EMBO/sd-character-level-ner huggingface dataset:
Figure 1Outline of endocytic cholesterol transport. After endocytosis by the LDL receptor, LDL are delivered to early and then late endosomes. Free cholesterol is released in late endosomes and then exported to its cellular destinations. Example of a 384-well plate treated with compounds. The panel shows the staining with filipin (free cholesterol in green pseudo-colour) and anti-LBPA antibodies (red). The leftmost column shows control wells treated with DMSO only, and the right penultimate column shows wells treated with U18666A. Effects of U18666A, thioperamide and trimeprazine, compared with controls. Cells treated with the indicated compounds or with DMSO alone for 18h at 37°C, were processed for immunofluorescence microscopy. Low magnification views are shown after staining with anti-LBPA antibodies (red), filipin (green pseudo-color) and DAPI (blue). The scale bar is 20 µm. Screen data plot. The plot shows the integrated intensity of LBPA vs. filipin (cholesterol) staining in all HeLa cells of each well analyzed in the screen (each dot is the average of replicates in the duplicate plates, and ≥600 cells were analysed per compound). Only compounds that showed less than 20% toxicity are plotted. The cells treated with U18666A, thioperamide maleate and trimeprazine tartrate are indicated, as well as the DMSO controls. Integrated intensity of cholesterol and LBPA staining. The integrated intensity of cholesterol (filipin in green) and LBPA (red) staining in all cells of the indicated samples is quantified after normalization to the DMSO controls. (n=3 independent experiments, 64 images analysed per experiments, error bars = SD, two ways ANOVA; **=p<0.01; ***=p<0.005).
Figure 7A. Immunofluorescence staining of F-actin in PTENε or PTENε Y210L overexpressed Hela PTEN-/- cells and control cells. PTENε-GFP, PTENε Y210L-GFP, or mock construct was introduced into HeLa PTEN-/- cells respectively. The transfected cells were stained with Phalloidin and DAPI before being imaged with confocal microscopy. The scale bars represent 5 μm (left panel). White arrows shown in the magnified immunofluorescence images indicate the filopodia in the cell membrane. B. Quantification of the number of filopodia per cell by FiloQuant software and the data are presented as mean ± SD of three independent experiments and were analyzed with the unpaired t-test. ****, p<0.001. C. Representative images of transwell migration and invasion assays in Hela PTEN-/- cells and H4 cells stably transfected with PTENε, PTENε Y210L, or an empty control (magnification, 200x). The scale bars represent 50 μm. D. Statistical analysis of transwell migration and invasion assays in Hela PTEN-/- cells and H4 cells stably transfected with PTENε, PTENε Y210L or an empty control (magnification, 100x; five random per well). Data are presented as the mean ± SEM based on three independent experiments and were analyzed with the unpaired t-test. *, p<0.01. ****, p<0.001. E. Adenine Base Editing mediated endogenous PTENε knockout in Hela cells. Immunoblotting analyses were used to verify the level of PTENε loss in Hela cells. F. Representative images of transwell migration and invasion assays in Hela PTENε+/- cells at 200× magnification (left panel, the scale bars represent 50 μm). (right panel) Migrated or invaded cells were counted at five random fields at 100× magnification per well to assess cell migration and invasion ability. G. Left panel: immunoblotting analyses were used to verify the level of overexpressed PTENε or PTENε Y210L in B16 cells. Middle panel: representative images showing the lung metastasis of PTENε or PTENε Y210L overexpressed B16 cells or control cells in the mouse pulmonary metastasis model (n=4). H & E, hematoxylin-eosin staining. The scale bars represent 600 mm (upper panel) and 500μm (lower panel) separately. Right panel: the representative bar graphs showing the quantification of tumor metastatic ability was displayed by measuring the number of tumor nodules (data are presented as mean ± SD based on three independent replicates). The overexpression of PTENε attenuated tumor metastatic ability compared with the vector control group. ****, p<0.0001 in the unpaired t-test.
Figure 6A HeLa cells were treated with 0.4 µM APH for 24 hours, fixed and processed for IF microscopy after staining with anti-TIAR and anti-CDK1 antibodies. Intensity profiles along the white line in the merged image are presented on the right side; yellow arrows mark GMGs. B IF microscopy analysis as in (A) using anti-Cyclin B antibody. C HeLa cells transfected with control or TIAR siRNAs were treated with 0.4 µM APH 24 hours prior to fixation. Cells were processed for IF microscopy after staining with anti-PRP19 and anti-CDK1 antibodies. Intensity profiles from staining of the same cells with anti-TIAR and anti-CDK1 antibodies are depicted on the right side. D CDK1 was immunoprecipitated from HeLa cells transfected with control or TIAR siRNAs, tested for in vitro kinase activity using recombinant histone H1 as substrate, and visualized by Western blot analysis and autoradiography. The mean CDK1 activity ± SE was quantified from n = 5 experiments. Data information statistical significance was determined by unpaired Student's t-test. ; *, p < 0.05; ***, p < 0.001. E CDK2 activity was analyzed following immunoprecipitation as in (D) (mean ± SE, n = 3). Data information statistical significance was determined by unpaired Student's t-test. *, p < 0.05; ***, p < 0.001. F HeLa cells were transfected with control or TIAR siRNAs for 72 hours before measuring expression levels of p(S22)-Lamin A by Western blot analysis (mean ± SE, n = 3). Data information statistical significance was determined by unpaired Student's t-test. ; *, p < 0.05; ***, p < 0.001. G p(S22)-Lamin A levels were analyzed by HTM in si-RNA transfected HeLa cells (n = 3, 1000 cells examined per experiment and condition). Data information statistical significance was determined by Wilcoxon rank-sum test; *, p < 0.05; ***, p < 0.001.
Figure 2A. MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with serum-free medium. sEVs released from the apical and basolateral sides of MDCK cells were purified by ultracentrifugation. Apical and basolateral sEVs (P100) were analyzed by silver staining. B. P100 were analyzed by LC-MS/MS. Venn diagrams represent the number of proteins detected in each sample with minimum three independent peptides. C. MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. sEVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation. Cell lysates and sEV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated. GPRC5C was detected only in the apical sEV samples D. PEG pellets were subjected to OptiPrep flotation analysis. GPRC5C was detected only in the apical sEV samples E. MDCK cells were cultured sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. GPRC5C was detected only in the apical sEV samples F. MDCK cells stably expressing human CD63 were cultured sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD63 antibody.
Intrinsic polarity is dependent upon Elongator's function on tRNAs and is impaired upon proteasome inhibition.Figure 5(A-B) Polarized enrichment of LGN at the cortices of dividing HEK293T cells is disrupted upon Elp3 or Alkbh8 knockdown. (A) HEK293T cells transfected with siRNAs for Elp3, Alkbh8 or control and immunolabelled with LGN, acetylated alpha-tubulin and DAPI. Scale bar=5µm. (B) The mean fluorescence intensity profile of LGN across the radii perpendicular to division in HEK293T cells was quantified (siRNA Control/Elp3/Alkbh8: n=27/30/27; pooled from 3 independent experiments; mean±SEM). (C-D) The length of the primary cilium is reduced in non-dividing HEK293T cells upon Elp3 or Albh8 knockdown. (C) Control, Elp3 or Alkbh8 siRNA-treated HEK293T cells were immunolabelled for basal body marker ALMS1, axoneme marker Arl13b and DAPI. Scale bar=5µm. (D) Primary cilium length of non-dividing HEK293T was measured and pooled data were plotted (siRNA Control/Elp3/Alkbh8: n=94/81/90; data collected from 3 independent experiments; One-way ANOVA, Tukey's multiple comparisons test, ***p<0.001, F=21.46, DF=4; minimum, first quartile, median, third quartile and maximum presented). (E-F) Elp3 knockdown increases the amount of polyubiquitinated conjugates in HEK293T cells (n=3; One-way ANOVA, Tukey's multiple comparisons test, **p<0.01, *p<0.05, F=16.51, DF=2; mean±SD). (G) Increased formation of aggresome-like structures in Elp3- or Alkbh8-depleted HEK293T. Aggresomes (red); DAPI (blue). Enlarged images depict examples of non-dividing (left) and dividing (right) cells. Scale bar=10µm. Inducing aggresomes with MG-132 disrupts the polarized enrichment of LGN at the cortices of dividing HEK293T cells. (H) HEK293T cells treated with DMSO vehicle or MG-132 and stained for aggresomes (left panels) or LGN, F-actin and DAPI (green, red and blue, respectively in right panels). Scale bar=10µm. Inducing aggresomes with MG-132 disrupts the polarized enrichment of LGN at the cortices of dividing HEK293T cells. (I) Fluorescence intensity profile of LGN across the radii perpendicular to division in dividing HEK293T cells. Mean fluorescence values per percentage of cell length with SEM are plotted. Fluorescence intensity profiles collected from 3 independent experiments (DMSO/MG-132: n= 24/24). (J) Proteostat staining of MG132-treated whole-mounted P0 organs of Corti reveals the presence of aggresome-like structures in HCs. Orthogonal slices (Orthog) reveal aggregated proteins localize at the apical surface of MG132-treated HCs. Scale bars=5µm. #IHC; *OHC1; **OHC2; ***OHC3 (K) Localization of LGN at the lateral apical surface of E16.5 WT HCs is disrupted by MG-132. LGN (white/green) and F-actin (red). Scale bars=5µm; magnified views=2µm.
Figure 4(A) Western blots of lysates of primary neurons transduced with control lentiviruses (NT) or lentiviruses to knockdown (KD) or overexpress (OE) PRR7. PRR7 knockdown reduced (75.6 ± 6.9%) while PRR7 overexpression (OE) increased (157.6 ± 22.8%) c-Jun levels compared to controls. No change was detected in c-Fos levels. One-way ANOVA test was used to analyze results with Dunett's post hoc test (n=16 gels for c-Jun and 4 gels for c-Fos). (B) Control or PRR7 transfected HEK293 cells were treated with cycloheximide for the indicated time points and c-Jun protein levels were measured by Western blotting. PRR7 expression significantly stabilized c-Jun levels at 120 min (110.0 ± 7.5% vs control 68.7 ± 7.9%) and 240 minutes (83.6 ± 5.2% vs control 43.7 ± 3.4%). Values for c-Jun were normalized to their respective 0 min time point (n=4 gels for each group performed blind). T-tests between similar time points were used to analyze results. (C) PRR7, FBW7 and/or a control protein (PSD95) were transfected into HEK293 cells and c-Jun ubiquitination was measured by immunoprecipitating c-Jun and blotting for ubiquitin. Ubiquitination of c-Jun in the presence of FBW7 (178.3 ± 26.9%) was significantly reduced in the presence of PRR7 (103.7 ± 20.6%) but not a control protein (PSD95) (n=5 gels for all groups). (D) Western blots of c-Junubiquitination in hippocampal primary neuronal cultures. PRR7 knockdown (KD) elevated (149±10.62%, n=6 gels) c-Junubiquitination level in comparison to control neurons (NT). Statistical significance was determined by t-test. (E) Western blots showing knockdown (KD) or overexpression (OE) of PRR7 in primary hippocampal neurons do not alter levels of other FBW7 targets mTOR or Myc.
In vitro characterization of Mabs grown inside PF hydrogels.Figure 1A Phase contrast images of Mabs cultured into PF hydrogels (5 days): cells formed a thick three-dimensional myotube network with numerous thick myofibres (arrows). Scale bar: 50 μm. B Confocalimmunofluorescence analysis with an antibody against myosin heavy chain (MyHC) (red) and SYTOX green which labels nuclei (green), showing multi-nucleated, mature musclefibres (arrows) developed from Mabs in PF (5 days). Scale bar: 50 μm. C, D Western blot and densitometry analysis of different protein extracts of Mabs differentiated inside PF constructs (PF) showing expression levels comparable to adult TA (Ctrl). Group of n = 6 of Mabs differentiated inside PF and host TA has been tested, densitometric analysis from n = 3 different Western blots gauging the level of expression of muscle markers is presented as means ± standard error.
Figure 1(A) Representative single immunofluorescent staining for dsDNA at 6 h, 24 h, and 3 d of reperfusion after MCAO in the penumbra as well as in the sham brain. Scale bars, 5 μm. (B) Representative images of double-immunofluorescent stained for 53BP-1 and dsDNA (upper panel), Iba-1 and dsDNA (middle panel), and for GFAP and dsDNA (lower panel). Scale bars, 5 μm. (C) Western blot analysis of cGAS and STING using lysates prepared from the indicated tissues of mice brain following MCAO. (D) Quantitative analysis for Western blot analysis. (E) Immunohistochemical staining for cGAS at 3 d of reperfusion after tMCAO in peri-ischemic area as well as in the corresponding regions of sham control brains. Scale bars, 50 μm, 20 μm in insets. (F) Quantitative analysis of cGAS immunohistochemical staining. n = 6 mice per group. **P<0.01, two-tailed unpaired Student's t test. (G) Representative double immunofluorescent stainings for cGAS (red) and 53BP1 (green). n=6 mice per group.
Figure 6A. The Venn diagram of overlapping genes targeted by hTrmt13, FOSL1, and E2F1 in MDA-MB-231 cells. Among 1136 (220+32+371+513=1136) hTrmt13 binding sites, ~35% were also bound by FOSL1(32+371=403), ~78% were also bound by E2F1 (513+371=884), while 92% of hTrmt13 and FOSL1 co-binding sites (403) also have E2F1 binding (371). B. Interaction of FLAG-hTrmt13 with endogenous FOSL1, E2F1, and USF1. Whole-cell lysates from MDA-MB-231 cells stably expressing FLAG-hTrmt13 were prepared and immunoprecipitation was performed with anti-FLAG followed by immunoblotting with antibodies against indicated proteins. C. Interaction of endogenous hTrmt13 with FOSL1, E2F1, and USF1.Whole-cell lysates from MDA-MB-231 cells were immunoprecipitated with antibodies against hTrmt13 or IgG followed by immunoblotting with the antibodies against the indicated proteins. * and ** are nonspecific bands. D. A graphical scheme illustrating PLA analysis of protein-protein interaction. E. PLA analysis showing representative images obtained from imaging confocal microscopy of the interaction of hTrmt13 and FOSL1, hTrmt13 and E2F1, hTrmt13 and USF1. Red dots indicate that the detected protein interacts with hTrmt13, and DAPI staining was included to visualize the cell nucleus (Blue). The anti-hTrmt13 and IgG was used as a negative control. F. ChIP-qPCR analysis of the selected promoters was performed using antibodies against indicated proteins in control and hTrmt13 knockdown MDA-MB-231 cells. Results are represented as fold change over control IgG. Statistical analysis was performed using t-tests. Error bars indicate mean ± SD for three independent experiments.
a–f) Immunofluorescence for PSA-NCAM (a–c) or NeuN (d–f) in the dentate gyrus (DG), SVZ, RMS and olfactory bulb (OB) of P28 mice treated with NAC or vehicle (Veh) control.figf8(a-f) Immunofluorescence for PSA-NCAM (a-c) or NeuN (d-f) in the dentate gyrus (DG), SVZ, RMS and olfactory bulb (OB) of P28 mice treated with NAC or vehicle (Veh) control. Representative images are shown for PSA-NCAM (a) and NeuN (d). Dotted lines indicate boundaries of the DG (a,d), SVZ (a) and layers of the OB (d). Arrows mark PSA-NCAM+ cells in a. Mean ± s.e.m. of the number of PSA-NCAM+ cells in SVZ (b) and DG (normalized to 1 mm2 area) (c), and the number of NeuN+ cells in DG (e) and IGL of OB (per mm2) (f) per section are shown (n = 5 mice for each; ≥4 sections for each mouse). cKO(n), NAC non-responder cKO mice; GZ, granular zone; LV, lateral ventricle; ML, molecular layer; ST, striatum; IGL, internal granular layer; MCL, mitral cell layer; EPL, external plexiform layer; GL, glomerular layer. **P 0.01.
PGCs survival is compromised between E10.Figure 3A, B IF for MVH+ germline cells (red) in (A) male, and (B) female gonads at E13.5. In males, sertoli cells are also marked with SOX9 (green). Dashed circle indicates a testis cord. Scale bar, 50 μm. C Higher power image of (A) at E13.5. Dashed circle indicates a cross section through a testis cord. Arrows point to PGCs, which were identified by MVH+staining. Scale bar, 20 μm. D IF of the indifferent gonads at E11.5 with PGCs identified by staining for STELLA (green) and MVH (red). Arrows indicate a small PGC cluster in the PCKO mutants. Scale bar, 100 μm. Note that PCKO mutant PGCs are STELLA‐positive but not MVH‐positive at this magnification. E IF at E9.5 with PGCs identified by co‐staining for STELLA (green) and OCT4 (red). Scale bar, 100 μm. F Higher power image of (E) at E9.5. Arrows indicate OCT4 and STELLA double‐positive PGCs. Scale bar, 20 μm. G Quantification of germ cell number between control and PCKO at different gestational stages. H E9.5 embryos from control and PCKO. Scale bar, 1 mm. I E11.5 embryos of control and PCKO. The majority of PCKO embryos are equivalent size to the control at E11.5. Scale bar, 1 mm.
Figure 3Histochemical visualization of β-galactosidase (β-gal) reporter activity revealed SPPL2c expression within seminiferous tubules. Cryo-sections from PFA-fixed wild type and SPPL2c-/- testis were stained with X-Gal as β-gal substrate. SPPL2c protein levels were analysed in germ cell populations from testis of wild type mice sorted for their DNA content which was determined by Hoechst 33342 staining as depicted in Fig EV4D. While for 4C, 2C and 1C fractions 10 µg of protein were loaded, for the testis control samples (=total lysates) 20 µg of protein were subjected to electrophoresis. *, non-specific band. Immunohistochemical detection of SPPL2c was performed on cryosections of Bouin-fixed wild type and SPPL2c-/- testis. The SPPL2c antiserum directed against a C-terminal epitope of the protease and diaminobenzidine as peroxidase substrate were employed. Nuclei were stained with hematoxylin. Scale bar, 100 µm (upper panel) or 50 µm (lower panel). SPPL2c levels were compared in total lysates prepared from testis of wild type mice sacrificed at the age of 4, 6, 8 or 12 weeks by Western blotting. A testis lysate from an adult SPPL2c-/- mouse was used as negative control. The weight of both testicles of wild type and SPPL2c-/- mice (n=12 per genotype) was determined and normalised to the body weight. Paraffin sections from Bouin-fixed wild type and SPPL2c-/- testis were stained with hematoxylin & eosin (H&E). Scale bars, 100 µm. In H&E stained paraffin-sections from Bouin-fixed wild type and SPPL2c-/- testis, the mean number of elongated spermatids per seminiferous tubule was determined. Cells were counted in 20 tubuli in sections from n=6 mice per genotype. SPPL2c is not present in mature spermatozoa. Western blot analysis of total lysates of wild type (+/+) and SPPL2c-/- testis, epididymis and spermatozoa recovered from the cauda epididymidis/vas deferens for SPPL2c. Mature sperm were isolated from the epididymis of wild type or SPPL2c-deficient mice and analysed by phase-contrast microscopy. Representative pictures of morphologically normal spermatozoa are depicted. Scale bars, 10 µm. Reduced motility of SPPL2c-/- sperm cells. Spermatozoa were recovered from the cauda epididymidis of wild type and SPPL2c-/- mice (n=3) and subjected to computer-assisted sperm analysis. Average path velocity (VAP), straight line velocity (VSL) and curvilinear velocity (VCL) were determined in micrometer per second. Mean litter sizes for different breeding schemes. The data depicted are based on n=12 (+/+X+/+), n=57 (+/-X+/-), n=11 (-/-X-/-), n=10 (-/-X+/+) and n=11 (+/+X-/-) different breedings, respectively.
Figure 6Heatmap shows the association between the plasma level of clinical variables including liver fat, ALT, AST, uric acid, and creatinine with A) plasma level of 10 most significant metabolites, Heatmap shows the association between the plasma level of clinical variables including liver fat, ALT, AST, uric acid, and creatinine with , B) plasma level of 10 most significant inflammation related proteins Heatmap shows the association between the plasma level of clinical variables including liver fat, ALT, AST, uric acid, and creatinine with , C) the abundance of the species in gut microbiome Heatmap shows the association between the plasma level of clinical variables including liver fat, ALT, AST, uric acid, and creatinine with D) the abundance of the species in oral microbiome. AST, Aspartate aminotransferase; ALT, Alanine transaminase. Asterisks indicate statistical significance based on Spearman correlation analysis. p < 0.05; Cor.Coeff: Correlation coefficient.
EPG-7 colocalizes with SQST-1 aggregates in autophagy mutants and directly interacts with SQST-1.Figure 3.(A) EPG-7 colocalizes with SQST-1 aggregates in epg-8 mutant embryos. Colocalization was defined as two aggregates showing >70% overlap in their fluorescence signals. Insets show a magnified view. (B) Percentage of EPG-7 aggregates colocalized with SQST-1 in indicated autophagy mutants. (C) A coimmunoprecipitation assay reveals that EPG-7 associates with SQST-1. (D) EPG-7 interacts with SQST-1 in a pull-down assay. (E) EPG-7 self-associates in a pull-down assay. (F) SDS-PAGE and blue-native PAGE of purified EPG-7(520-714) proteins. Lane M shows molecular weight marker. Lanes 1, 2, 3, and 4 contain 10, 5, 2.5, and 1.25 µg of proteins, respectively. (G) An EPG-7 fragment containing amino acids 531-631 self-interacts. (H and I) SQST-1 aggregates accumulate in epg-7 mutant embryos carrying an epg-7(del 531-631)::gfp transgene. (H) DAPI image of the embryo shown in I. (J and K) EPG-7(del 531-631)::GFP is weakly expressed and diffusely localized in epg-7(tm2508) embryos. Its expression is not elevated in lgg-1(RNAi) animals. H-K show embryos carrying the same transgene. Images in J and K were taken using the same exposure time. (L and M) SQST-1 self-interacts (L) and the self-interaction is mediated by a fragment containing amino acids 26-181 (M). (N) Levels of SQST-1(del 26-181)::GFP are dramatically elevated in epg-7 and atg-3 mutants. (O and P) SQST-1(del 26-181)::GFP is weakly expressed and diffusely localized in sqst-1(ok2892) mutant embryos. (O) DIC image of the embryo shown in P. (Q and R) SQST-1(del 26-181)::GFP is expressed at dramatically elevated levels and accumulates into a large number of aggregates in lgg-1 (Q) and epg-7 (R) mutants.
Figure 7Scheme of the experiment verifying if the HO-1+/+ niche is able to reverse phenotype of HO-1-/- LT-HSCs. Transplantation of HO-1-/- HSCs provides lower chimerism among HSPC fractions in primary recipients. Data are shown as mean ± SEM, n = 7-8 mice/group. Transplantation of the same number of donor-derived BM cells from primary recipients provides the same (C) PB chimerism Transplantation of the same number of donor-derived BM cells from primary recipients provides the same BM LT-HSC chimerism. Analysis pipeline used to determine whether transplantation of HO-1-/- HSCs to the HO-1+/+ recipients reverses their transcriptional alterations. Overlap between DEGs in young HO-1-/- HSCs and DEGs changed by transplantation alone. 145 overlapping genes were excluded from further analysis. GSEA analysis based on 145 excluded genes indicates processes that cannot be analyzed with the pipeline. Only 1 out of 267 DEGs identified in non-transplanted HO-1-/- LT-HSCs was still dysregulated in HO-1-/- LT-HSCs transplanted twice to the wild-type HO-1+/+. Comparison of gene log fold changes in non-transplanted HO-1-/- LT-HSCs and HO-1-/- LT-HSCs transplanted twice to the wild-type HO-1+/+ showed that transplantation of HO-1-/- LT-HSCs twice to the wild type niche reverses their transcriptional alterations. GSEA analysis based on genes that were altered in non-transplanted HO-1-/- LT-HSCs, but were normalized by double transplantation to the wild-type niche.