EMBO/sd-character-level-ner huggingface dataset:
Figure 6(A) Per2 is known to act as an intermediary between the light detector and the melatonin synthesis pathway. Box plot shows per2 mRNA levels measured by qPCR in 6 dpf larvae placed under a low (light blue box) or medium intensity (dark blue box) LD regime. Under low intensity light a significant reduction in the per2 transcript levels is detected in the light phase. Bar under the chart indicates the 12:12 hour LD interval. (B) Box plot as in A shows mRNA levels of arntl1b, which is in part regulated by per2. Under low intensity light a significant reduction in the arntl1b transcript level is detected at ZT3 and an increase at ZT21. (C) Plot as in A shows mRNA levels of per1a, which is regulated by the Arntl-Clk heterodimer. Under low intensity light a significant reduction in the per1a transcript level is detected at ZT21 and an increase at ZT9. (D) Box plot shows similar per2 mRNA levels between wild-type (blue box) and opn4 dko (grey box) under a medium intensity LD regime at 6th dpf. (E) Box plot as in D, shows similar arntl1b mRNA levels. (F) Box plot as in D, shows similar per1a mRNA levels. (G) Box plot as in D, shows similar clk1a mRNA levels. (H) Box plot as in A shows ddc mRNA levels. Similar levels were detected between wild-type larvae under a low or medium intensity LD regime. (I) Box plot as in A shows asmt mRNA levels. Under a low intensity LD regime a significant increase in the asmt transcript level is detected at ZT3. Data information: boxplot divides the data in quartiles: the box indicates the interquartile range, with the horizontal line in the box denoting the median of the data set, the whiskers extend to the minimum and maximum, and meet the box at the median of the lower (quartile 1) and median of the upper (quartile 3) half of the dataset. Black dots indicate biological replicates (n=12), the red dot indicates the mean, red error bars indicate the 95% confidence interval and asterisks indicate significance (0.01<p(✱)<0.05, 0.001<p(✱✱)<0.01, p(✱✱✱)<0.001, ns = not significant). Expanded
Figure 1(A, B) Actively dividing cells labelled by BrdU and Ki67 are reduced in the SGZ of DSCR1 mutants compared to that of wild type mice. Brain sections were prepared 1 day after BrdU injection. The white box area is magnified in the lower panels: DAPI (blue), Ki67 (green), and BrdU (red). Arrow heads indicate BrdU and Ki-67 double positive cells in the SGZ. (C, D) Differentiating cells are identified by staining with BrdU and DCX. Brain sections were prepared 10 days after BrdU injection. (E, F) Maturation of progenitor cells are assessed by staining with BrdU and NeuN. Brain sections were prepared 21 days after BrdU injection Each hippocampal section was 40 μm in thickness, and a total of 24 sections were obtained from one hippocampus. Scale bars:100 μm in the large panel, and 10 μm in the magnified images. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 animals for each condition, *P< 0.05, **P< 0.01.
Figure 3(A) MYC2, PDF1.2A and NIMIN1 expression in ANAC032 transgenics compared to WT after spraying with Pst DC3000 (24 hpi) normalised to their respective controls. FCh, fold change. Means ± SD (n = 3; 'n' represents independently performed experiments, each including the rosette leaves of at least three plants grown in individual pots). (B) EMSA showing binding of ANAC032 to MYC2, PDF1.2A and NIMIN1 promoter regions (in 5´-DY682-labelled double-stranded oligonucleotides) harbouring ANAC032 binding sites. 1, labelled probe only; 2, labelled probe plus GST protein 3, labelled probe plus ANAC032-GST protein; 4, labelled probe, ANAC032-GST protein and 100 x competitor (unlabelled oligonucleotide containing ANAC032 binding site). (C) Confocal microscopy image showing nuclear localization of ANAC032-GFP fusion protein expressed from the ANAC032 promoter in ANAC032prom:ANAC032-GFP/anac032-1 seedlings treated with Pst at 6 hpi. Left, bright field; right, chlorophyll auto-fluorescence (red) and GFP fluorescence (green) under bright field. (D) Expression of MYC2, PDF1.2A, NIMIN1 and ANAC032 in two-week-old ANAC032prom:ANAC032-GFP/anac032-1 seedlings compared to WT at 6 hpi with Pst, normalised to their respective controls. FCh, fold change. Means ± SD (n = 3; 'n' represents independently performed experiments, each including the rosette leaves of at least three plants grown in individual pots). Asterisks indicate a significant difference from WT, normalised to their respective controls (p < 0.05; Student´s t-test). (E) ChIP-qPCR shows enrichment of MYC2, PDF1.2Aand NIMIN1 promoter regions containing ANAC032 binding site compared to a promoter region lacking the ANAC032 binding site (AT5G09810; Neg Cont). Means ± SD (n = 3; 'n' represents independently performed experiments, each including the rosette leaves of at least three plants grown in individual pots). Asterisks indicate a significant difference from negative control (p < 0.01; Student´s t-test).
Increasing VEGF doses inhibit NEM recruitment and endothelial Sema3A expression.Figure 3Immunofluorescence staining of endothelial cells (CD31, in red) and NEM (CD11b, in green) on cryosections of limb muscles 1 week after injection with V Low, V Med, and V High myoblast clones. Scale bar = 100 μm.Quantification of the number of NEM/cm of vessel length in sites of new angiogenesis by increasing VEGF doses. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group: V Low, n = 48; V Med, n = 39; V High, n = 94; *P < 0.05 and ***P < 0.001 by one-way ANOVA with Bonferroni multiple comparisons test: V Low versus V Med P = 0.0008; V Low versus V High P < 0.0001; V Med versus V High P = 0.0304. Endothelial cells (CD31+ quadrant) and NEM (CD11b+ quadrant) were sorted by FACS from muscles 1 week after injection with the same myoblast clones. Relative gene expression was quantified in the ex vivo FACS-purified endothelial cells (CD31+) and NEM (CD11b+) and expressed as fold-change versus the V High group. Data represent individual values, with mean ± SEM (n = 3-7, as indicated); **P < 0.01 by one-way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic transformation; Sema3A: V Low versus V High P = 0.0157.
Figure 3(A) Western blotting analysis of NCoR1 in neonatal rat ventricular myocytes (NRVMs) transfected with siControl or siNCoR1 for 3 days. siControl indicates control siRNA; siNCoR1, NCoR1 siRNA. (B) Representative immunofluorescence staining of α-Actinin in NRVMs transfected with siRNA for 48h and then treated with vehicle (H2O) or phenylephrine (PE) for another 48h. Scale bar: 50μm. (C) Quantification of the surface area of α-Actinin-positive NRVMs with or without NCoR1 knockdown. A total of 40 NRVMs was randomly chosen from 4 replicate coverslips for each group and used for statistical analysis. (D) qRT-PCR analysis of Acta1 and Nppa in NRVMs with or without NCoR1 knockdown. n=4:4. (E) Western blotting analysis of NCoR1-flag in NRVMs infected by control lentivirus (Control) or NCoR1-flag lentivirus (NCoR1-OV) for 4 days. (F) Representative immunofluorescence staining of α-Actinin in NRVMs infected with lentivirus for 48h and then treated with vehicle or PE for another 48h. Scale bar: 50μm. (G) Quantification of the surface area of α-Actinin-positive NRVMs with or without NCoR1 overexpression. A total of 30 NRVMs was randomly chosen from 3 replicate coverslips for each group and used for statistical analysis. (H) qRT-PCR analysis of Acta1 and Nppa in NRVMs with or without NCoR1 overexpression. n=4:3. Data represent three independent experiments. Two-way ANOVA followed by Bonferoni post-tests was used for statistical analysis.
Figure 5(A) Localization of GFP-tagged Pgc1 derivatives in which the hydrophobic hairpin (aa275-321) was replaced by membrane anchor of Scs2 (Pgc1Scs2MA) or Bos1 (Pgc1Bos1MA). ER and LDs were visualized by expression of Sec63-Cherry and MDH staining, respectively. Scale bar: 5 µm. (B) The degradation of 3HA-Pgc1 or the indicated chimeras in wt and doa10Δ cells was analyzed as in Fig. 1A. The blot is representative of at least three independent experiments. (C) The degradation of 3HA-GFP-Pgc1275-321 in wt and doa10Δ cells was analyzed as in Fig. 1A. The graph shows the average of four independent experiments; error bars represent the standard deviation. (D) Localization of 3HA-GFP-Pgc1275-321 in doa10Δ cells. The ER was visualized by expression of Sec63-Cherry. Scale bar: 5 µm.
SseL interacts with p62 and autophagic substrates.Figure 7SseL interacts with p62 and autophagic substrates.(A and B) Co-immunoprecipitation from RAW264.7 macrophages infected with ΔsseL mutant bacteria expressing SseL-HA or SseLC/A-HA for 10 h. (A) Infectedcell lysates and immunoprecipated fractions were probed with anti-HA, anti-ubiquitin and anti-p62 antibodies. (B) At 7 h post-infection, infected cells were subjected to mock, 3-MA or starvation (Stv) treatments for 3 h before harvesting and processing as described in (A). Cell lysates and immunoprecipiations were probed with anti-HA and anti-ubiquitin antibodies.
Loss of p53 tumor suppressor function and constitutive activation of NFATc1 correlates with highly metastatic and dedifferentiated PDACStatistical analysis of NFATc1 expression with respect to tumor grading in KPC mice tissues.Figure 1A Statistical analysis of NFATc1 expression with respect to tumor grading in KPCmice tissues. B Immunohistochemistry detection of NFATc1 and p53, illustrating G1‐G2 and G3 in human PDAC tissues. Scale bars, 100 μm. C Statistical illustration of TMA analysis demonstrating NFATc1 strong nuclear positive tumors (n = 161 patients) with respect to functional status of p53 expression in human PDAC tissues. D Representative H&amp;amp;E‐stained sections as well as immunohistochemistry detection of HA‐NFATc1 and p53 in well‐ and poorly differentiated pancreatic tissues of KNCmice. Scale bars, 100 μm.E Quantification of p53 expression with respect to the tumor grading by evaluating both the intensity of immunostaining and the number of cells with overexpression by means of an immunoreactivity score (IRS) in KNCmice (every dot represents one mouse). F Incidence of livermetastases in KNCmice (mean values ± SD).
CEP55 regulates genomic instability and aneuploidy.Figure 2(A) Polyploidy analysis (>4N DNA contents) determined using FACS in control and CEP55 knockdown MDA-MB-231 cells (left panel). Representative corresponding cytogram showing different phases of cell cycle and the polyploidy subpopulation analyzed using ModFit LT 4.0 software (right panel). Yellow peaks represent subpopulation of polyploidy/aneuploidy. Graph represents the mean±SEM of three independent experiments. (B) Analysis of chromosome number in control and CEP55 knockdown MDA-MB-231 cells (sh#8) by Giemsa staining of metaphase spreads (left panel). Representative images showing chromosome spreads assessed by Zeiss AxioScope2 microscopy (right panel). n= number of metaphase spreads counted (mean ±95% confidence interval of two independent experiments). (C) Representative SNP array plots showing chromosomal alterations following CEP55-knockdown in MDA-MB-231 cells. BAF is B allele frequency, value between 0 and 1 and represents the proportion contributed by one SNP allele (B) to the total copy number: BAF is an estimate of NB/(NA+ NB), where NA and NB are the number of A and B alleles, respectively. (D) Percentage of genome altered evaluated using amplified copies (6-8 copies across genome) in both parental and CEP55 knockdown cells determined using data from SNP arrays. Graph represents the mean±SEM of two independent experiments. (E) SNP array plots showing chromosomal alterations following CEP55 overexpression in MCF10A cells. Red box indicates an extra chromosome 20 in CEP55-overexpressing cells. (F) Correlation between average copy number alterations of genome (CNAs)/tumor and CEP55 mRNA expression in the METABRIC dataset, n=997 patients. (G) Kaplan-Meier survival analysis of METABRIC data showing the relationship between CEP55 expression and survival. Cases were subdivided into tertiles. Log-rank (Mantel-Cox) test, P< 0.0001.
Figure 1A Quantitative RT-PCR (RT-qPCR) results showing relative expression levels of mouse Pabpn1l in somatic tissues, oocytes, and preimplantation embryos. n = 3 biological replicates. Error bars indicate S.E.M. B Western blot results of PABPN1L, BTG4, and phosphorylated ERK1/2 (pERK1/2) in oocytes and embryos. Total protein from 400 oocytes or embryos is loaded in each lane. DNA damage-binding protein 1 (DDB1) is blotted as a loading control. C Western blot results of PABPN1L in MII oocytes of wild type (WT) and Pabpn1l−/− females. DDB1 is blotted as a loading control. D Cumulative pup numbers of WT and Pabpn1l-/- female mice. n = 5 for each genotype. Error bars, S.E.M. ***P < 0.001 by two-tailed student's t-test. E Quantification of preimplantation embryos derived from WT and Pabpn1l-/- females when WT embryos reached the corresponding stages. The number of analyzed embryos is indicated (n). Error bars, S.E.M. ***P < 0.001 by two-tailed Student's t-test. F Representative images of the embryos collected from the oviducts of mated WT and Pabpn1l-/- females at the indicated time points after hCG injection. Scale bar = 100 μm.
SNX18 is a positive regulator of autophagy and its function is conserved in D.Figure 10.(A) During starvation, Rab11-positive recycling endosomes are recruited to the SNX18-positive perinuclear area dependent on SNX18 itself. SNX18 and Rab11 are required for the perinuclear localization of Atg16L1 to omegasomes marked by DFCP1. LC3 lipidation and autophagosome formation proceeds. In the absence of SNX18, recruitment of recycling endosomes to the perinuclear site is inhibited, Atg16L1 fails to localize to DFCP1-positive omegasomes, and WIPI2 accumulates. LC3 lipidation and autophagosome formation is inhibited. Upon SNX18 overexpression, Rab11-, TfR-, and LC3-positive membranes accumulate at a perinuclear site, and extensive membrane tubules with SNX18, Atg16L1, LC3, and TfR form from perinuclear recycling endosomes. LC3 lipidation and autophagosome formation is increased. Based on the experimental observations summarized in this model, we propose that SNX18 facilitates LC3 lipidation via Atg16L1 and provides membrane from recycling endosomes to forming autophagosomes. (B and C) RNAi against SH3PX1 (GFP-positive clones) decreases formation of LTR punctae in response to 4 h of starvation in 20% sucrose (B) and inhibits formation of mCherry-Atg8a-positive autophagosomes in response to 2 h starvation (C). Genotypes: (B and D) hs-flp; UAS-dicer/+; Act>CD2>GAL4 UAS-GFPnls/UAS-SH3PX1 RNAi; (C and E) hs-flp; UAS-dicer/+; r4:mCherry-Atg8a Act>CD2>GAL4 UAS-GFPnls/UAS-SH3PX1 RNAi; (D and E) RNAi against SH3PX1 inhibits formation of LTR (D) and mCherry-Atg8a-positive structures (E) at early stages of developmental autophagy in fat bodies of 110-h-old larvae. Genotypes: (B and D) hs-flp; UAS-dicer/+; Act>CD2>GAL4 UAS-GFPnls/UAS-SH3PX1 RNAi; (C and E) hs-flp; UAS-dicer/+; r4:mCherry-Atg8a Act>CD2>GAL4 UAS-GFPnls/UAS-SH3PX1 RNAi; (F) There was no change in mCherry-Atg8a in SH3PX1 mutant clones (GFP negative, outlined) in well-fed larvae. Genotypes: (F and G) hs-flp; Cg-GAL4 UAS-mChAtg8a/+; FRT80B UAS-2XeGFP/FRT80B SH3PX1HK62b. (G) Decreased formation of mCherry-Atg8a-positive autophagosomes in SH3PX1 mutant clones (GFP negative, outlined) in fat bodies from larvae starved in 20% sucrose for 3 h. Bars, 20 µm. (H) The graph shows quantification of total mCherry-Atg8a spot intensity per cell for WT and SH3PX1 mutant cells. The graph shows mean ± SEM (error bars); ***, P < 0.001. Genotypes: (F and G) hs-flp; Cg-GAL4 UAS-mChAtg8a/+; FRT80B
Figure 1(A) Schema of the analysis of gene expression in islets isolated from control larvae and larvae subjected to β-cell ablation. β cells were ablated by exposing nitroreductase (NTR)-expressing transgenic larvae to metronidazole (MTZ) from 3-4 dpf. Islets were then isolated, and their RNA extracted and analyzed by microarray. Out of the 470 genes that were upregulated more than 50%, 33 genes encoded proteins that harbored a signal peptide for secretion (according to the algorithm of SignalP). Excluding genes that encode enzymes or proteins with a transmembrane (TM) domain, we selected 11 genes for overexpression studies in zebrafishlarvae (C-E). (B) Microarray heat map showing the upregulation in expression of the 11 candidate genes in β cell-ablated versus control islets. Igfbp1a and b were the genes whose expression increased the most after β-cell ablation. (C) Schema of the construct used for overexpression of the candidate genes (under the control of the beta-actin promoter), and expression of GFP in the heart (as an internal control for genomic integration). (D-E) Representative images at 6 dpf of Tg(ins:kaede);Tg(ins:CFP-NTR) transgenic larvae that had been injected at the 1-2-cell stage with transposase mRNA (control) or transposase mRNA + bactin:igfbp1a (bactin:igfbp1a), subjected to β-cell ablation by metronidazole (MTZ) during 3-4 dpf, and subsequently allowed to regenerate for 2 days. The GFP+ heart (arrowhead) visualizes successful integration of the construct. Islets are indicated by white arrows. Scale bars: 100 μm. (F) Quantification of β-cell regeneration at 6 dpf in control (n=23), bactin:igfbp1a-overexpressing (n=13), and bactin:igfbp1b-overexpressing (n=8) Tg(ins:kaede);Tg(ins:CFP-NTR) larvae; ***P=0.0002, ns= nonsignificant (P=0.3106). (G) Immunohistochemistry showing Igfbp1 protein expression in 6 dpf Tg(ins:GFP) following β-cell ablation between 3-4 dpf. Scale bar: 50 μm. (H) Both liver-specific (lfabp promoter; n=46) and widespread (bactin promoter; n=18) overexpression of igfbp1a increase β-cell regeneration when compared to control (n=77). (I-M) Quantification of β cells with or without β-cell ablation and igfbp1a overexpression by confocal microscopy, which detects even weakly insulin-expressing β cells. Scale bars: 15 μm. See also Figure EV1.
CDK7i induced inhibition of MITF and SOX10 and release of GATA6 expressionA-B.Figure 5A-B. Gene track of CDK7 occupancy at MITF (a) or SOX10 (b) loci in 501melBIO-FLAG:CDK7 cell line. Gene tracks of H2A.Z, BRG1, MITF, SOX10 and H3K27ac (GSE94488 and GSE61967) at the same loci in parental 501mel are indicated. SE is denoted by a red opened square. H3K27ac deposition is also shown in Hair Follicle Melanocytes (HFM) (GSE94488). C-E. qRT-PCR analysis showing average TBP-normalized fold expression of MITF (C), SOX10 (D) and GATA6 (E) in 501mel treated either with DMSO/THZ1 (50nM) (upper) or DMSO/JQ1 (10μM) (lower) for 24h. Data information: data are presented as mean values + SD for six replicates (n=6). The p-value (Student's t-test) is indicated, *<0.05.
Figure 3(A) Body weights of hamsters treated with antibodies were measured at indicated days after inoculation with SARS-CoV-2. The mean % of starting weight ± s.e.m. (n = 5 biological replicates) are plotted. (B-E) SARS-CoV-2 viral RNA (B and D) or infectious virus (C and E) was detected in the lung (B and C) and nasal turbinates (D and E). The mean copy number or the mean infectious titer is shown, error bars represent s.e.m. (n = 5 biological replicates). (F, G) Percentage of inflamed lung tissue (F) and percentage of lung tissue expressing SARS-CoV-2 antigen (G) estimated by microscopic examination in different groups of hamsters at four days after SARS-CoV-2 inoculation. Individual (symbols) and mean (horizontal lines) percentages are shown. Error bars represent s.e.m. (n = 5 biological replicates).
A) 293 cells were transfected with nonsense (nons), bag1 or bag3 siRNAs for 48 h and then treated for 2 h with the lysosomal inhibitors pepstatinA and E64 (both 10 μg/ml; Pep.Figure 3(A) 293 cells were transfected with nonsense (nons), bag1 or bag3 siRNAs for 48 h and then treated for 2 h with the lysosomal inhibitors pepstatinA and E64 (both 10 μg/ml; Pep.A/E64) or DMSO as control, followed by immunoblot analysis of the indicated proteins. (B) 293 cells were transfected for 48 h with the indicated siRNAs and BAG3 expression plasmid (BAG3‐N1) or vector control (N1) followed by the same analysis as in (A). (C) Diagram shows the autophagic flux of 293 cells with differently modulated BAG1 and BAG3 levels as described in (A) and (B). Autophagic flux was determined by the strength of LC3‐II accumulation in a 2‐h treatment period with Pep.A/E64. Therefore, normalised LC3‐II levels in the absence of inhibitors were subtracted from corresponding levels obtained in the presence of Pep.A/E64. Values are expressed as mean±s.e.m. *P0.05 versus control‐transfected cells, n=3. (D) I90 cells were transfected with GFP fused to LC3 (pGFP.LC3) and co‐transfected either with BAG3‐N1 or N1. After transfection for 48 h, cells were treated as in (A) and levels of indicated proteins were detected by immunoblot analysis. (E) I90 cells, transfected as in (D) for 24 h, were microscopically analysed for GFP‐LC3 fluorescence. Representative pictures are shown. Bar: 20 μm. Values expressed in diagram are mean±s.e.m. from three independent experiments (50 cells were counted per experiment). *P0.05 versus N1, n=3.
Figure 5(A) Heatmap of the altered Hippo related genes in colons from control and cKO mice one day after 5-day DSS treatment. The parameter of the color key indicated the fold changes converted to log2 of signal value normalized. (B) qRT-PCR analysis validates the altered Hippo-related genes in control and cKO mice. n=4. (C) Western blotting for pMst1/2, Mst1, pMob1 and Mob1 in colon epithelium from control and cKO mice two days after 5-day DSS treatment. β-actin was used as a loading control. The quantification of pMst1/2 versus Mst1 and pMob1 versus Mob1 were shown under the corresponding protein bands. (D) Western blotting for Yap1 in nuclear and cytoplasmic proteins isolated from intestinal epithelial cells from control and cKO mice two days after 5-day DSS treatment. Histone H3, a positive control for nuclear proteins. α-Tubulin, a positive control for cytoplasmic proteins. The quantification of Yap1 versus Histone H3 in nuclear proteins was shown under the corresponding band. (E and F) Double immunofluorescence for Yap1 and β-catenin in the colons from control (n=4) and cKO (n=4) mice two days after DSS removal (E), and in AOM/DSS tumors from control (n=4) and cKO (n=4) mice (F). The percentage of nuclear Yap1+ cells versus epithelial cells was quantified. Scale bar: 50 μm. (G) Western blotting for Yap1 and pYap1 in colon epithelial cells from control and cKO mice two days after 5-day DSS treatment. β-actin was used as a loading control. The ratio of Yap1/pYap1 were quantified. n=3. (H) The growth curve of HCT116 colorectal cancer cells cultured in the supernatant from WT or cKO IMCs, concomitantly transfected with PCDH empty vector or PCDH-YAP1-5SA vector. n = 4 technical replicates. (I) Pearson correlation analysis of ISLR and CTGF (P < 0.001; R = 0.69), ISLR and FSTL1 (P < 0.001; R = 0.81), as well as ISLR and CYR61 (P < 0.001; R = 0.65) in human colorectal cancer based on TCGA RNA-seq database.
Genetic ablation of Usp42 renders mouse intestinal organoids Wnt hypersensitive.Figure 6A, Representative images of mouse small intestinal organoids generated with empty (Non-edited) or USP42gRNA containing CRISPR-concatemers, and cultured for 3 days in matrigel with the indicated growth factors, or the Wnt secretion inhibitor IWP-2. EN, EGF and noggin. Note that knockout of Usp42 (Usp42 gRNAs) rendered the organoids insensitive to RSPO1 withdrawal. Scale bar = 1 mm. B, Quantification of the number and subtype of organoids from (A). C, qPCR analysis of Lgr5 and Axin2 expression levels in first passage mouse intestinal organoids cultured in EN + 5% RSPO1.