa cells were treated with 100 nM wortmannin for 1h, and processed for confocal microscopy using anti-UVRAG and anti-PDI antibodies (top panel). UT, untreated. Insets highlight the relative localization of UVRAG at the ER. Confocal co-localization is indicated (bottom left panel). Data are mean±s.d.; n = 75 cells for each group from three independent experiments; ***P0.001. Endogenous UVRAG and PDI expression before and after wortmannin treatment were confirmed by immunoblotting (bottom right panel). (c) Mutation of the C2 domain leaves UVRAG unable to associate with the ER. HeLa cells expressing Flag-UVRAG (WT) or its K78A/R82A mutant were fixed and processed for confocal microscopy for co-localization with the ER marker PDI (top panel). Insets highlight the relative distribution of UVRAG at the ER. UVRAG and endogenous PDI expression were confirmed by immunoblotting (bottom panel). See Supplementary Fig. S9 for uncropped data. Scale bars, 10 μm.
Figure 3A Thermodynamic characterization of the RBPJ/L3MBTL3 interaction. Representative thermograms (raw heat signal and nonlinear least squares fit to the integrated data) for L3MBTL3-(31-70) binding to RBPJ-(53-474). B/C NOTCH1 ICD outcompetes L3MBTL3 for binding to RBPJ in a dose-dependent manner. IPs were performed in CRISPR/Cas9-mediated L3MBTL3 knockout (KO) HEK293T cells. (B) SBP-FLAG-RBPJ and HA-L3MBTL3-Δ(SAM) in the presence of an increasing amount of HA-NOTCH1 ICD. The L3MBTL3-Δ(SAM) mutant construct was used instead of the L3MBTL3 WT construct in order to allow the analysis of both NOTCH1 ICD and L3MBTL3 proteins in the same Western blot. CRISPR/Cas9 sg-L3MBTL3-resistant plasmids were used to express HA-L3MBTL3-Δ(SAM). The experiment was independently replicated thrice. WB: Western blot; IP: immuno-precipitation. B/C NOTCH1 ICD outcompetes L3MBTL3 for binding to RBPJ in a dose-dependent manner. IPs were performed in CRISPR/Cas9-mediated L3MBTL3 knockout (KO) HEK293T cells. (C) SBP-FLAG-RBPJ and HA-NOTCH1 ICD in the presence of an increasing amount of HA-L3MBTL3-Δ(SAM). The L3MBTL3-Δ(SAM) mutant construct was used instead of the L3MBTL3 WT construct in order to allow the analysis of both NOTCH1 ICD and L3MBTL3 proteins in the same Western blot. CRISPR/Cas9 sg-L3MBTL3-resistant plasmids were used to express HA-L3MBTL3-Δ(SAM). The experiment was independently replicated thrice. WB: Western blot; IP: immuno-precipitation.
Figure 7A, Yeast two-hybrid (Y2H) assay of interactions between the Gal4 DNA binding domain BD-NF-YB2/3, BD-NF-YC3/4/9 and Gal4 activation domain AD-TOC1/5X fusions. Yeast growth shown on leucine-, tryptophan-deficient plate indicates successful plasmid transformation, and yeast growth shown on leucine-, tryptophan-, histidine-deficient and 3-AT plate indicates positive protein interactions. B, Quantification of interactions between TOC1/5X-TAP and NF-YC proteins in presence or absence of NF-YB subunits. Proteins were transiently expressed in N. benthamiana. TOC1/5X were immunoprecipitated with IgG resins followed by HRV-3C protease digestion. TOC1/5X and NF-YB/C were detected by α-MYC and α-HA, respectively. Protein band intensity was measured by ImageJ and interactions were exhibited as ratio of co-immunoprecipitated NF-YC to immunoprecipitated TOC1/5X to NF-YC input. Results from 3 independent trials were averaged. Error bars indicate SEM, different letters denote significant differences (p<0.05, Student's t test). C, ChIP-qPCR assay of native promoter driven TOC1-GFP and 5X-GFP's residence at target promoters in toc1 vs. nf-yc3/4/9 toc1 (c349t1) mutants. 3-d-old SD-grown seedlings were harvested at ZT14, TOC1 and 5X were immunoprecipitated by α-GFP and magnetic protein G beads. Data from 3 independent trials were averaged. Error bars indicate SEM, asterisks indicate significant differences (* p<0.05, *** p<0.0001, Student's t test), n.s= not significant.
Figure 4Figure 4. Mmi1 drives Rrp6-dependent heterochromatin gene silencing at pericentromeric regions. (A) Upper part, TNAAAC-rich sequence present in nam5, 6 and 7 lncRNAs. Lower part, schematic representation of S. pombe three centromeres showing the different pericentromeric DNA repeats susceptible to produce nam5, 6 and 7 (green arrows). Figure 4. Mmi1 drives Rrp6-dependent heterochromatin gene silencing at pericentromeric regions. (B) Northern blot showing the level of nam5/6/7 lncRNA population in mmi1-ts3, dcr1∆, clr4∆ single mutant cells and in mmi1-ts3 dcr1∆ and mmi1-ts3 clr4∆ double mutant cells, at the permissive (25ºC) and restrictive (36ºC) temperatures. Figure 4. Mmi1 drives Rrp6-dependent heterochromatin gene silencing at pericentromeric regions. (C) RT-PCRs monitoring the accumulation of nam7 lncRNAs and nam7-L read-through transcripts, in the same cells and conditions as in (B). Red arrow heads point to the expected PCR products while the other bands correspond to non-specific PCR products. From the scheme and the agarose gels: black arrows, primers used for the three different reverse transcriptions; black lines and red numbers, expected PCR products of the three different RT-PCRs; M, DNA ladder Markers; tub1, tubulin control. Figure 4. Mmi1 drives Rrp6-dependent heterochromatin gene silencing at pericentromeric regions. (D) RT-qPCRs showing the levels of nam5/6/7 lncRNA population in the double mutant rrp6∆ dcr1∆ cells, relative to the single mutant rrp6∆ and dcr1∆ cells. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (D, E). P value was calculated using a two-tailed Student's t-test. Figure 4. Mmi1 drives Rrp6-dependent heterochromatin gene silencing at pericentromeric regions. (E) RNA-IPs showing that Rrp6-Myc13 binds to nam5/6/7 lncRNAs in a Mmi1-dependent manner. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (D, E). P value was calculated using a two-tailed Student's t-test.
Figure 4A: Relative mRNA copy number dynamics in WT and Cnot6l-/- samples at the indicated stages. Error bars indicate values of the two biological replicates B: Box plot showing gene expression levels of WT, Cnot6l-/- and Btg4-/- oocytes at the MII stage. Genes were divided into 10 bins according to their expression levels in the WT MII oocytes. The box indicates upper and lower quantiles, the thick line in the box indicate the median, and the whiskers represent 2.5th and 97.5th percentiles. ***P < 0.001 by two-tailed Student"s t test. n.s.: non-signif C: qRT-PCR results showing the relative expression levels of Cnot6l in oocytes collected from WT and Cnot6l-/- mice (Cnot6l-/-/WT). Error bars, s.e.m. ***P < 0.001 by two-tailed Student"s t test. n = 3 biological repli D: Scatter plot comparing transcripts between WT and Cnot6l-/- oocytes (at GV, MI and MII stages) and zygotes derived from these oocytes. Transcripts decreased or increased more than five folds in Cnot6l-/- oocyte samples were high-lighted with blue or red, respectively E: Heatmap showing transcripts that were degraded during meiotic maturation (GV/Zygote > 10) in WT oocytes. The definition of cluster I-III is described in the text F: Relative mRNA copy number dynamics of the three gene clusters in WT, Cnot6l-/- and Btg4-/- samples G: Venn diagrams showing the overlap of transcripts that were significantly degraded during GV-to-MII transition in WT, Cnot6l-/- and Btg4-/- oocytes (GV/MII > 10) H: Venn diagrams showing the overlap of transcripts that were stabilized during GV-to-MII transition in Cnot6l-/- and Btg4-/- oocytes (GV/MII > 10 in WT, but <10 in KO) I: Venn diagrams showing the overlap of transcripts that were destabilized in GV-MII transition (FPKM > 1; fold change (GV/MII) > 10) of WT oocytes and the genes upregulated in this process after Cnot6l knockout (FPKM > 1; fold change (Cnot6l−/−/WT at MII) > 10)
Stress alters HRPA-1 assembly in vivo.Figure 5A) mScarlet was added between the RNA recognition motifs (RRMs) and LC of HRPA-1 and the third exon was replaced with the corresponding human sequence; disease mutation D290V was introduced. B) Glutamatergic touch neurons ALM and PLM expressing mec-4 were scored for number of spots in processes. C) Representative images of mec-4 neurons with and without HRPA-1mScarlet spots. (various HRPA-1mScarlet containing genotypes.) D) After 22 hours of paraquat induced oxidative stress, both HRPA-1mScarlet and HRPA-1HsLCWTmScarlet assemble more than when unstressed (ANOVA p<0.0001). No significant differences were found within the no stress (p=0.8) stress groups (p=0.45) by ANOVA, indicating that there is no difference in assembly between HRPA-1mScarlet and HRPA-1HsLCWTmScarlet. N = 12 animals per genotype. E) Fyn expression constructs including activated Fyn (Fyn*, Y531F) and empty control Fynempty. F) There is no significant difference in number of spots in HRPA-1HsLCWTmScarlet lines with or without Fyn, although there seems to be a trend of reduced spots in HRPA-1HsLCD290VmScarlet lines in animals also expressing Fyn*; this difference is not statistically significant (ANOVA). N = 12 animals per genotype. G) After exposure to 22 hours paraquat induced oxidative stress there is no significant difference between HRPA-1HsLCWTmScarlet lines with or without Fyn, although there seems to be a trend of reduced spots in HRPA-1HsLCD290VmScarlet lines in animals also expressing Fyn*; this difference is not statistically significant (ANOVA). N = 12 animals per genotype.
Figure.Figure 8(A) YY1 controls the activation of SCs by acting as a switch of metabolic reprogramming. In Ctrl SCs, its expression is increased during SC activation upon injury induced muscle damage and it transcriptionally suppresses mitochondrial gene expression through direct binding to their TSS regions together with PRC2 repressive complex. Meanwhile, it also stabilizes Hif1α protein to facilitate its trans-activating of glycolytic genes. As a result, successful activation of glycolytic pathways occurs, allowing SCs to activate and proliferation to drive the repairing of the damaged muscle. When YY1 is deleted in the YY1iKO mouse, Hif1α level is decreased due to protein destabilization, leading to a reduction in glycolytic gene expression and glycolytic rate. Meanwhile, although the expression of mitochondrial genes is up-regulated, OXPHOS could not be increased likely because of reduced flow of pyruvate substrate into mitochondria. As a result of the inhibited metabolic reprogramming, SC activation is repressed and the regeneration is compromised. Quiescent SCs are defined by expression of Pax7 and lack of MyoD expression (Pax7+MyoD-), activated and proliferating myoblasts by the concomitant expression of Pax7 and MyoD (Pax7+MyoD+), differentiating myocytes by expression of MyoG (Pax7-MyoG+), and differentiated myotubes are marked by expression of Myosin heavy chain (MyHC). Error bars represent s.d.'s of the mean. Student's t-test (two-sided): *P<0.05, **P<0.01, ***P<0.001.
Figure 4Mice were treated as described in Fig.1A. LP cells were isolated from each mouse. The proportions of IL-22+ cells in CD3-RORγt+, CD3-RORγt-, CD3+RORγt- and CD3+RORγt+ were determined by flow cytometry. Mice were treated as described in Fig.1A. LP cells were isolated from each mouse. The proportions of IL-22+ cells in NKp46-RORγt+, NKp46+RORγt- and NKp46+RORγt+ were determined by flow cytometry. WT and Dectin-3-/- mice were intraperitoneally treated with anti-CD90 antibody or anti-IgG antibody during AOM-DSS administration (n=5, each group). Mice were euthanized on Day 100, feces were collected in colon from each mouse. Total fungal burden in feces were quantified using 18S rDNA qPCR. Mice were treated as described in Fig.4E and colonic LP cells were isolated. Numbers of IL-22+ cells in LP cells were determined by flow cytometry. Mice were treated as described in Fig.4E. Tumor number, tumor size, tumor load and histological score in colons were measured.
Figure 2A) The upper panel indicates the FACS strategy followed to purify the different mammary epithelial subpopulations found in the mouse mammary gland (basal, luminal HR-, luminal HR+, and MaSCs). The lower panel shows the normalized expression of miR-424(503) and miR-503 revealing that MaSCs express the highest levels. The error bars show the standard deviation. FACS was performed at two different stages of the ovarian cycle (estrus and diestrus). Carmine-red staining of the structure of the mammary tree at the time of purification is also provided. The scale bar indicates 5 millimeters (mm). B) The bar graphs show the number of PROCR+ in 1-year-old female WT and miR-424(322)/503 KO mice during diestrus. The error bars show the standard deviation. C) The bar graphs indicate the number of primary organoids formed when FACS sorted WT or miR-424(322)/503 mammary epithelial basal and luminal cells from female mice at diestrus (4-6 months old) were cultured as organoids. The error bars show the standard deviation. D) The western blot shows the difference in nuclear accumulation of β-catenin when dox-induced (100ng/ml for 3 days) miR-cluster-Dox-MCF-10A cells (miR) and miR-control-Dox-MCF-10A cells (EV, empty vector) were exposed to of WNT3a ligand (100ng/ml for 6 hours). The numbers below the blots indicate the quantitation by densitometry of protein expression relative to EV without WNT3A. Histone H1 staining is shown to control for nuclear-cytosolic purification. The upregulation of miR-424 and miR-503 when dox was added to the media was quantified by QRT-PCR and is shown in the bar graph. The error bars show the standard deviation. E) The panel shows the lack of induction of bona fide WNT targets in miR-over expressing cells from panel C compared with EV control. UT indicates samples untreated with WNT3.
Figure 9Data were fitted with an allosteric sigmoidal model in GraphPad Prism software and used to determine the kinetic parameters associated with cNMP dependent phosphodiesterase activity.Data points for both sets of results are the average of three experimental replicates with the standard deviation shown by intersecting bars. Results are representative of at least three independent experiments.
Figure 3CD8+ HER-2 CAR-T cells generated from Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl;Lck+/- splenocytes were stained for intracellular p(Y418)-SFK and p(Y418)-SFK MFIs were determined by flow cytometry. HER-2-specific Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl;Lck+/- CAR-T cells were incubated with HER-2 expressing 24JK sarcoma cells (24JK-HER-2) or HER-2 negative 24JK sarcoma cells and CD44, CD25, PD-1 and LAG-3 MFIs on CD8+ CAR-T cells were determined by flow cytometry. Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl, or Lck-Cre;Ptpn2fl/fl;Lck+/- HER-2 CAR-T cells were incubated with 24JK-HER-2 or 24JK sarcoma cells and the proportion of CD8+IFNγ+ versus CD8+IFNγ+TNF+ CAR-T cells determined by flow cytometry. Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl or Lck-Cre;Ptpn2fl/fl;Lck+/- HER-2 CAR-T cells were incubated with 5 μM CTV-labelled (CTVbright) 24JK-HER-2 and 0.5 μM CTV-labelled (CTVdim) 24JK sarcoma cells. Antigen-specific target cell lysis was monitored for the depletion of CTVbright 24JK-HER-2 cells by flow cytometry. HER-2-specific Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl and Lck-Cre;Ptpn2fl/fl;Lck+/- CAR-T cells were incubated with plate-bound α-CD3 and CD25 MFIs on CD8+CD44hiCD62Llo versus CD8+CD44hiCD62Lhi CAR-T cells determined by flow cytometry.
Sodium butyrate inhibited proliferation and induced apoptosis in colorectal cancer cells.Fig 1HCT-116 (A) and HT-29 (B) cells were treated with the indicated concentrations of sodium butyrate (NaB) for 24 (grey bars), 48 (pink bars), or 72 (yellow bars) hours and cell proliferation was assessed. HCT-116 (C, E) and HT-29 (D, F) cells were treated with the indicated concentrations of NaB for 24 h. C.D. The percentage of apoptotic cells in HCT-116 (C) and HT-29 (D) cells was quantified in three independent experiments using an annexinV/PI assay (annexinV+/PI-) by flow cytometry and expressed as mean ± SD. One-way ANOVA was used to compare between the control cells and NaB treatments. *p<0.05, ** p<0.01 compared to control. E.F. Representative Western blots of full-length and cleaved PARP products in HCT-116 (E) and HT-29 (F) cells.
Figure 6(A) Localization of the basal-specific optogenetic anchor CIBN-PatJ::GFP-pm during ventral furrow formation. Embryos expressing CIBN-PatJ::GFP-pm were imaged using two-photon excitation during ventral furrow formation. Transverse cross section (z-projection) at a stage when cells were already apically constricted and elongated, and the ventral tissue already internalized. Embryos co-expressing CIBN-PatJ::GFP-pm, RhoGEF2-CRY2 and Sqh::mCherry (B,C were mounted with the ventral tissue facing the objective. After cells underwent apical constriction and cell elongation the basal volume of the ventral cells was photo-activated using two-photon illumination and the myosin-II signal was recorded in alternation. (B) myosin-II distribution in a cross section (z-projection) 3 min after photo-activation. myosin-II was locally recruited to the base as indicated by the red arrowheads in the activated region (red line). (C) Top view (sum of slice of 4 µm-sized image stack) showing basal myosin-II recruitment in the photo-activated region (red dashed square) Data information: Scale bars, 20 µm Embryos co-expressing CIBN-PatJ::GFP-pm, RhoGEF2-CRY an the membrane marker GAP43::mCherry (D-I) were mounted with the ventral tissue facing the objective. After cells underwent apical constriction and cell elongation the basal volume of the ventral cells was photo-activated using two-photon illumination and the myosin-II signal was recorded in alternation (D) Cross section (z-projection) showing the plasma membrane and (E) top view showing the apical plasma membrane at the initial time point before photo-activation. At this stage, the central mesodermal tissue has started to internalize and cells were constricted at the apical surface. In an alternating fashion, the base of the cells in the anterior half of the embryo (left) was photo-activated for 100 sec and the GAP43::mCherry signal in the entire field of view was recorded. The furrow further ingressed and closed completely 3 min (F) and 6 min (G) after initial activation, respectively. In the activated region, the tissue failed to internalize and to form a closed furrow both at 3 min (H) and 6 min (I) after initial photo-activation. The width of the central tissue forming the ventral furrow at the initial point of the experiment is 40 µm (D). In the non-activated region 6 min after initial activation the width of the tissue shrank to about 25 µm (G), whereas it remained constant at 40 µm in the activated region (I). Data information: Scale bars, 20 µm
Figure 7A. Violin plot showing the expression of MCT family members (monocarboxylate transporters) in brain pericytes from scRNAseq database (Brain cell atlas; 1,100 endothelial cells) B. Barplot showing the expression of MCT12 across brain cells from the scRNAseq database (vSMC: venous smooth muscle cells, aaSMC: arteriolar SMCs, aSMC: arterial SMCs, MG: Microglia, FB1-2 : Fibroblast subtypes, OL : Oligodendrocytes, EC1-3 : EC subtypes, vEC : venous ECs, capiEC: capillary ECs, aEC: arterial ECs, AC : astrocytes) (1,100 endothelial cells, mean±SD) C. Relative mRNA expression level of MCT12 in HBVPs (blue) and BMECs (orange). (n=3 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.) D. Western blot analysis showing expression level of MCT12 and β-actin in HBVPs and BMECs. E. Western blot analysis for siRNA efficiency of MCT12. F. Quantification of [13C3]-lactate using LC-MS/MS in siCON (blue) or siMCT12 (orange) treated HBVPs after 20mM [13C3]-Lactate treatment (2hr). (n=3 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.) G. Representative images showing mTFP (cyan) and Venus (yellow) fluorescence in control (upper panel) or MCT12 (lower panel) siRNA treated HBVPs in a 2 well chamber assay system with BMECs. VECAD (purple) immunostaining was used for endothelial staining. H. Quantification of the mTFP/Venus fluorescence ratio of Laconic in control (blue) or MCT12 (orange) siRNA treated HBVPs. (n=8 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test. I. Isotope carbon tracing with [13C3]-lactate treatment (20mM, 24hr) in siCON or siMCT12-treated HBVPs showing the extent of downstream metabolites and incorporation rate of labeled carbons. (F-6-P : Fructose 6-phosphate, G-3-P : Glycerol-3-phosphate, Lac : Lactate, Cit : Isocitrate and aKG : α-ketoglutarate) (n=3 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.
HOTAIR expression in Epi cells induces drastic changes in expression of protein-coding genes, majorly dependent on the presence of both, PRC2- and LSD1-interacting domains.Figure 2Number of up- and down-regulated protein-coding genes (PCG) defined as differentially expressed in Epi-HOT, -HOTΗP and -HOTΗP cells compared to Epi-CTR by RNA-seq and DESeq analysis (fold change > 2 and adj. P-value < 0.05), including those associated with EMT (Gröger set, Dataset EV2) and already identified as HOTAIR/PRC2 targets (Gupta set, Dataset EV2); nd stands for non-determined. 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). KEGG pathways identified by DAVID as significantly enriched (adj. P-value < 0.05) for HPL-neg and HPL-pos sets of protein-coding genes, ranked by enrichment score calculated as -log10(P-value).