EMBO/sd-character-level-ner huggingface dataset:
Figure 4Decreased pTyr levels, but normal total levels of IL1RAPL1 protein, in the Ptprd-/- brain (P21-27), as revealed by immunoprecipitation of IL1RAPL1 from whole-brain lysates followed by immunoblotting for total IL1RAPL1 protein and pTyr levels (4G10 antibody). Decreased levels of synaptic IL1RAPL1 protein in the Ptprd-/- brain (P21-27), as revealed by immunoblotting of crude synaptosomal (P2), synaptic plasma membrane (SPM), and PSD (PSD II) fractions. Analysis of immunoblots reveal significant decrease of IL1RAPL1 in the SPM and PSD fractions of Ptprd-/- brain samples. (n = 3 mice [WT and KO] for each fraction [P2, SPM, and PSD], mean ± SEM, **p<0.01, Student's t-test). Schematic diagram showing that the PTPδ-meA splice insert is important for the interaction between PTPδ and IL1RAPL1. Note that the meA splice insert in the Ig2 domain of PTPδ interacts with the Ig1 domain of IL1RAPL1. Schematic diagram showing the strategy for generating mice carrying a deletion of the PTPδ-meA splice insert encoded by exons 15 and 16 (Ptprd-meA-/-). Normal levels of the PTPδ protein in whole-brain lysates of Ptprd-meA-/- mice (P21-27), which contrasts with the Ptprd-/- brain, where PTPδ protein is undetectable. Strongly decreased levels of tyrosine-phosphorylated PTPδ protein in whole-brain lysates of Ptprd-meA-/- mice (P21-27), as shown by the immunoprecipitation of IL1RAPL1 protein from whole-brain lysates followed by immunoblotting for total and tyrosine-phosphorylated PTPδ protein. Decreased levels of synaptic IL1RAPL1 protein in the Ptprd-meA-/- brain (P21-27), as revealed by immunoblotting P2, SPM, and PSD (PSD II) fractions. Analysis of immunoblots reveal significant decrease of IL1RAPL1 in the SPM and PSD fractions of Ptprd-meA-/- brain samples. (n = 3 mice [Ptprd-meA+/+ and Ptprd-meA-/-] and [P2, SPM, and PSD], mean ± SEM, *p<0.05, ***p < 0.001, Student's t-test). Note that, while unchanged in the P2 and SPM fractions, PTPδ is significantly decreased in the PSD of Ptprd-meA-/- samples. Decreased basal excitatory synaptic transmission in the hippocampal SLM region of Ptprd-meA-/- mice, as shown by the I/O ratio at the TA-SLM pathway (P20-24). (n = 15 slice from 5 mice [Ptprd-meA+/+], 12, 4 [Ptprd-meA-/-], mean ± SEM, *p < 0.05; ***p<0.001, two-way RM ANOVA with Holm-Sidak test). Normal presynaptic release in the hippocampal SLM region of Ptprd-meA-/- mice, as shown by the PPF ratio at the TA-SLM pathway (P20-24). (n = 15 slice from 5 mice [Ptprd-meA+/+] and 13, 4 [Ptprd-meA-/-], mean ± SEM, ns, not significant, two-way RM ANOVA).
Figure 8(A) MITOL did not merge with Hsp60 in control siRNA-treated cells, whereas most MITOL co-localized with Hsp60 in p97/VCP knockdown cells in response to mitophagy stimuli. HeLa cells stably expressing 3Flag-MITOL and HA-Parkin were transfected with control or p97/VCP siRNA, treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-Flag, anti-catalase, and anti-Hsp60 antibodies. Scale bars, 10 µm. (B) HeLa cell lysates treated with control or p97/VCP siRNA were immunoblotted with anti-p97/VCP and anti-tubulin antibodies. (C) Overexpression of an p97/VCP ATP hydrolysis-defective mutant, E305Q/E578Q (p97QQ), blocked MITOL redistribution from mitochondria to peroxisomes, while overexpression of wild-type p97/VCP had no effect on MITOL redistribution. HeLa cells stably expressing GFP-Parkin were transfected with MITOL-HA and Flag-p97/VCP wild-type or p97QQ, treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-HA and anti-catalase antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm. Arrowheads indicate representative examples of MITOL-peroxisome co-localization that was only observed in the presence of a functional VCP. (D) NMS-873, a specific inhibitor of p97/VCP, prevented MITOL translocation following CCCP-treatment. HeLa cells stably expressing HA-Parkin were transfected with 3Flag-MITOL, treated with 15 µM CCCP in the presence or absence of 10 µM NMS-873 for 3 hours, and then subjected to immuno-staining with anti-Flag, anti-catalase, and anti-HA antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm. Arrowheads indicate representative examples of MITOL-peroxisome co-localization that was only observed in the presence of a functional VCP. (E) Correlation statistics for the localization of 3Flag-MITOL and catalase in the presence of NMS-873. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.
Network analysis provides insight into hierarchy and relations among TFs and their downstream transcriptional programmes.Figure 3A A representation of the pairwise degree of overlap in targets (size of the circle) and correlation in gene expression changes (colour) among overlapping targets for indicated TF perturbations. Red indicates positive correlations and blue negative correlations. B Network view of (A) showing relations amongst TFs based on their target correlation/anti-correlation. Edge width is proportional to the absolute value of the correlation. To increase readability, connections with |correlation| < 0.4 are not plotted; all correlation values are shown in A. C Network view of TF-TF cross-regulation. Directed edges indicate how transcription factors regulate each other's expression. Edge width is proportional to the magnitude of gene expression change (for clarity capped at a value corresponding to absolute log2(Fold Change) of 0.8). D Identification of target gene modules - groups of genes with common regulatory patterns by TFs. Colour indicates the fold change (adjusted for significance) of gene expression following each TF perturbation. Rows (perturbed TFs) and columns (target genes) are hierarchically clustered. 44 modules for target genes are shown. Below examples. Selected modules with highlighted overall regulation pattern and example genes listed below.
a, b) H1299 cells were transfected with an empty control plasmid or with the Flag–FoxO1(WT) plasmid.figf5(a, b) H1299 cells were transfected with an empty control plasmid or with the Flag-FoxO1(WT) plasmid. At 24 h after transfection, the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-mTOR, Beclin 1, Vps34 or Atg1 (a) and with Atg3, Atg4, Atg10, Atg7 or LC3 (b). (c) H1299 cells were co-transfected with Myc-Atg7, with Flag-FoxO1(WT) or with an empty control plasmid. At 24 h after transfection, the cytosolic lysate was extracted for Flag with anti-Flag, followed by probing with anti-Myc. HCT116 cells were serum-starved for 24 h (d) and the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-Atg7 (e) Different Myc-tagged FoxO1 fragments and Flag-tagged Atg7 were co-transfected into H1299 cells, and the cytosolic lysate was extracted for co-immunoprecipitation with anti-Myc, followed by probing with anti-Flag. (f) HCT116 cells were transfected with an empty control plasmid, an SIRT2(WT)-expressing plasmid or an SIRT2(168A)-expressing plasmid. At 24 h after transfection, cells were serum-starved for 24 h, and the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-Atg7. HCT116 cells weretreated with AGK2 for 24 h (g), and the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-Atg7
a) Electron micrographs of the IM (black arrowheads) associated with the ER (black arrows) from Atg4BC74A-expressing NIH 3T3 cells.figf1(a) Electron micrographs of the IM (black arrowheads) associated with the ER (black arrows) from Atg4BC74A-expressing NIH 3T3 cells. The IM appears to expand between the two ER cisternae and forms an autophagosome. The white arrowhead indicates vesicular clusters visible in the vicinity of the IM. The white arrow indicates the autophagosome. (b) The ratio of ER-associated autophagic structures (ER-IM complexes) to total autophagic structures. Data are expressed as the mean ± s.d. of triplicates from representative experiments. (c) Localization of GFP-DFCP1 with Atg16L1 in both control and Atg4BC74A-expressing cells. Insets show enlarged images of boxed regions. (d-g) Gold-enhanced immunogold electron microscopy revealed that GFP-DFCP1 localized to ER-IM complexes in both control (d, e) and Atg4BC74A-expressing cells (f). Immunogold particles are indicated by small arrows. No specific labelling was observed on ER-IM complexes in cells expressing GFP (g). Large arrows and arrowheads indicate the ER and the IM, respectively. Scale bars, 200 nm in a and d-g, an d 10 μm in c.
Figure 4Six- to eight-weeks old WT and Nedd4l cKO mice were treated with IMQ for 6 consecutive days and at the beginning of the treatment, the mice were injected intra-epidermally with 10 μg anti-GP130 antibody or with same dosage of isotype on every other day. Representative pictures of the mice treated with IMQ. Six- to eight-weeks old WT and Nedd4l cKO mice were treated with IMQ for 6 consecutive days and at the beginning of the treatment, the mice were injected intra-epidermally with 10 μg anti-GP130 antibody or with same dosage of isotype on every other day. Erythema, scaling and thickness of the back skin was scored daily on a scale from 0 to 4, and the cumulative score (Erythema plus scaling plus thickness) is depicted. Data information: Data are shown as mean ± s.e.m., and are representative of two independent experiments. Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Mann-Whitney U test Six- to eight-weeks old WT and Nedd4l cKO mice were treated with IMQ for 6 consecutive days and at the beginning of the treatment, the mice were injected intra-epidermally with 10 μg anti-GP130 antibody or with same dosage of isotype on every other day. Representative images of Ki67 staining in back tissues are on the left. The number of Ki67-positive cells per HPF is shown on the right. Data information: Data are shown as mean ± s.e.m., and are representative of two independent experiments. Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test C, control, n=5, IMQ, n=6. * P＜0.05, ** P＜0.01, *** P＜0.001. NS, no significance. Six- to eight-weeks old WT and Nedd4l cKO mice were treated with IMQ for 6 consecutive days and at the beginning of the treatment, the mice were injected intra-epidermally with 10 μg anti-GP130 antibody or with same dosage of isotype on every other day. Keratinocyte proliferation associated genes in the skin tissues are detected using real-time PCR analysis. Data information: Data are shown as mean ± s.e.m., and are representative of two independent experiments. Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test D, control, n=3, IMQ, n=6. * P＜0.05, ** P＜0.01, *** P＜0.001. NS, no significance.
Figure 4Heatmap, Red: up-regulated genes; green: down-regulated genes. qPCR Data are expressed as relative fold-change compared with the expression in scr-shRNA cells after normalization to the housekeeping gene TBP immunoblots showing the differentially expressed cell cycle related genes between scr-shRNA and sh-TFEB ECs. Immunoblots of total lysates from scr-shRNA and sh-TFEB ECs probed with specific Abs. The bar graph shows the densitometric analysis expressed as the ratio between the cell cycle genes and α-tubulin Modulation of cell cycle genes expression in the lung ECs derived from control and TfebiEC-/- mice. Data are expressed as relative fold-change compared with the expression in ECs of control mice after normalization to the housekeeping gene TBP Analysis of TFEB binding and modulation of the CDK4 promoter in human ECs. Representative snapshot of TFEB binding on CDK4 in human ECs. Analysis of TFEB binding and modulation of the CDK4 promoter in human ECs. ChIP was performed using digested chromatin from control ECs and TFEBS142A ECs incubated with IgG (indicated in the bar graph as "+IgG") or with Ab anti-TFEB (indicated in the bar graph as "+Ab anti-TFEB"), followed by qPCR for CDK4. Bar graph shows the percent enrichment (n=3, mean±SD). Analysis of TFEB binding and modulation of the CDK4 promoter in human ECs. Analysis of TFEB modulation of CDK4 promoter in human ECs. Bar graph shows the relative luciferase activity % evaluated in control and TFEBS142A human ECs after transfection of CDK4 full promoter and CDK4 promoter deleted of TFEB binding site Modulation of Rb protein in sh-TFEB ECs. Immunoblots of total lysates from scr-shRNA and sh-TFEB ECs probed with phospho-Rb and total Rb Abs. The bar graph shows the densitometric analysis of the immunoblotting expressed as % of sh-TFEB vs scr-shRNA and the ratio between phospho-Rb and total Rb in the different conditions
Figure 4A-L IF of integrin β1, ERK1, and RUNX2 in miniature pig canine frontal sections at embryonic day 60 (E60), E90, and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days; (A'-L') are magnifications of boxed regions in the corresponding figure panels. M Relative IF expression levels of integrin β1, ERK1, and RUNX2 during E60, E90, and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days. Data information: Data represent the means ± SEM. n = 3. Scale bars = 25 µm (A', G') and 50 µm (other panels). Unpaired t-tests for M , *P < .05, **P < .01, ***P < .001; NS, not significant. N Morphology of the human PC at weeks 18-19 (H&E staining); (N') is magnification of the boxed region in (N). DC, deciduous canine; PC, permanent canine; IEE, inner enamel epithelium; OEE, outer enamel epithelium. Mesenchyme between DC and PC is indicated with a green arrow. O-Q ISH expression patterns of ITGB1, ERK1, and RUNX2 in human PCs. Green arrows indicate the mesenchyme between DC and PC. R IF of RUNX2 in human PC. Mesenchyme between DC and PC is indicated with a red arrow. Red arrow indicates the mesenchyme between DC and PC. S PC epithelium areas in E60, E90, and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days. Data information: Data represent the means ± SEM. n = 3. , *P < .05, **P < .01, ***P < .001; NS, not significant. T Illustration of force exertion on cultured dental follicle cells (DFCs). IF of RUNX2 and phospho-ERK1/2 (p-ERK1/2) upon force loading with 0 or 1.0 g/cm2 for 2 h or after force was removed and the cells cultured for an additional 1, 2, and 4 hours. V Relative IF expression levels of RUNX2 and p-ERK1/2 between groups Data information: Data represent the means ± SEM. n = 3. One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for V , *P < .05, **P < .01, ***P < .001; NS, not significant. W Western blots of p-ERK1/2 and RUNX2 levels after 1.0 g/cm2 was applied for 2 h, with or without anti-integrin β1 antibody and ERK1 inhibitor (U0126). X Relative expression levels of p-ERK1/2, RUNX2, and Actin in (W). Data information: Data represent the means ± SEM. n = 3. Unpaired t-tests for M; One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for X, *P < .05, **P < .01, ***P < .001; NS, not significant.
Figure 6Electron microscopy of wild-type and Fam134 single knockout MEFs (ER=endoplasmic reticulum). Scale bar: 500nm. Immunofluorescence images stained for endogenous Calnexin (Canx) or Climp63 in wild-type (WT), Fam134 knockout MEFs, and Fam134 knockout MEFs reconstituted with the respective wild-type or ∆LIR mutant Fam134 protein. Scale bar: 10μm. Inset scale bar: 5μm. Representative Western blots of total cell lysate derived from MEFs used in (B) and detected for endogenous Fam134 and Actin, respectively. Quantification of (B). At least n=10 independent cells across three independent experiments each condition. The statistical significance was estimated by unpaired Student's t test. All data are represented as mean ± s.e.m. and the statistical significance is defined as * p<0.05, ** p<0.01, *** p<0.001.
Figure 1Ribbon diagrams of the (M/E)2 dimer extracted from the structure on virions (PDB:5O6A) [10], in side (left panel) and bottom (right panel) views with M in brown and E colored according to domains: domain I, red; domain II, yellow; domain III, blue; stem, green; transmembrane (TM) segments, grey; fusion loop (FL), orange. The
Plasma Membrane Localized WIPI2b Promotes LC3 Lipidation through Binding Atg16L1 Independently of FIP200.Figure 6(A) HEK293A cells transiently expressing the indicated mCherry constructs were treated in either full medium (F), starvation medium (S), or starvation medium with wortmannin (W) for 2 hr before immunoblot analysis.(B) Statistical analysis of (A) was performed by one-way ANOVA with Tukey's post hoc test. SEM for n = 3. ∗p < 0.05. (C) Complexes from HEK293A cells transiently expressing the indicated mCherry constructs were immunoprecipitated using RFP-Trap before analysis by immunoblotting. (D) 2GL9 cells transiently expressing the indicated mCherry constructs treated as in (A) were fixed and visualized by confocal microscopy. (E) CLEM of MCF7 cells expressing GFP-LC3 and mCherry-WIPI2b CAAX. Bright-field and confocal image merged (left), confocal of expressing cell (middle), low-magnification TEM of cell of interest (right). Boxed area indicates plasma membrane region showing colocalization of GFP-LC3 and mCherry-WIPI2b. Scale bar, 10 μm (n = 3).(F) High-magnification TEM showing plasma membrane region boxed in (E). Magnified insets, in top right, show small vesicular clusters under the plasma membrane detected in mCherry-WIPI2b CAAX, GFP-LC3 expressing cells but not in untransfected cells from a control experiment. (G) HEK293A cells treated with either RISC-free (RF), Atg16L1 siRNA, or FIP200 siRNA for 72 hr before transfection with HA-WIPI2b-CAAX were incubated in full medium (F), EBSS (S), or EBSS with wortmannin (W) for 2 hr before immunoblot analysis. Please note that all WIPI2 constructs are FTTG mutants. See also Figure S6 and Table S1.
Figure 5Combined median fold-change in RNA (A) levels between NUP58 mutant clones and WT cells (calculated using n=4 RNA-seq and n=3 mass spectrometry datasets). Data were visualized using Cytoscape. White: comparable average expression to WT; red: upregulated relative to WT; blue: downregulated relative to WT. Nodes belonging to the same subcomplex are grouped; edges represent physical interactions Combined median fold-change in protein (B) levels between NUP58 mutant clones and WT cells (calculated using n=4 RNA-seq and n=3 mass spectrometry datasets). Data were visualized using Cytoscape. White: comparable average expression to WT; red: upregulated relative to WT; blue: downregulated relative to WT. Nodes belonging to the same subcomplex are grouped; edges represent physical interactions A stable cell line carrying KPNB1-KPNA4-eGFP and a WT cell line were transfected with eRFP Cas9 plasmid carrying dual gRNA. Cell populations were either treated with doxycycline (doxy +) or left untreated (doxy -) and individually sorted into 384-well plates. A total of 2 plates were prepared for each replicate. Cellular viability expressed as number of viable colonies following Cas9-induced gene editing of NUP58 in WT cell line and in a cell line stably overexpressing KPNB1-KPNA4-eGFP (KAPs).
Figure 4(A RNA-Seq analyses of different time points in primary CD4+ T-cells infected in vitro with HIV-1 or mock infected. Data were analyzed by Fisher test (number of donors =3 biological replicates). Boxplots in depict median and 25-75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1.5 * IQR (inter-quartile range) of the hinge. Data beyond the end of the whiskers are outliers and plotted as points. For each time point, dots below boxplots illustrate the pathway enrichment analysis of genes up-regulated in infected vs matched mock infected controls. Dots are color-coded based on the enrichment q-values and their size indicates the fraction of differentially expressed genes in the pathway; grey dots are not statistically significant. The gene set considered for the analyses is Biocarta_PML_pathway (17 genes, M4891). B) proteomic analyses of different time points in primary CD4+ T-cells infected in vitro with HIV-1 or mock infected. Data were analyzed by Fisher test (number of donors =3 biological replicates). Boxplots in depict median and 25-75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1.5 * IQR (inter-quartile range) of the hinge. Data beyond the end of the whiskers are outliers and plotted as points. For each time point, dots below boxplots illustrate the pathway enrichment analysis of proteins up-regulated in infected vs matched mock infected controls. Dots are color-coded based on the enrichment q-values and their size indicates the fraction of differentially expressed proteins in the pathway; grey dots are not statistically significant. (C Western blot of PML protein in HIV-1 infected vs mock infected CD4+ T-cells over time. Raw data were normalized using actin (protein) as housekeeping control and expressed as Log2 fold change expression in infected vs mock infected cells. D) relative expression PML mRNA in HIV-1 infected vs mock infected CD4+ T-cells over time. Raw data were normalized using GAPDH or 18S (mRNA) as housekeeping control and expressed as Log2 fold change expression in infected vs mock infected cells. Data were analyzed by two-way ANOVA followed by Tukey´s post-test (mean±SEM of 3 and 4 biological replicates for PML mRNA (E) Western blot and quantification of PML protein in two different clones of J-Lat cells left untreated or treated with TPA for 24h. Data were analyzed by unpaired t-test (mean±SEM; n=3 technical replicates for each cell line). (F PML protein expression in CD4+ T-cells infected with HIV-1 or mock infected at 7dpi as measured by western blot. Cells were left untreated or treated with the antioxidant compound NAC (10mM, F) G) PML protein expression in CD4+ T-cells infected with HIV-1 or mock infected at 7dpi as measured by western blot. Cells were left untreated or treated with the iron chelator deferiprone (L1, 50μM, G).
The GBS hemolytic pigment/lipid toxin induces caspase 1 activation and pyroptosis.Figure 6THP-1macrophages were treated with GBSpigment, and caspase 1 activation was measured by flow cytometry using a FLICA reagent. Pigment treatment of the scrambled shRNA control cell line induces more caspase 1 activation compared to the shNLRP3cell line, demonstrating that the pigment activates caspase 1 exclusively through the NLRP3 inflammasome. Results are representative of three independent experiments. WT THP-1macrophages were treated with the caspase 1 inhibitor Z-YVAD-FMK, the caspase 3/7 inhibitor Z-DEVD-FMK, or DMSO only prior to treatment with the GBSpigment. YVAD is able to significantly decrease cytotoxicity in cells treated with GBSpigment, demonstrating that caspase 1 is required for GBSpigment-mediated cell death, characteristic of pyroptosis, while DEVD had no effect. Data are average of three independent experiments, error bars ± SEM. Significance was determined using Bonferroni's multiple comparison test following ANOVA (n = 3, ****P < 0.0001, ***P = 0.0002, *P = 0.014).