EMBO/sd-character-level-ner huggingface dataset:
NCOA5 represses Abca1 expression following TLR3–LXR signal crosstalk by inhibiting RNAPII function A, B Unmodified/pSer5 RNAPII (A) or RNAPII pSer2 (B) ChIP assays from primary BMMs stimulated for 4 h with 1 μM T0901317, 1 μM T0901317 + 6 μg/ml PolyIC, or vehicle control.Figure 6A, B Unmodified/pSer5 RNAPII (A) or RNAPII pSer2 (B) ChIP assays from primary BMMs stimulated for 4 h with 1 μM T0901317, 1 μM T0901317 + 6 μg/ml PolyIC, or vehicle control. qPCR was performed for the Abca1 TSS. Error bars represent ± SEM for n = 6-9 (**P = 0.0001 in A, **P = 0.002 in B versus T0901317). C, D RNAPII ChIP assays as in (A, B) but performed from LXR−/− BMMs. Error bars represent ± SEM for n = 5-9 (**P = 0.000000001, *P = 0.02 versus T0901317). E RT-qPCR of Abca1 expression from LXR+/+ versus LXR−/− BMMs. Ligand stimulations were performed for 4 h with vehicle control, 1 μM T0901317, or 1 μM T0901317 together with 6 μg/ml PolyIC. Fold changes are shown relative to vehicle‐stimulated LXR+/+. Error bars represent ± SEM for n = 4 (**P = 0.004, *P = 0.012 versus T0901317). F RNAPII pSer2 ChIP assays from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Ligand stimulations were performed for 4 h with vehicle control, 1 μM T0901317, or 1 μM T0901317 + 6 μg/ml PolyIC. qPCR was performed for the Abca1 TSS. Note RNAPII pSer2 returns to baseline occupancy following TLR3 stimulation in shControl but not shNcoa5 BMMs (**P = 0.004 versus baseline). Error bars represent ± SEM for n = 3 from 11 mice.
Figure 1Immunoblot analysis of mouse intestinal crypts treated with Wnt3a (60 ng/mL) for indicated time. The relative protein level was quantified and shown under each band. Polysome profiles of mouse intestinal crypt treated with or without Wnt3a for 30 min. The right panels show the distributions of Ythdf1 and Actb in polysome fractions. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test). Immunoblot analysis of SW620 cells with or without APC overexpression. The relative protein level was quantified and shown under each band. Polysome profiles of SW620 cells with or without APC overexpression. The right panels show the distributions of YTHDF1 and ACTB in polysome fractions. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test). Dual-Luciferase assay wih a construct bearing the 5'UTR of YTHDF1 in SW620 cells with or without APC overexpression. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test). YTHDF1 protein expression in intestinal tissue from 4 pairs of Apc+/+ and Apcmin/+ mice. N, normal tissue. T, tumor tissue. A, tumor adjacent tissue. YTHDF1 staining in matched samples of human CRC tissue and adjacent non-tumor tissue. Scale bar, 450 μm. The right panel shows the statistics of IHC scores. Chi-square test; n = 75 for each group.
Stat3 regulates directly the mitochondrial DNA.Figure 2(A) Schematic representation of D-loop-Lux reporter construct used for luciferase assays. (B) Luciferase assay on ES cells transfected with D-loop-Lux reporter plasmid and Stat3 in the presence or in the absence of LIF for 48 hours; p53 was previously shown to activate a similar reporter construct (Heyne et al, 2004) and therefore was used as positive control. Increased expression of Stat3 enhances Luciferase activity. Mean and sem of 4 independent experiments. Unpaired t-test: * P<0.05. (C) Luciferase assay on EpiSCs transfected with D-loop-lux reporter plasmid and Stat3 in the presence of LIF for 48 hours. Mean and sem of 3 independent experiments. Unpaired t-test: * P<0.05. (D) Diagram highlighting available ChIP-seq results (GSM288353) of Stat3 binding on the D-loop. A,B and C indicate regions where primers for ChIP-PCR analysis were designed. (E) Chromatin immunoprecipitation (ChIP) was performed using anti-Stat3 or a rabbit control IgG antibody in Rex1-GFP cells cultured in LIF and serum conditions in the presence of Blasticidin to reduce heterogeneity of the culture. ChIP-PCR was performed with primers located on three regions of the D-loop (A, B, C), as indicated in Figure 2D. Mean and st.dev of two independent experiments are shown. (F) Representative confocal images of Stat3+/+ cells subjected to proximity-ligation-assay (PLA) by using anti-Stat3 and anti-ATAD3 antibodies (See Appendix Figure S2 for ATAD3 localisation in nucleoids). Only when the two proteins are close to each other an enzymatic reaction takes place, producing discrete fluorescent red dots in the nanometer range (bottom). Anti-Stat3 or anti-ATAD3 alone were used to assess the assay specificity (top and centre respectively). DAPI serves as a nuclear counterstain. Scale bar, 10µm.
Figure 7A. Scheme of the protocol designed for the combined treatment of HEK293 cells with H2O2 and DPTA. B. HEK293 cells overexpressing the wild type form of GSNOR (GSNORwt) or an empty vector (Empty) were subjected to combined treatment (200 μM H2O2 + 400 μM DPTA) in the presence or absence of the ATM inhibitor KU55933 (KU). Analysis of dead cells were performed with Trypan blue exclusion assay. Western blot analysis of GSNOR is shown as inset in the graph to substantiate transfection efficiency. Data, shown as fold change of dead cells relative to untreated cells (arbitrarily set to 1), represent the mean count ± SEM of n = 4 experiments done in duplicate *p<0.05; with respect to Empty cells. C. Representative optic microscopy image of GSNORwt and Empty cells upon 24 h treatment with H2O2 + DPTA in the presence KU. D. Western blot analysis of Caspase3 and PARP1 in GSNORwt and Empty cells treated as in panel C. Tubulin was used as loading control. E. HEK293 cells overexpressing the wild type form of GSNOR (GSNORwt) or an empty vector (Empty) were transfected with siRNA against Pakin (siParkin) or control siRNA (scramble, siScr). Afterwards, they were subjected to combined treatment (200 μM H2O2 + 400 μM DPTA) and viability assessed by Alamar blue (AB) fluorescent assay. Data, shown as fold change of viable cells, refer to AB fluorescence (relative to untreated cells, arbitrarily set to 1) and represent the means ± SD of n = 6 independent experiments. **, p<0.01; n.s., not significant. F. Western blot of Gsnor was assessed in mouse adult fibroblasts (MAFs), obtained from wild type (WT) and Gsnor-null (KO) mice treated with 200 or 400 μM H2O2 for 24 h. WT and Gsnor KO MAFs were subjected to treatment with 200 μM H2O2, or 400 μM DPTA, or a combination of both. Cell viability was evaluated by LIVE/DEAD assay. H. WT and Gsnor KO MAFs were subjected to treatment with 200 μM H2O2, or 400 μM DPTA, or a combination of both. Cell viability was evaluated by LIVE/DEAD assay. Scale bar = 50 µm. Data, shown as % of dead (red) cells, represent the mean count ± SD of n = 3 different fields of three independent experiments. *p<0.05; n.s., not significant with respect to WT MAFs.
Figure 8In brief, nuclear IκBα is required for the PRC2-mediated timely repression of fetal ISC genes during development and activation of adult ISC genes. In the absence of IκBα, expression of fetal ISC genes is sustained after birth resulting in defective ISC maturation (upper panel). During regeneration (lower panel), specific signals such as IFN impose a temporary fetal ISC phenotype that is required for subsequent tissue regeneration (Nusse et al, 2018, Yui et al, 2018). Next, nuclear IκBα massively accumulates at regeneration areas to repress fetal ISC gene expression thus facilitating ISC maturation and tissue repair. Paradoxically, IκBα KO mice do not show evidence of colonic ulceration after DSS treatment suggesting that fetal-like ISC (imposed by IκBα deficiency) are intrinsically resistant to injury.
Figure 6Double immunolabeling of SYCP3 (red) and the cohesin subunits SMC3 (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm. Double immunolabeling of SYCP3 (red) and the cohesin subunits REC8 (green in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm. Double immunolabeling of SYCP3 (red) and the cohesin subunits SMC1β (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm. Double immunolabeling of SYCP3 (red) and the cohesin cofactors Sororin (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm. Double immunolabeling of SYCP3 (red) and the cohesin cofactors WAPL (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.
Figure 3A. Sequence alignment of CENP-CCR and CENP-Cmotif regions from different mammals. Conserved residues involved in CENP-A binding are highlighted in blue (electrostatic interactions) and pink (hydrophobic interactions). Residues identified in this study to contribute higher affinity of CENP-CCR comparing to CENP-Cmotif, are boxed. B. CryoEM density map of the human CENP-A nucleosome, color-coded for histones, in complex with CENP-CCR (magenta). CENP-CCR binds CENP-A nucleosome through hydrophobic region (big box) and arginine anchor (small box). Interacting residues in each of the regions are shown in (C). C. Ribbon diagram showing interactions of the CENP-CCR hydrophobic region (left) and CENP-CCR arginine anchor (right) with the CENP-A nucleosome. D. Overlay of the CENP-A C-terminal tail before (gray) and after (red) binding of CENP-CCR. E. Interactions between H3-GIEGGL and rat CENP-Cmotif as observed in PDB 4X23. F. Schematic diagram of mutated CENP-CCR sequences used to test importance of CENP-CV532 and CENP-CV533 for generation of CENP-A/CENP-CCR complexes. G. Native gels showing the impaired ability of mutated CENP-CCR to form complexes with the CENP-A nucleosome. The molar ratio of CENP-CCR/CENP-A nucleosomes is shown above each lane.
Cytotoxic CD8 T lymphocytes infiltrate macrophage depleted tumours.Figure 4A-B MC38 (A) and KPC (B) tumours were harvested 5 days following 10Gy IR ± aCSF as indicated. The left panels show the percentage of CD45+ CD3+ CD8+ T cells in these tumours after the indicated treatments. Representative flow cytometry plots from the irradiated groups are shown in the right panels. Tumours that did not receive irradiation were harvested when tumours reached 500mm3. Data are presented as mean SEM and analysed by one-way ANOVA with Tukey post-hoc adjustment (A) and Kruskal-Wallis test with Dunn's multiple comparison (B) (n=6 mice/group, 3 independent experiments). C, Flow cytometric analysis of Ki67 expression in the CD8+ T cells in MC38 tumours from A. Data are presented as mean SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=6/group). D, Flow cytometric analysis of PD-1 expression on CD8+ T cells from MC38 in tumours as in A. Data are presented as mean SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=6/group). E-F Flow cytometric analysis of IFN-γ expression in CD8+ T cells isolated from MC38 tumours in A. Data presented as mean SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=6 mice/group, 3 independent experiments). E-F, Flow cytometric analysis of Granzyme B expression in CD8+ T cells isolated from MC38 tumours in A. Data presented as mean SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=6 mice/group, 3 independent experiments). G-H, Immunofluorescent staining of MC38 (G) and KPC (H) tumour sections, blue=DAPI, green = CD8. I-J, Tumour growth in CD8 depleted C57BL6 wild-type mice bearing MC38 (I) and KPC (J) tumours receiving treatment as indicated (n=6 mice/group). K, Flow cytometric quantification of intra-tumoural CD8 T cells following anti-CD8 treatment. Data presented as mean SEM and analysed by unpaired t-test (n=5 mice/group). L-M, Tumour growth in athymic nude mice bearing MC38 (I) and KPC (J) tumours receiving treatments as indicated (n=6 mice/group). Data are presented as mean tumour volume ± SEM and analysed by one-way ANOVA with Tukey post-hoc adjustment. *P <0.05 **P <0.01 ***P <0.001.
Figure 2A The different stages of seizures based on increased severity. B Average seizure score of KA-treated WT and Fxr2 KO mice over time from t=0 (injection time) to 2 h. * p < 0.05 compared to WT mice (two-way Repeated Measures ANOVA; interaction effect of time and genotype; F1,666 = 10.05, p = 0.0016). KA-treated WT mice (n=16), KA-treated Fxr2 KO mice (n=12). Data are presented as mean ± Standard Error of the Mean (SEM). D Average seizure score of pilocarpine-treated WT and Fxr2 KO mice over time from t=0 (injection time) to 2 h (two-way Repeated Measures ANOVA). Pilocarpine-treated WT mice (n=6), pilocarpine-treated Fxr2 KO mice (n=6). Data are presented as mean ± SEM. C Responsiveness to KA of WT (black, n=16) and Fxr2 KO mice (magenta, n=12). *** p < 0.001 compared to Fxr2 KO mice (two-tailed Student's t-test; t26 = 5.72, p < 0.001). Data are presented as mean ± SEM. E Responsiveness to pilocarpine (PC) of WT (n=6) and Fxr2 KO mice (n=6). (two-tailed Student's t-test). Data are presented as mean ± SEM.
Figure 1(A) Stable RNAi screening of cancer-related genes was conducted to identify gene knockdowns that enhance reprogramming of MEFs into iPSCs. The graph shows the 10 shRNAs that were most strongly, positively selected in iPSCs compared to non-reprogrammed cells on day 14 of reprogramming (n=3 biological replicates). (B-E) Mouse embryonic fibroblasts (MEFs) were co-infected with shRNAs against Tnfaip2 or a scramble shRNA and reprogramming factors. The co-transduced cells were sorted and were reprogrammed for 14 days: (B) Representative images and (C) quantification of alkaline-phosphatase-positive (AP+) iPSC colonies on day 14 of reprogramming by transfection of 4 reprogramming factors (=4F: Oct4, Sox2, Klf4, c-Myc = OSKM) or 3 factors (=3F: OSK). [n=3 biological replicates per group; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by one-sided t-test analyzed by one-sided t-test with Holm-Sidak correction for multiple testing]. Mouse embryonic fibroblasts (MEFs) were co-infected with shRNAs against Tnfaip2 or a scramble shRNA and reprogramming factors. The co-transduced cells were sorted and were reprogrammed for 14 days: (D) Representative FACS profiles (E) percentage of Oct4-GFP+ iPSCs obtained after 14 days of reprogramming of MEFs from Oct4-eGFP mice using 4F-transduction on day zero that were infected with shRNAs targeting Tnfaip2 or a scrambled shRNA control [n=5 biological replicates; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by one-sided t-test with Holm-Sidak correction for multiple testing]. ]. (F) mRNA of Tnfaip2 was measured by RT-qPCR at the indicated days of reprogramming by 4F-transduction on day zero [n=3 biological replicates; log-transformed data were not normally distributed (P>0.05 as per Shapiro-Wilk test), P value for the upregulation of Tnfaip2 on consecutive test days-9,-12 and -14 was calculated starting with the probability of maximum rank-based difference, i.e. having two groups of three data points perfectly separating, p1,b = 0.1 (bidirectional) and p1,u = 0.05 (unidirectional); the probability of finding a triple series of max. difference starting at some timepoint is Ptriple,tp = p1,b · p1,u · p1,u, and finding such a triple somewhere across the 5-step time series is Ptriple = Ptriple,tp + 2 · (1- p1,u) · Ptriple,tp = 0.0007.
Nrf2 transcription factor–binding sites are enriched among Ub-associated genes.Figure 4.(A) Enrichment of predicted transcription factor (TF)-binding sites in the promoter region (−5,000 to 300) of Ub-associated genes versus its representation genome wide. The top 10 most enriched transcription factor-binding sites predicted among Ub-associated genes are labeled, with their enrichment scores (or −Log10-transformed Benjamini-Hochberg-adjusted p-values) plotted against its occurrence. (B) Elevated Ub mRNA levels from Atg5-deficient brains. Total RNA was extracted from control Atg5 littermates (Atg5flox/+;nestin-Cre) at 16 wk or Atg5 KO (Atg5flox/flox;nestin-Cre) at 16 wk (n = 4 for each genotype; **, P ≤ 0.01) and the amount of transcript from Ubc, Ubb, Uba52, and Uba80 genes was quantified using real-time RT-PCR and normalized to actin. Error bars indicate SEM. au, arbitrary unit. (C) Gene expression analysis of Ub-associated Nrf2 targets mapped to mouse orthologues. Probes detecting transcripts exhibiting differential expression (Benjamini-Hochberg-adjusted P < 0.05) in at least one of the four comparisons are shown. Heat map represents Log2 (fold changes) with respect to either wild type (WT) or Atg7 KO (as indicated). Labels show the symbol, RefSeq (accession numbers from the NCBI Nucleotide database), gene probe name, and the gene row and gene column (i.e., array coordinates).
Figure 6The phosphatase PP2ACdc55 lies at the center of a hyperosmotic stress signaling cascade. Under non-stress conditions (left) the balanced activities of PP2ACdc55 and counteracting kinase(s) result in a steady state of low phosphorylation of shared substrates. Hyperosmotic stress signaling transmitted through the Greatwall Kinase/Endosulfin module activates the PP2ACdc55 inhibitors Igo1 and Igo2 via phosphorylation at key regulatory sites (right). The resulting inhibition of the phosphatase is essential to tip the balance towards increased net phosphorylation.
Figure 2Bubble graphs representing, for each feature, the number of strains with a score value, s, beyond 3, 4, 5 or 6 times the interquartile range away from the median of the WT distribution (240 replicates), corrected by a factor 1.35 to express the deviation in terms of standard deviations (iqr ≈ 1.35 x σ for a normal distribution). The size of the circles (bubbles) reflects the number of strains with a score beyond a specific, color-coded threshold for s, as indicated. The "reference" bubble graph illustrates the expectations from a dataset of the same size (4,227 strains), assuming a standardized normal distribution of scores (with a mean of 0 and a standard deviation of 1).
Fig.Figure 4HEK293 cells were transfected with α11.2, α2δ-1, and β2A plus, if indicated, α-actinin-1 before cell attached patch recording. Representative single CaV1.2 channel recordings of WT CaV1.2 alone or with WT or E847K/E851K mutant α-actinin-1. Mean assemble averages for all experiments for each combination. Dot plots and means + SEM for availability (c), NPo (d), Po (e), unitary current amplitude i (f), and the mean of the current of a single channel calculated from the ensemble averages of each experiment (g) (*p<0.05, **p<0.01, ****p<0.001; unpaired t-test; n = 13 to 35; see Table 6 for more details).
Figure 4(A) RPE1 cells were transfected with siRNAs as indicated, followed by transfection with wild-type pGFP-Rab8a (WT-Rab8), constitutively active pGFP-Rab8a (Q67L) (CA-Rab8), or DN Rab8 dominant-negative pGFP Rab8a (T22N), incubated in serum-starved media for 12 h, and immunostained for ARL13B (Red), GFP-Rab8 (green), and DAPI (blue). Scale bar, 10 μm. (B) Quantification of the percentage of ciliated cells Data represent mean ± SD (n=3 experiments), 200 GFP-positive cells were scored per condition per experiment, *P < 0.05, Student's t-test. (C) (Upper panel) Cells were transfected as indicated and the cell lysates were incubated with purified proteins, including GST or GST-JCF1 (RBD). The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by western blot with Rab8 antibody. (Lower panel) Intensity of the bands was quantified by Image J software. The amount of GTP-Rab8 were normalized to the control level. Bar graph represents mean ± SD (n=3 experiments). *P < 0.05, Student's t-test. (D) Cells were transfected and cell lysates were incubated with purified GST-JCF1 (RBD) fusion protein. The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by Western blot with Rab8 antibody. (Lower panel) Cells were transfected and cell lysates were incubated with purified GST- protein. The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by Western blot with Rab8 antibody.
a) Immunoblot analysis of caspase-1 and IL-1β in lysates (Lys) and supernatants (Sup) of peritoneal macrophages obtained from Map1lc3b−/− mice and Becn1+/− mice and the corresponding wild-type littermate mice, then left unstimulated (−) or stimulated with LPS alone (LPS) or LPS followed by 30 min of ATP treatment (L + A).figf1(a) Immunoblot analysis of caspase-1 and IL-1β in lysates (Lys) and supernatants (Sup) of peritoneal macrophages obtained from Map1lc3b-/- mice and Becn1+/- mice and the corresponding wild-type littermate mice, then left unstimulated (-) or stimulated with LPS alone (LPS) or LPS followed by 30 min of ATP treatment (L + A). p10, mature form of caspase-1. Right margin, molecular size in kilodaltons (kDa). (b) Enzyme-linked immunosorbent assay (ELISA) of cytokine secretion by peritoneal macrophages stimulated with LPS, followed by treatment for 1 h with ATP. (c) Immunoblot analysis of caspase-1 and IL-1β in lysates (left) and ELISA of cytokines in supernatants (right) of Map1lc3b+/+ and Map1lc3b-/- BMDMs incubated with LPS, followed by 30 min (immunoblot) or 1 h (ELISA) of ATP treatment. (d) ELISA of IL-1β secretion by LPS-primed Map1lc3b-/- mice and Becn1+/- BMDMs incubated for 1 h with z-YVAD-fmk (z-YVAD; 10 μM) or vehicle (dimethylsulfoxide (DMSO)), followed by stimulation for 1 h with ATP. *P 0.05 (Student's t-test). Data are representative of three or four experiments (mean and s.d. in b-d).
Figure 3 Figure 3. 14-3-3 outcompetes ITSN for binding to phosphorylated DENND2B A Purified GST-14-3-3 WT or K50E were incubated with lysates of HEK-293T cells expressing GFP-DENND2B WT or S30A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively. B HEK-293T cells expressing Flag-DENND2B were treated with DMSO or 250 nM OA and cell lysates were incubated with GST-14-3-3. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively. C Quantification of B. where bound DENND2B is normalized to the SM. Mean +/- SD. Welch's t-test **p=0.006 n=8 for both treatment groups from 5 independent experiments D HEK-293T cells expressing Flag-DENND2B were treated with 250 nM OA alone or with 50 µm PKD inhibitor CID755673 and cell lysates were incubated with GST-14-3-3. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively. E Quantification of D. where bound DENND2B is normalized to the SM. Mean +/- SD. Welch's t-test *p=0.040. n=5 for both treatment groups from 3 independent experiments F GST-SH3A was incubated with lysates of HEK-293T cells expressing GFP-DENND2B WT or S30A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively. G Lysates of HEK-293T cells expressing Flag-DENND2B were pre-incubated with increasing concentrations of purified 14-3-3 WT and subsequently incubated with GST-SH3A (top panel) or GST-Grb2 (bottom panel). Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively. H Regulation of the ITSN-DENND2B interaction. In the absence of phosphorylation, DENND2B binds the SH3A (A) domain of ITSN. Phosphorylation of DENND2B by PKD occurs on Ser-30 residue within the first proline-rich domain (PRD). Phosphorylation of Ser-30 recruits 14-3-3 which outcompetes ITSN binding.