EMBO/sd-character-level-ner huggingface dataset:
is required for CMG ubiquitylation and replisome disassembly during S-phase in mouse ES cells.Figure 3(A) Cell extracts were generated after the indicated treatments and then incubated with anti-GFP beads. Factors co-purifying with GFP-SLD5 were monitored by immunoblotting. (B) TAP-SLD5 cells were subjected to the indicated treatments. The level of LRR1 and VHL was assessed by immunoblotting. The asterisk denotes a non-specific band. (C) For the experiment described in (B), TAP-SLD5 was isolated from cell extracts and the indicated factors monitored by immunoblotting. (D) The activity of CUL2LRR1 was inhibited in GFP-SLD5 cells , by treatment with a combination of LRR1 siRNA and MLN4924 Fixed cells were analysed Arrows indicate the accumulation of GFP-SLD5 on constitutive heterochromatin (49 % cells, n = 125). (E) Cells were treated as in (D) and the localisation of GFP-SLD5 was compared with CLASPIN by immunofluorescence. Arrows indicate the co-localisation of GFP-SLD5 and CLASPIN on constitutive heterochromatin (in 90 % cells with GFP-SLD5 patches, n = 103).
a, Cherry::APE1 GFP::ATG8 cells were grown and shifted to YPD, nitrogen starvation (SD-N) or YPD+VPA medium for 3 h.f3a, Cherry::APE1GFP::ATG8 cells were grown and shifted to YPD, nitrogen starvation (SD-N) or YPD+VPA medium for 3 h. Samples were processed for microscopy. The table shows numbers corresponding to the experiment. Percentage of fluorescence signals is presented and error bars represent the s.d. obtained from three independent experiments. DIC, differential interference contrast. Scale bars, 3 µm. b, pho8Δ60 and pho8Δ60 atg1Δ; cells were grown as in a. Pho8Δ60 activity was calculated by measuring alkaline phosphatase levels. Error bars represent s.d. calculated from five independent experiments. c, GFP::ATG8 and GFP::ATG8atg1Δ; cells were grown as in a. Cell samples were processed for western blot using anti-GFP antibody. Quantification is presented in Supplementary Fig. 3.
Figure 3A Gadd45 proteins significantly improve reprogramming efficiency. The reprogramming efficiencies were compared with SKO or SKOM, respectively. We normalized the numbers of SKO+Factor to SKO+Flag (1 as control); also we normalized the numbers of SKOM+Factor to SKOM+Flag (1 as control). (**p≤0.01; ***p≤0.001; n=3) B The efficiencies of SKO- and SKOM-induced reprogramming were tested in over expression of two or three Gadd45 proteins. The reprogramming efficiencies were compared with SKO or SKOM, respectively. (**p≤0.01; ***p≤0.001; n=3) C SKO-MEFs or SKOM-MEFs infected with DOX-inducible Gadd45a were either treated with DOX immediately after infection, and DOX was removed at different time points (left panel) or treated with DOX from different time points until the end of the experiment (right panel). The reprogramming efficiencies were compared with DOX-free treatment. (n=3)
Foxd1Cre::Pdgfrb+/J mice show similar gene expression profiles like human patients with diabetic or hypertensive nephropathy (A) Gene expression arrays were performed in 6 week old Foxd1Cre::Pdgfrb+/J mice compared to wt mice.Figure 8Gene expression arrays were performed in 6 week old Foxd1Cre::Pdgfrb+/J mice compared to wt mice. Listed are genes with log2 fold changes higher or lower than 0.6. Out of these 46 genes, 17 are IFN-regulated genes, 5 are involved in autophagy and 5 in matrix remodeling and fibrosis. Same genes were analysed in microdissected glomeruli (G) and tubulointerstitium (T) of patients with diabetic (DN, G: n=14, T: n=18), hypertensive (HT, G: n=15, T: n=21) nephropathy and living donors (LD, G: n=42, T: n=42) as controls, revealing a similar expression profile. Heatmap depicting pathway activities in Foxd1Cre::Pdgfrb+/J mice and human CKD patients according to PROGENy. Enrichment analyses of nodes in the inferred causal regulatory signaling networks of Foxd1Cre::Pdgfrb+/J mice and human CKD patients using the Biocarta database. Based on the gene array datasets transcription factor activities were estimated by using DoRothEA. Depicted are the most regulated ones regulated both in Foxd1Cre::Pdgfrb+/J mice and in human CKD patients.
Figure 5A) Expression of the 21U sensor in the indicated genetic backgrounds. Gonads are outlined by a dashed line. The mCherry signal is represented in pseudo-colours [LUT fire (ImageJ)] to reflect differences in the intensity of the signal. The panels on the right are DIC images of the respective animals. Scale bar: 25 μm. B) 22G RNA coverage of the 21U sensor(RNAe) in the indicated genetic backgrounds. C) MA-plot of log2 fold changes (on the Y-axis) versus the mean of normalized counts of 22G RNAs (on the X-axis) for pid-4 mutants, compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2. D) Heatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in pid-4 mutants. Colour scheme represents the odds ratio of overlaps representing the strength of association. MA-plot of log2 fold changes (on the Y-axis) versus the mean of normalized counts of 22G RNAs (on the X-axis) , compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2. E) for pid-5 mutant Heatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in F) pid-5 mutant MA-plot of log2 fold changes (on the Y-axis) versus the mean of normalized counts of 22G RNAs (on the X-axis) compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2. G) for pid-4;pid-5 double mutant Heatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in H) pid-4;pid-5 double mutant I, J) Line plots representing fertility over generations for the indicated genetic backgrounds at 25oC. Six L2-L3 larvae for each of the indicated backgrounds were hand-picked to a fresh plate every four or six days, counting as two or three generations, respectively, until no larvae were present on the plate to be picked. *: two plates of N2 were contaminated and were excluded from the assay.
Interaction of purified type IV pili with cells(A) Time-lapse of a cell-adhered pilus fluorescently labeled with the F10 nanobody at three different focal planes.Figure 5Time-lapse of a cell-adhered pilus fluorescently labeled with the F10 nanobody at three different focal planes. Initiation of flow corresponds to t=0. Scale bar: 1 µm. Schematic representation of the experimental setup. Changes in pili position were visualized by imaging different confocal planes (z1-3) using spinning-disk confocal microscopy. Quantification of side adhesion of pili to HUVEC as a function of the shear stress applied. Cell-adhered pili were subjected to gradually increasing flow rates. Each increase was followed by flow arrest and the number of pili adherent along their sides determined. The probability for pili to newly adhere along their side at the ith flow was then calculated as $P_{i} = 1 - \frac{N_{i}}{N_{i - 1}}$, where Ni is the number of nonadherent pili after the application of the ith flow. N=3 independent experiments. The red curve corresponds to the fit based on the physical model described in the Appendix. Error bars correspond to mean ± SEM. Schematic representation of the principles of the physical model of a pilus adhering via its tip submitted to shear stress. θ is the angle relative to the vertical position that fluctuates around the equilibrium angle θ0 due to thermal fluctuations (blue arrow). Probability for the pilus to reach the angle θ during the 30-second application of shear stress τ as determined by the physical model fitted to the data in panel (C). The red line is the equilibrium angle θ0 and the purple shading the thermal fluctuations. Spinning-disk confocal immunofluorescence of pili stained with both SM1 (magenta) and F10 (green). The thick appearance of TFP is due to the primary and secondary reagents used to detect them as well as diffraction of light. In each field of view, a substrate- and a cell-bound pilus are shown. Upper panels: z-projection from the stacks in the xy plane; bottom panels: side view in the z plane. Scale bar: 2 µm. White arrows correspond to SM1 staining at the tip of pili. Quantification of adhesion after infection of HUVEC cells for 30 minutes, expressed as the number of adherent bacteria over the number of cell nuclei. Prior to infection, bacteria were incubated at 37°C with or without antibody or nanobody for 15 minutes. Mean values ± SEM are indicated for each strain. N=5 independent experiments. Paired t-test. p<0.05 (*).
a) Frequency of CD138hiB220lo cells (numbers above outlined areas, bottom) among the IRF4hi population (numbers above outlined areas, top), assessed by flow cytometry (left).figf7(a) Frequency of CD138hiB220lo cells (numbers above outlined areas, bottom) among the IRF4hi population (numbers above outlined areas, top), assessed by flow cytometry (left). 488, Alexa Fluor 488. Right, quantification of bone marrow PCs (each symbol represents an individual mouse; small horizontal lines indicate mean (and s.e.m.). (b) Quantitative RT-PCR analysis of mRNA encoding IRF4 and Blimp-1 in CD138hiB220lo bone marrow PCs and B220hiCD138lo spleen B lymphocytes (control) sorted from Atg5f/f and Atg5f/fCD19-Cre mice, to confirm PC phenotype; results are presented relative to that of histone H3 mRNA (loading control). (c) Quantitative PCR analysis of genomic DNA assessing undeleted and deleted loxP-flanked Atg5 alleles in sorted B cells and PCs (as in b; presented as in Fig. 5c). (d) Immunofluorescence of endogenous LC3 (left, top row) and immunoglobulin content (left, bottom row) in bone marrow PCs sorted from Atg5f/f and Atg5f/fCD19-Cre mice, and quantitative RT-PCR analysis of Atg5 mRNA in bone marrow PCs and spleen B cell populations (right) sorted from Atg5f/f and Atg5f/f CD19-Cre mice. Nuclei (left) are stained blue with Hoechst. Scale bar (left), 5 μm. *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).
Figure 2A NDUFC2 mRNA transcript levels in fibroblast cell lines of controls (white), Subject 1 (grey) and Subject 3 (black). Quantification of NDUFC2 mRNA transcript levels (normalised to ACTB) showed diminished transcript levels in Subject 1 while transcript levels were unaffected in Subject 3. Bars correspond to standard deviations of two independent experiments, performed in duplicate and analysed by Student's t-test; *p<0.05. B Assessment of respiratory chain enzyme activities, normalised to citrate synthase (CS), in fibroblast cell lines from controls (white), Subject 1 (grey) and Subject 3 (black). Mean residual enzyme activities of control fibroblasts (n = 8) are set at 100% and error bars represent 1 standard deviation of control activities. Asterisks (*) denote enzyme activity below control range. C Assessment of respiratory chain enzyme activities, normalised to citrate synthase (CS), in skeletal muscle from controls (white) and Subject 3 (black). Mean residual enzyme activities of control skeletal muscle (n>100 samples) are set at 100% and bars are 1 standard deviation of control activities. Asterisks (*) denote enzyme activity below control range. D Respiration of Subject 3 and Subject 1 derived fibroblasts (black and grey, respectively) compared to control fibroblasts (white) using high-resolution respirometry. ROX-corrected analysis of basal respiration, Oligomycin inhibited LEAK respiration and maximum uncoupled respiration (ETS). Both subject fibroblasts exhibited a severe mitochondrial respiration defect as measured by oxygen consumption. Data are mean ± SD from at least 3 experiments and analysed by Student's t-test. **p<0.01, ***p<0.001.
breast cancer model(A) Representative staining images (N = 5) of tissue from TNC high (WT/shC) and TNC low (KO/shTNC) tumors (N = 5 per condition) with the indicated antibodies.Figure 2(A) Representative staining images (N = 5) of tissue from TNC high (WT/shC) and TNC low (KO/shTNC) tumors (N = 5 per condition) with the indicated antibodies. Arrows point at the respective immune subtype. Scale bar, 50 µm. (B, C) Quantification of CD8 TIL upon tissue staining as total number per field (B) and in the stroma or tumor cell nest (C), expressed as relative ratio, *** p = 0.0002, Fisher's exact test and in TNC high (N = 5) and TNC low tumors (N = 5). 3 sections per tumor, 8 random fields per section. Mean ± SEM. (D, E) Apoptosis measurement upon tumor staining for cl. casp3 (D) and signal quantification per field (E) in TNC high and TNC low tumors (N = 7 per condition), 2 sections per tumor, 5 random fields. *** p = 0.0006, Mann-Whitney test, Mean ± SEM. Scale bar, 50 µm. (F) Representative staining images (N = 5 tumors, 3 sections per tumor) for the indicated molecules. Scale bar, 50µm. Arrows point at CD8 TIL. (G, Gzmb expression as determined by qRT-PCR. N = 6 tumors per condition. ** p = 0.0022 for both graphs, Mann Whitney test. Mean ± SEM. H) Perforin expression as determined by qRT-PCR. N = 6 tumors per condition. ** p = 0.0022 for both graphs, Mann Whitney test. Mean ± SEM. (I, J) Detection (I) and quantification (J) of CD8 TIL in lung metastasis of NeuNT WT and TNCKO mice (N = 3 mice, n = 7 and 12 lung metastases, respectively). Scale bar, 100 µm. ** p = 0.0097, Mann Whitney test. Mean ± SEM.
Figure 2(A) U2OS cells were transfected with control (mixture of control DS scrambledNeg, siLuci, and siGFP) or GRWD1-targeting (mixture of siGRWD1-3 and 4 or siGRWD1-1) siRNAs for 24 h. Whole cell extracts were then analyzed by immunoblotting with the indicated antibodies. B) U2OS cells treated as above were stained with propidium iodide and subjected to flow cytometry. (C) HCT116 cells treated with actinomycinD (5nM) or vehicle (DMSO) for 12h were first extracted with Triton X-100 to remove nucleoplasmic proteins, double-immunostained with anti-GRWD1 (green) and anti-fibrillarin (red) antibodies as a marker for nucleoli, and counterstained with DAPI. Scale bar, 20 um. (D) HCT116 cells were transfected with control (mixture of control siLuci and siGFP) or GRWD1-targeting (mixture of siGRWD1-3 and 4 or siGRWD1-1) siRNAs for 24 h and then immunostained as above. Scale bar, 20 um.
Sequential 5-AzadC + HDACI treatments synergistically activate HIV-1 gene expression and production in latently-infected cells.Figure 4A-F J-Lat 8.4 (A, C, E) and 15.4 (B, D, F) cell lines were mock-treated or treated with 5-AzadC. At 48 h post-treatment, HDACIs were then added for 24 h. At 72 h 5-AzadC post-treatment samples were harvested and analysed as follows: viral p24 production in cell supernatant was measured (A and B) A-F J-Lat 8.4 (A, C, E) and 15.4 (B, D, F) cell lines were mock-treated or treated with 5-AzadC. At 48 h post-treatment, HDACIs were then added for 24 h. At 72 h 5-AzadC post-treatment samples were harvested and analysed as follows:FACS analyses were performed and the percentages of GFP+ cells are presented as histograms (C and D) A-F J-Lat 8.4 (A, C, E) and 15.4 (B, D, F) cell lines were mock-treated or treated with 5-AzadC. At 48 h post-treatment, HDACIs were then added for 24 h. At 72 h 5-AzadC post-treatment samples were harvested and analysed as follows: initiated (primers TAR) or elongated (primers tat) transcripts were quantified by RT-qPCR and results were normalized using the β-actin gene primers and are presented as histograms representing fold-inductions compared to the mock-treated condition (E and F). Means and standard errors of the means from three independent biological duplicates (n=6) are indicated. The result obtained with mock-treated cells was arbitrarily set at a value of 1 (E, F).
Expression of Cwh43 mRNA and protein in the mouse brain.Figure 2mRNA in situ hybridization images showing expression of Cwh43 mRNA in the mouse brain. Enclosed areas containing potions of the ventricle, hippocampus and dorsal thalamus are shown at higher magnification on the right. Arrowheads point to choroid plexus. Scale bar is approximately 400 µm. Fluorescence immunohistochemistry of the ependymal surface of the lateral ventricle of the mouse brain. Cilia are visualized using an antibody for acetylated alpha tubulin (green). Cwh43 is visualized using a specific anti-Cwh43 antibody (red). Nuclei are counterstained using DAPI (blue). Arrowheads point to motile cilia and scale bar is approximately 5 µm. Confocal fluorescence immunocytochemistry images of a single cultured mouse ciliated ependymal cell. Cilia were visualized using an antibody for acetylated alpha tubulin (green). Cwh43 immunoreactivity was visualized using a specific anti-Cwh43 antibody (red). Scale bar (left column) is approximately 4 µm. Images in the column on the right represent a Z-stack reconstruction of confocal images showing localization of Cwh43 immunoreactivity in cilia of a mouse ependymal cell. Scale bar (right column) is approximately 5 µm.
Figure 4A Graphs show the quantification of different parameters in mice striatal MSNs nuclei (Ctip2+/NeuN+). N = 4 for both genotypes. An average of 5,000 nuclei was analysed for each sample. Representative images are shown. Scale bar 7 μm. B Graphs show the quantification of different parameters in mice striatal interneurons (Ctip2-/NeuN+). N = 4 for both genotypes. An average of 5,000 nuclei was analysed for each sample. Representative images are shown. Scale bar 7 μm. C Graphs show the quantification of lamin B1 intensity and circularity in 30-week-old R6/1 mouse striatal neurons nuclei with (mHtt +) or without (mHtt -) nuclear inclusions. N = 6. EM48 antibody was used to label mHtt inclusions. Representative images are shown. Scale bar 7 μm. D Graphs showing lamin B1 intensity and circularity in MSNs nuclei from the putamen of HD patients at different stages of the disease (VS: Vonsattel grade) and corresponding controls (CTL: non-affected individuals). N = 6 for CTL, N = 3 for VS I-II and N = 4 for VS III-IV. An average of 50 nuclei was examined for each sample. Representative images (maximal Z-projections) are shown. Scale bar 7μm. E Graphs show the quantification of different parameters in hippocampal CA1 ( Ctip2+/Prox1-) and DG (Ctip2+/Prox1+) neurons nuclei. N = 4 for each genotype. Representative images are shown. Scale bar 7 μm.
Figure 6A. Total quantified proteins at day one, three and seven upon primary cell culture in hepatocytes (hHEP), hepatic stellate cells (hHSC) and liver sinusoidal endothelial cells (LSEC). In all cases, number of biological replicates is 3. Values are presented as mean ± s.d. No additional replications of the experiment was done in laboratory. B. PCA showing proteome dynamics of primary cells upon cell culture. C. Pair-wise Pearson correlation of proteomes during cell culture. D. Significantly changing proteins during primary hHEP cell culture, indicating significantly enriched GO terms on the cluster of down-regulated proteins. Significance was calculated by Fisher's Exact test corrected for multiple hypothesis testing with FDR < 0.05. E. Significantly changing proteins during primary hHSC cell culture, indicating significantly enriched GO terms on the cluster of up-regulated proteins. Significance was calculated by Fisher's Exact test corrected for multiple hypothesis testing with FDR < 0.05. F. Protein expression patterns over the course of primary hHSC culture (n=3). Values are presented as mean ± s.d. Significance was calculated by ANOVA followed by Benjamini-Hochberg correction for multiple hypothesis testing (FDR < 0.05) with a significance level of **P < 0.01, and ***P < 0.001.
Figure 4(A) 1 μg of each of the indicated recombinant proteins was incubated with amylose resin for 60 min, the beads were pelleted by centrifugation, and proteins in the pellet (P) and supernatant (S) were compared to the protein input (I) by means of western blotting with the appropriate antibodies. (B) Bovine liver glycogen and potato starch-derived amylopectin were incubated for 60 min with the indicated recombinant proteins, centrifuged for 90 min at 100,000 × g, and protein in the resulting supernatants (S) and pellets (P) analysed by western blotting with the appropriate antibodies. Protein without polysaccharide was used as a negative control (Buffer). (C) Amylose binding assays of full-length (1-1072) and truncated HOIP protein, visualised by immunoblotting. Shown on the right are schematic representations of the truncated HOIP constructs used. Images not drawn to scale. PUB, peptide N-glycanase and UBA- and UBX-containing protein domain; ZF, zinc finger domain; NZF, Npl4 zinc finger domain; UBA, ubiquitin-associated domain; RBR, RING-in-between-RING domain; LDD, linear ubiquitin chain determining domain. (D) Amylose binding assays of full-length (1-387) and truncated Sharpin mutants along with schematic representations of the Sharpin constructs used. Images not shown to scale. PH, pleckstrin homology domain; LTM, LUBAC-tethering motif; UBL, ubiquitin-like domain. (E) Diagrams of amylose, amylopectin, and glycogen, indicating differences in polymer chain-length and degree of branching.
Figure 4: Wild type and mps1-3 cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the presence (A) of nocodazole (t=0). Cells were collected at the indicated time points for western blot analysis of the indicated proteins. Equal amounts of protein extracts were loaded on two different gels, for western blot of Spc105-3PK and Clb2/Pgk1 respectively. Clb2 was used as mitotic marker and Pgk1 as loading control. Cyc: cycling cells. Wild type and mps1-3 cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the absence (B) of nocodazole (t=0). Cells were collected at the indicated time points for western blot analysis of the indicated proteins. Equal amounts of protein extracts were loaded on two different gels, for western blot of Spc105-3PK and Clb2/Pgk1 respectively. Clb2 was used as mitotic marker and Pgk1 as loading control. Cyc: cycling cells. Cells with the indicated genotypes expressing Bub1-GFP and the kinetochore marker Mtw1-Tomato were grown in SD glu 2% and then shifted to 34°C for 1 hour before filming; they were then filmed every 4 minutes by time lapse fluorescence microscopy at 34°C. Note that BUB1-GFP is synthetic lethal with mps1-3. Thus, we used mps1-3 GALs-MPS1 BUB1-GFP cells that were grown in -His RG medium at 30°C; glucose was added to the culture for 30 minutes to shut off GALs-MPS1, followed by shifting cells to SD medium at 34°C for 1 before imaging in the same medium. Montages show representative cells (C). Arrowheads indicate Bub1-GFP signals at kinetochores. DIC: differential interference contrast. Scale bar: 5 μm. GFP signals (Bub1) co-localizing with Tomato signals (kinetochores) were scored on the movies (D). Wild type and mps1-3 cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the presence (E-F) of nocodazole (t=0). Cells were collected at the indicated time points for western blot analysis of the indicated proteins and for FACS analysis of DNA contents Equal amounts of protein extracts were loaded on two different gels, for western blot of Bub1-3HA/Cdc5 and Clb2/Pgk1 respectively. A white asterisk indicates a phosphorylated isoform of Bub1 that in wild type cells correlates with lack of chromosome biorientation. Cdc5 and Clb2 were used as mitotic marker and Pgk1 as loading control. Cyc: cycling cells. Wild type and mps1-3 cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the absence (G-H) of nocodazole (t=0). Cells were collected at the indicated time points for western blot analysis of the indicated proteins and for FACS analysis of DNA contents (F, H). Equal amounts of protein extracts were loaded on two different gels, for western blot of Bub1-3HA/Cdc5 and Clb2/Pgk1 respectively. A white asterisk indicates a phosphorylated isoform of Bub1 that in wild type cells correlates with lack of chromosome biorientation. Cdc5 and Clb2 were used as mitotic marker and Pgk1 as loading control. Cyc: cycling cells.