EMBO/sd-character-level-ner huggingface dataset:
F mutant.Figure 4A schematic illustration of the co-transcriptional RNAPII-spliceosome complex. plaNET-seq mainly detects splicing intermediates corresponding to 5' splicing site (5'SS) co-purified with engaged RNAPII complex. Bar charts showing the fractions of 3' and 5' splicing intermediate reads from plaNET-seq in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Genome browser snapshots illustrating enhanced intron splicing in NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F, red) compared to NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT ,blue). Scale bars denote 0.5 Kb. The fraction of RNA-seq intronic reads calculated for all genes (n=24912) in NRPB2WT +/+ nrpb2-2 -/- and NRPB2Y732F +/+ nrpb2-2 -/-. Two-sided Mann-Whitney U test: **** denotes p-value<2.2e-16. The solid horizontal lines and box limits represent medians, lower and upper quartiles of data values in each group. The upper and lower whiskers extend to the largest or smallest value, respectively, no further than 1.5 * IQR from the relevant quartile. Differentially expressed (DE) exons and introns in NRPB2Y732F +/+ nrpb2-2 -/- compared to NRPB2WT +/+ nrpb2-2 -/- based on RNA-seq results. Numbers of DE exons and introns were shown in plot. Quantification (log fold change of FPKM from RNA-seq) of differentially expressed (DE) exons and non-DE exons in NRPB2Y732F +/+ nrpb2-2 -/- compared to NRPB2WT +/+ nrpb2-2 -/-. ** denotes p-value <0.01 by Wilcoxon signed-rank test. The solid horizontal lines and box limits represent medians, lower and upper quartiles of data values in each group. The upper and lower whiskers extend to the largest or smallest value, respectively, no further than 1.5 * IQR from the relevant quartile. Genome browser snapshots illustrating enhanced exon skipping in NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F, red) compared to NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT ,blue). Scale bars denote 0.5 Kb. Quantification (log fold change of FPKM from RNA-seq) of differentially expressed (DE) introns and non-DE exons in NRPB2Y732F +/+ nrpb2-2 -/- compared to NRPB2WT +/+ nrpb2-2 -/-. **** denotes p-value <0.0001 by Wilcoxon signed-rank test. The solid horizontal lines and box limits represent medians, lower and upper quartiles of data values in each group. The upper and lower whiskers extend to the largest or smallest value, respectively, no further than 1.5 * IQR from the relevant quartile.
Figure 2Analysis of TAPBPL protein expression on immune cells. Splenocytes from C57BL/6 mice were freshly harvested and analyzed for TAPBPL protein expression on resting immune cells. To obtain activated T cells, the splenocytes were incubated with anti-CD3 (1 µg/ml) and anti-CD28 (0.5 µg/ml) antibodies for 3 days. To obtain activated B cells, DCs, monocytes and macrophages, the splenocytes were incubated with LPS (10 µg/ml) for 3 days. The resting and activated immune cells were stained with anti-TAPBPL or isotype antibody (Ab), as well as anti-CD4, CD8, CD11b, F4/80, CD11c, CD19 or B220 antibody to identify immune cells. Representative flow cytometric profiles showing the expression levels of TAPBPL protein on resting and activated immune cells (n=3). Significance was calculated by two‐way ANOVA with Tukey test. * P<0.05 compared with isotype Ab; ** P<0.05 compared with resting cells. Analysis of TAPBPL protein expression on immune cells. Splenocytes from C57BL/6 mice were freshly harvested and analyzed for TAPBPL protein expression on resting immune cells. To obtain activated T cells, the splenocytes were incubated with anti-CD3 (1 µg/ml) and anti-CD28 (0.5 µg/ml) antibodies for 3 days. To obtain activated B cells, DCs, monocytes and macrophages, the splenocytes were incubated with LPS (10 µg/ml) for 3 days. The resting and activated immune cells were stained with anti-TAPBPL or isotype antibody (Ab), as well as anti-CD4, CD8, CD11b, F4/80, CD11c, CD19 or B220 antibody to identify immune cells. statistical analysis showing the expression levels of TAPBPL protein on resting and activated immune cells (n=3). Significance was calculated by two‐way ANOVA with Tukey test. * P<0.05 compared with isotype Ab; ** P<0.05 compared with resting cells. Determination of TAPBPL protein expression in normal and tumor human tissues by immunohistochemistry using anti-TAPBPL or isotype Ab. Statistical analysis of the expression levels of TAPBPL on tumor tissues. The data were presented as relative expression levels of TAPBPL on tumor tissues versus relative normal tissues (each group was normalized with isotype Ab first) (n=5). Significance was calculated by two‐tailed Student's t‐test. * P<0.05 compared with normal tissue. (E, F) Analysis of TAPBPL protein expression on the indicated cancer cell lines. Representative flow cytometric profiles and (F) statistical analysis showing the expression levels of TAPBPL protein on the cancer cells (n=3). Significance was calculated by two‐tailed Student's t‐test. * P<0.05 compared with isotype Ab.
WT mice and their BMDM are restrictive to parent L.Figure 1(A) BMDMs were infected with L. pneumophila JR32 or the legA9 mutant with MOI of 0.5. CFUs were scored at 1, 24, 48, and 72 h. (B) BMDMs were infected with JR32 or the legA9 mutant harboring empty PL, or legA9+ legA9 pBC‐KS+ PL. (A, B) Data are presented as means ± SD of at least three independent experiments. Asterisks indicate significant differences, (*p < 0.05, **p < 0.01, ***p < 0.001; two‐tailed t‐test). Four female C57BL/6 mice/group received 1 × 106 of JR32 or legA9 mutant bacteria intratracheally. Lungs were homogenized and plated for CFUs counting at (C) 4 or (D) 48 h postinfection. (C, D) Data are shown as mean + SD of n = 4 and are representative as one of two independent experiments. Asterisks indicate significant differences (**p < 0.01 two‐tailed t‐test)
Figure 5Western blots showing activity changes in both USP7 and USP47 upon treatment with nigericin. MDMs were either unprimed or LPS-primed (1 μg/mL, 4 hrs.) and then treated with nigericin (10 μM) for differing lengths of time, as indicated. Relative, activity-based labelling of USP7 and USP47 was analysed by western blot after incubation with ABPs. Higher panels show lysates incubated with ABPs (labelled), with the active DUB bound to ABP indicated by the suffix '-VME'. Lower panels, without probe labelling, show total USP7 or USP47 levels. (Unlabelled). Western blots are representative of 3 independent MDM donors. Western blots showing activity changes in both USP7 and USP47 upon treatment with CPPD. MDMs were either unprimed or LPS-primed (1 μg/mL, 4 hrs.) and then pre-incubated with either 0.1% DMSO or P22077 (2.5 μM) prior to incubation with CPPD crystals (250 µg/mL, 3 hrs.). Blots are labelled as in B and are representative of 3 independent MDM donors. Western blots showing activity changes in both USP7 and USP47 in LPS-primed (1 μg/mL, 4 hrs.) MDMs pre-incubated with different P22077 concentrations, as indicated, prior to activation by nigericin treatment (10 μM; 45 mins.). Blots are labelled as in B and are representative of 3 independent MDM donors. Western blots showing activity changes in both USP7 and USP47 upon activation in high extracellular potassium (High K+) conditions. MDMs were LPS-primed (1 μg/mL, 4 hrs.) and then changed to media that either had normal or high concentrations of K+, and subsequently treated with nigericin (10 μM; 45 mins.). Blots are labelled as in B and are representative of at least 2 independent MDM donors. Pro-IL-1β (31 kDa) and mature IL-1β (mIL-1β, 17 kDa) in the supernatants of these cells are shown as a control. Non-specific bands are highlighted with an asterisk.
Figure 5A Sucrose density gradient fractionation allowed purification of ribosome subunits and polysomes. Polysomal or total cellular mRNA fractions were isolated following dsRNA treatment and RNA sequencing libraries were prepared. B Sedimentation analysis showing the relative abundance of 40S, 60S, and 80S ribosomes in cells treated with a gfp-dsRNA (control) or porthos (pths)-dsRNA. Profiles were aligned by the 40S ribosome peak's position. Black: control, Red: pths-KD. n=3 independent biological replicates. C Scatter plot of Translational efficiency (TE) from pths-dsRNA S2R+ vs control gfp-dsRNA cells. Red (down-regulated, DR) and green (up-regulated, UP) dots represent genes with log2TE changes that meet the 2 standard deviation cutoff. D Bioinformatic analysis shows that mRNAs whose TE is increased by Porthos have significantly shorter 5'UTRs than non-targets (p=1.69e-35). Box and whisker plot of log2(Mean 5'UTR length) of each genotype. Middle line of box represents median, upper and lower bounds of box represents 1st and 3rd quartiles of data. Whiskers represent 1.5*IQR (inner quartile range), points outside of 1.5*IQR are plotted explicitly. The corresponding table shows median/mean 5'UTR lengths in base pairs (bp) for all mRNAs expressed in S2 cells (non-targets) and for the subset whose TE is enhanced by Porthos (Porthos targets). Targets were defined using n=3 biological replicates of polysome-seq. 5'UTR length comparison is between n=8451 non-target genes and n=204 Porthos targets. Data information: Mean±SEM, ns=p>0.05, *p<0.05, ****p<0.0001. Welch's t-test E-F Confocal images of early Stage 12 embryos from the controls, from lines expressing nlacZ and RNAis against atos or pths in macrophages (mac>) as a second control, and lines expressing RNAis targeting atos or pths along with individual components of the dTORC1 pathway in macrophages, including Nrpl2, Iml1, and TSC1. Germband edge: dotted white line. Data information: Scale bar: 30µm G-H Quantification of the number of germband macrophages in embryos from the controls and upon RNAi knockdown of the genes in E-F. Expression of different RNAis against Nrpl2, Iml1, and TSC1 in atos or pths-depleted macrophages rescues macrophage invasion into the germband while expression of lacZ does not. In (G) control (n=21 embryos) vs. atos RNAi (n=30) p<0.0001; control vs. atos RNAi rescued with Nrpl2 RNAi (n=32) p=0.019, with Iml1 RNAi (n=24) p=0.03, and with TSC1 RNAi (n=15) p=0.04. In (H) control (n=29 embryos) vs pths RNAi (n=26) p<0.0001; control vs. pths RNAi rescued with Nrpl2 RNAi (n=27) p=0.01, with Iml1 RNAi (n=17) p=0.02, and with TSC1 RNAi (n=18) p=0.02. Data information: Mean±SEM, ns=p>0.05, *p<0.05, ****p<0.0001. one-way ANOVA with Tukey I Western blot of protein extracts from control and pths KD S2R+ probed with p-4EBP1 antibody. Tubulin serves as a loading control and quantification normalized to loading control. N=3 biological replicates for both control and pths KD, p=0.69. Data information: Mean±SEM, ns=p>0.05, *p<0.05, ****p<0.0001. unpaired t-test
Loss of TREM2 reduces the DAM signature of Grn-/- miceA.Figure 2A. Heatmap of 65 DAM-associated gene transcripts analyzed by NanoString in FCRLS- and CD11b-positive Grn-/- (n=6), Trem2-/- (n=5) and Double-/- (n=6) microglia in comparison to WT (n=7) microglia isolated from 6-month-old male mice. The expression corrected and housekeeping gene normalized RNA counts for each gene and sample were normalized to the mean value of WT followed by a log2 transformation. B. Volcano plot presentation of the differently expressed transcripts in FCRLS- and CD11b-positive Grn-/- (n=6) in comparison to WT (n=6) microglia isolated from 6-month-old male mice. 35 out of 65 analyzed genes are significantly changed more than 20%, with 22 genes upregulated (purple) and 13 genes downregulated (blue). Data information: For statistical analysis the unpaired, two-tailed student`s t-test was performed Statistical significance was set at **, p < 0.01; ***, p < 0.001; ****, p < 0.000; ns, not significant. C. Volcano plot presentation of the differently expressed transcripts in FCRLS- and CD11b-positive Trem2-/- (n=5) in comparison to WT (n=6) microglia isolated from 6-month-old male mice. 17 out of 65 analyzed genes are significantly changed more than 20%, with 7 genes upregulated (purple) and 10 genes downregulated (blue). Data information: For statistical analysis the unpaired, two-tailed student`s t-test was performed Statistical significance was set at **, p < 0.01; ***, p < 0.001; ****, p < 0.000; ns, not significant. D. Volcano plot presentation of the differently expressed transcripts in FCRLS- and CD11b-positive Double-/- (n=6) in comparison to WT (n=6) microglia isolated from 6-month-old male mice. 21 out of 65 analyzed genes are significantly changed more than 20%, with 14 genes upregulated (purple) and 7 genes downregulated (blue). Data information: For statistical analysis the unpaired, two-tailed student`s t-test was performed Statistical significance was set at **, p < 0.01; ***, p < 0.001; ****, p < 0.000; ns, not significant. E. Expression profiles of selected DAM genes, whose mRNA levels are rescued in Double-/- (n=6) vs. Grn-/- (n=6) microglia. mRNA expression normalized to the mean of the WT cohort. Data represent mean ± SEM. Data information: For statistical analysis one-way ANOVA with Dunnett`s post hoc test was used to compare Grn-/-, Trem2-/- and Double-/- microglia. Statistical significance was set at **, p < 0.01; ***, p < 0.001; ****, p < 0.000; ns, not significant.
Figure 7A, B IF of Lef1 in mandible slices at embryonic day 60 (E60) infected with RUNX2 overexpression lentiviral vector (overexpression) or overexpression control lentiviral vector (O-control). (A'-B') are magnifications of boxed regions in the corresponding figure panels. Dashed lines mark the epithelium of PC. C Relative IF expression levels of Lef1 in epithelium (Epi.) and mesenchyme (Mes.) of the overexpression and O-control groups. Data information: Data represent the means ± SEM. Scale bars = 100 µm. n = 3 for all experiments. Unpaired t-tests for C , **P < .01, ***P < .001; NS. not significant. D, E IF of Lef1 in E60 mandible slices subjected to 3 or 0 kPa stress for two days. (D', E') are magnifications of boxed regions in the corresponding figure panels. Dashed lines mark the epithelium of PC. F Relative IF expression levels of Lef1 in the epithelium and mesenchyme after applying 3 and 0 kPa pressure. Data information: Data represent the means ± SEM. Scale bars = 100 µm. n = 3 for all experiments. Unpaired t-tests for , *P < .05, **P < .01, ***P < .001; NS. not significant. G Western blots of Lef1, non-phospho-β-catenin, and RUNX2 after DFCs were infected with control lentiviral vector or RUNX2 overexpression lentiviral vector. (G') Relative expression levels between control vector and RUNX2 overexpression groups. Data information: Data represent the means ± SEM. Unpaired t-tests , *P < .05, **P < .01, ***P < .001; NS. not significant. H Western blots of Lef1, non-phospho-β-catenin, and RUNX2 after DFCs were infected with scrambled shRNA (scramsh) or RUNX2 knockdown shRNA (knockdown) lentiviral vectors. (H') Relative expression levels between scramsh and knockdown groups. I Western blots of nuclear non-phospho-β-catenin and Lamin B in DFCs of RUNX2 overexpression (O), control vector (V), RUNX2 knockdown (K), scramsh (S), compressed force (F; 1.0 g/cm2), and control (C; 0 g/cm2) groups. Sample were loaded in a gradient (7.5, 15, 30, and 60 μg) showing a linear relationship for each group. J Western blots of nuclear non-phospho-β-catenin and Lamin B in DFCs treated with IWR-1-endo. Relative expression levels were compared between force and no force groups and between overexpression and control groups with or without IWR-1-endo treatment. K Diagram illustrating the biomechanical stress regulation of Wnt/β-catenin signaling in the mesenchyme between the deciduous (DT) and permanent tooth (PT) via the integrin β1-ERK1-RUNX2-Wnt/β-catenin pathway. L Diagram illustrating the biomechanical stress-associated downregulation of RUNX2-Wnt/β-catenin pathway in the mesenchyme, inducing upregulation of Wnt signaling in the epithelium, which triggers PT development.
A palmitoylation-dependent interaction between DHHC5 and Golga7b.Figure 1Immunoblots of acyl-biotin exchange purifications from Hela cells transiently transfected with WT Golga7b or a palmitoylation-deficient mutant form of the protein with cysteines replaced with alanine residues. The hydroxylamine sensitivity of the Golga7b signal in the + HA condition of the WT pull-down and not in either the - HA condition or the + HA condition of the mutant shows that Golga7b is a palmitoylated protein. Immunoblots of acyl-biotin exchange purifications from Hela cells treated with either DHHC5 siRNA or control non-targeting siRNA. Immunoblots showing rescue of Golga7b palmitoylation in DHHC5 depleted cells after transfection of WT DHHC5 but not by the DHHC5 catalytic mutant. A significant reduction in the Golga7b signal is observed in the + HA condition of the pull-down when DHHC5 is depleted with siRNA. The graph displays mean +/- s.e.m. N=4, *=p<0.05, paired t-test. Immunoblots of WT and non-palmitoylatable mutant Golga7b from immunoprecipitations of WT DHHC5 and a pair of DHHC5 mutants (DHHS5 = catalytically inactive mutant where catalytic cysteine has been mutated to serine, C-terminal mutant = mutant where the 3 C-terminal palmitoylation sites of DHHC5 have been mutated to alanine residues.). WT and DHHS5 immunoprecipitates both WT and mutant Golga7b but the C-terminal mutant is unable to immunoprecipitate either form of Golga7b showing that the palmitoylation of the C-terminus of DHHC5 is essential for the stability of this interaction.
Figure 4A, Immunofluorescence of Nrl::GFP and ROSAmT/mG retina co-cultures after 3 days in vitro (DIV) shows cells expressing cytoplasmic GFP only (green arrow), membrane RFP only (red arrow) or both (yellow arrow). Nuclei are stained with Hoechst (blue). B-E, Flow cytometric analysis and quantification of cytoplasmic and membrane protein exchange in co-cultures. Material transfer is quantified as the percentage of GFP+mT+ CD73+-photoreceptors over the total population of fluorescent GFP+ and mT+ -CD73+-photoreceptors in the cultures (0 DIV, n=17; 3 DIV, n=15). Nrl::GFP photoreceptors do not accumulate mT after co-culture with lysed ROSAmT/mG cells (3 DIV, n=3; 3 DIV lysed, n=3) (B). Material transfer increases over time in co-cultures (n=3 per timepoint) (C), does not occur when Nrl::GFP and ROSAmT/mG photoreceptors are separated by a transwell insert (control, n=3; transwell, n=3) (D) and also occurs in cultures of purified photoreceptors (n=3) (E). F, G, C57BL/6J cells with mitochondria labelled with MitoTrackerTM-Green (MTG) or MitoTrackerTM-Red (MTR) exchange mitochondria after 3 DIV (0 DIV, n=6; 3 DIV, n=6). White dashed line delimitates both cell somas and the protrusion connecting them (F). No exchange is observed when the two photoreceptor populations are separated by a transwell (n=3), nor in co-cultures of live MTG-labelled and lysed MTR-labelled cells (n=3) (G).
Figure 7(A) T47D-MTVL cells treated or not with hormone were subjected to ChIP-seq assay with a-FOXA1 (see materials and methods). The number of genes with FOXA1 peaks found in different windows around the TSS is shown. Proximal promoter window: -/+ 1Kb around TSS. (*) P-value <0.05, ** P-value <0.01 and *** P-value <0.001. (B) FOXA1 is significantly enriched at PRBs localized in repressed genes compared with up- and non-regulated genes. Lower Panel: heat maps of the results shown in the upper panel. (***) P-value < 10-7. (C) Cells were transfected with control or FOXA1 siRNAs and treated with 10 nM R5020 as indicated, and the levels of repressed genes were measured by RT-PCR. * P-value <0.05. BC: BCAS1, KR: KRT23 and IG: IGFBP5 genes. The extent of knockdown was analyzed by western blotting using specific antibodies (right panel). (D) T47D-MTVL cells transfected with control or FOXA1 siRNAs were induced or not with hormone, and subjected to ChIP assays with a-BRG1 and IgG as control. Precipitated DNA was analyzed by PCR for the presence of sequences corresponding to the BCAS1, KRT23 and IGFBP5 genes. The histograms show the mean ± SD of 3 experiments performed in duplicate. * P-value < 0.05. (E) T47D-MTVL cells treated or not with hormone were lysed and immunoprecipitated either with a-FOXA1 specific antibody (upper panel) or BRG1 (lower panel). The immunoprecipitates (IP) were analyzed by immunoblotting with BRG1, PR and FOXA1 specific antibodies. The inespecific band in the control IgG of the FOXA1IP, which does not coincide with PRB or PRA is also shown in a shorter exposition (right panel).
Figure 7a, IHC staining with ISX (brown) or PCAF (brown) antibody in lung tumors from patients with lung cancer. N, normal tissue; T, tumor mass; Nuclei, hematoxylin (blue); Blue and red arrow, cytosol and nuclear ISX (PCAF). Scale bar, 500μm (400X); Scale bar, 2mm (100X). b, Confocal immunofluorescence detection of ISX (green) and BRD4 (red) in lung tumors from patients with NSCLC. Cell nuclei were visualized by DAPI (blue); Yellow arrows indicate co-localization; IgG, negative control. N, normal tissue; T, tumor mass. c, Confocal immunofluorescence detection of ISX (green) and PCAF (red) in lung tumors from patients with NSCLC. Cell nuclei were visualized by DAPI (blue); Yellow arrows indicate co-localization; IgG, negative control. N, normal tissue; T, tumor mass. Correlation analysis of mRNA expression for, (d) ISX and BRD4, in 157 lung cancer samples. e, Correlation analysis of mRNA expression for, (e) ISX and PCAF in 157 lung cancer samples. f, Protein expression of ISX, ISX(69Kac) and Vimentin in tumor and normal parts from patients with NSCLC by western blotting. N, normal tissue; T, tumor mass; Arrow, target protein; *, unspecific band. The Kaplan-Meier survival curve was used to analyze survival correlation between patients (n=157) with NSCLC and ISX (g) levels. On the basis of the cut-off values of fold differences, the study population was dichotomized into the "high" and "low" expression group. p‐values were calculated by log‐rank (Mantel-Cox) test comparing the two Kaplan-Meier curves. g and h, The Kaplan-Meier survival curve was used to analyze survival correlation between patients (n=157) with NSCLC and PCAF (h) levels. On the basis of the cut-off values of fold differences, the study population was dichotomized into the "high" and "low" expression group. p‐values were calculated by log‐rank (Mantel-Cox) test comparing the two Kaplan-Meier curves.