EMBO/sd-character-level-ner huggingface dataset:
Genetic ablation of microglia in the retina ameliorates photoreceptor degeneration in rd10 mice.Figure 6Retinalmicroglia were depleted in rd10/CreDTAmice by the oral administration of tamoxifen (in corn oil) to activate microglia-specific Cre-mediated recombination and diphtheria toxin expression; control CreDTA littermates were administered corn oil without tamoxifen.A-D Depletion of retinalmicroglia in the rd10 retina. Representative retinal section from a P28 control animal demonstrates Iba1+microglia in the retina, including those infiltrating the ONL (A), while a tamoxifen-administered littermate (B) was substantially depleted of retinalmicroglia. Scale bar, 40 μm. Microglial cell counts in the entire retina (C) and in the ONL only (D) confirmed efficient depletion of infiltrating microglia following tamoxifen administration (n = 8 control and 11 depleted animals from four litters, two-sided unpaired t-test).E-J Effect of microglial depletion on retinal degeneration at P28-29. ONL atrophy and thinning in control animals (E) was significantly more advanced relative to microglia-depleted littermates (F). Scale bar, 40 μm. Quantification of mean ONL thickness (G) and mean number of layers of ONLnuclei (H) at P28-29 demonstrate significantly greater ONL preservation in depleted retinas; the degree of ONL preservation correlated with the extent of microglia depletion (I). (J) The mean density of TUNEL+nuclei in the ONL was not significantly decreased in depleted vs. control animals (n = 8 control and 11 depleted animals from four litters, two-sided unpaired t-test).K-N Continuation of microglial depletion until P37-39 resulted in the persistence of morphological rescue (K-M), with a significant reduction in TUNEL+nuclei density (N) (n = 6 control and nine depleted animals from two litters, two-sided unpaired t-test).O-R Similar rescue effects as in (K-N) remained apparent when depletion was sustained until P50 (O-R), a time when rod degeneration in the rd10 model is relatively complete (n = 9 control and 10 depleted animals from three litters, two-sided unpaired t-test).S, T Functional rescue of photoreceptors was evident following microglial depletion until P50 in significantly increased dark- (S) and light-adapted (T) responses in ERG testing in depleted animals (green lines) relative to control animals (black lines), in both a- and b-wave amplitudes across multiple flash intensities (n = 9 control and nine depleted animals, *P < 0.05 in one-way ANOVA with Sidak's multiple comparison test).
Figure 1A, B MARS (A) and MARS2 (B) mRNA expression levels were measured by real-time PCR in blood samples with 2 and 3-4 copies. Relative expression levels were normalized to GAPDH levels, and data were quantified relative to the mRNA expression levels of 2 copy samples. Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Unpaired Student's t test C, D Immunoblotting analysis was performed to detect MARS protein levels in samples with 2 and 3-4 copies. Protein-level intensity shown in (D) was quantified using ImageJ and divided by the protein-level intensity of actin. The results were then graphed relative to samples with 2 copies. E, F MARS2 protein expression levels were detected (E) and quantified (F). Actin was used as a loading control. Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Unpaired Student's t test was used G Cell proliferation was assessed by Ki67 (red) immunofluorescent staining in mice neural tube sections at E10. MARS (green) was co-stained. Scale bar: 150 μm. H Double immunofluorescent staining for MARS (green) and Ki67 (red) in developing heart sections at E10. Scale bar: 150 μm. I MARS expression was detected in E80 rhesus monkey brain sections (n=3) by IHC. Local large magnification is shown as framed. Red arrow indicates germinar layer, blue indicates immature neurons. Scale bar: 1mm. MARS protein levels in embryonic chorion (C) cells and their corresponding maternal deciduae (D) cells were determined by western blot. Each D/C lane pair was quantified relative to D as shown at the bottom. MARS protein levels in embryonic chorion (C) cells and their corresponding maternal deciduae (D) cells were determined by western blot. The mean MARS levels in all D and C groups were quantified by ImageJ and shown in (K) (n=6). Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Wilcoxon matched pairs test was used L MARS2 mRNA expression in embryonic chorion (C) and maternal deciduae (D) were detected by qPCR (n=6). Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Wilcoxon matched pairs test was used M MARS protein quantification in the brains of foetal rats (F) and their corresponding mothers (M) from experiment (O) (n=6). Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Wilcoxon matched pairs test was used N MARS mRNA levels (n=6) in the brains of foetal rats (F) and their corresponding mothers (M) were determined by qPCR (n=6). Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Wilcoxon matched pairs test was used O MARS protein levels in foetal rats (F) and their corresponding mothers (M) were determined by western blot. Each M/F lane pair was quantified relative to M as shown at the bottom. P Representative result of MARS expression was detected by IHC in tumor tissues (T) and adjacent noncancer tissues (N) of UCC samples. Scale bar: 50 μm. Q Ki67 and MARS expression was analysed by IHC in HCC samples. Scale bar: 20 μm.
PPM1G-B56δ holoenzyme is required for adherens junction assembly.Figure 4(A) Cells transduced with the indicated shRNAs were fixed and localization of α-catenin and β-catenin was determined by imaging with a confocal microscope after staining with antibodies against α-catenin and β-catenin. Representative images from three independent experiments are shown. Scale bars, 10 μm. (B) Quantification of plasma membrane (PM) to cytoplasmic ratio of α-catenin intensity per each cell from figure 4A was plotted (n=40 cells). Error bars indicate SD. ***p < 0.001, (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test). (C) Control, PPM1G and B56δ shRNA transduced cells were transfected with SFB-α-catenin. Cell lysates were pulled down using streptavidin beads and interaction with β-catenin was determined by western blotting. (D) F-Actin in control and PPM1G depleted cells was stained using rhodamine labelled Phalloidin and the cytoskeleton arrangement was detected by using confocal microscope. Scale bars, 20 μm. E) Localization of α-catenin and β-catenin in control shRNA cells or B56δ depleted cells with or without expression of SFB-PPM1G and SFB-PPM1G ΔNLS was detected by imaging with confocal microscope after staining with specific antibodies. Representative images are shown, Scale bars, 10 μm. (F) Quantification of plasma membrane (PM) to cytoplasmic ratio of α-catenin intensity per each cell from figure 4F was plotted (n=30 cells). Error bars indicate SD. ***p < 0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).
a) Scheme of the experimental design to monitor CMA activity in cultured cells using the KFERQ–PA–mCherry1 reporter.f7(a) Scheme of the experimental design to monitor CMA activity in cultured cells using the KFERQ-PA-mCherry1 reporter. (b) Mouse fibroblasts (NIH3T3) stably expressing KFERQ-PA-mCherry1 were photoactivated and maintained in media supplemented (+) or not (−) with serum for 16 h. Left: Representative images. Nuclei are staining with DAPI. Right: Quantification of the number of red fluorescent puncta per cell. Values are mean+s.e. of three different experiments with >20 cells counted per experiment. *P=0.001, ANOVA-Bonferroni. (c) Temporal changes in the mCherry fluorescent intensity in cells stably expressing KFERQ-PA-mCherry1 at the indicated times after photoactivation using FACS analysis. Cells were maintained in media supplemented (white squares) or not (black circles) with serum, and fluorescence by the MFI was calculated on events gated on living cells. Values are expressed as percentage of the intensity at time 0 and are mean+s.e. of 3-4 individual experiments. (d) Representative FACS experiment showing the effect of serum removal at 6 h (top) and 42 h (bottom). PS, photoswitching. Scale bars, 5 μm.
Fig.Figure 3(A) Western blot analysis of DIV 5 cortical neurons transfected with siMecp2 or siControl (siCtl) and treated with 1 µM FK506 or vehicle for 1 h, and quantification of pS421 HTT (n=9 per group). Data are presented as means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, Mann-Whitney test. (B) Representative kymographs showing axonal trafficking of BDNF-mCherry-containing vesicles in cortical neurons transfected with siMecp2 or siControl (siCtl) and treated with 1 µM FK506 or vehicle for 1 h. Scale bar = 20 µm. (C) Quantification of anterograde, retrograde, mean velocity, linear flow rate and number of BDNF-mCherry-containing vesicles from the data in (A) (siMecp2+Vehicle: n=801 vesicles/95 axons; siMecp2+FK506: n=1020 vesicles/116 axons; siCtl+Vehicle: n=780 vesicles/83 axons; siCtl+FK506: n=1029 vesicles/102 axons). Data are presented as the mean ± SEM of at least three independent experiments (one-way ANOVA with Tukey's comparison). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.
Figure 3(A) In MGC80-3 and HGC-27 cells, lncPSCA-knockout accelerates wound-healing and the stably enforced lncPSCA expression inhibits wound-healing. The dashed lines indicate the edges of the cell layers. (B) lncPSCA inhibits invasion abilities of MGC80-3 and HGC-27 cells. Cells on the lower surface of the chamber were stained by crystal violet. Scale bar = 100 μm. (C) In MGC80-3 and HGC-27 cells, expression changes of different markers (β-catenin, ZO-1, SNAIL and VIMENTIN) of epithelial-to-mesenchymal transition were examined after overexpression or knockout of lncPSCA. (D) Fluorescent images of tumors in nude mice with tail vein injected MGC80-3 and HGC-27 cells with or without knockout of lncPSCA. (E) Representative images of hematoxylin and eosin-stained slides of lung metastatic nodules of MGC80-3 and HGC-27 with or without knockout of lncPSCA. Scale bar = 100 μm. (F) Incidence of distant metastases in nude mice (n = 8 per group).
Chromatin landscape in the ABCB1 promoter region upon LentiGuide vector integrationA) H3K9me3 ChIP-sequencing tracks from the q21.Figure 3A) H3K9me3 ChIP-sequencing tracks from the q21.12 arm of chromosome 7 in RPE-1 cells (Parental and Taxol-resistant clones). The ABCB1 gene region is shown in pink. Each colored track shows the H3k9me3 profile of a different Taxol-resistant clone. B) H3K9me3 ChIP-qPCR in the ABCB1 promoter region. Bar graph show primer pairs amplifying the sgRNA#6 break and spanning this region (+/- base pairs) for each Taxol-resistant clone compared to the parental. Bar plots show the mean of H3K9me3 relative enrichment. Each dot represents a technical replicate (n=3). C) Graphical representation of the ABCB1 promoter region. The location of the sgRNAs targeting the promoter are shown in blue. The location of the eleven CpG islands analyzed in the methyl array are shown in colored dots. D) Relative methylation status (1: methylated, 0: non-methylated) of the eleven CpG islands shown in C for RPE-1 parental and the Taxol-resistant clones.
A.Figure 5A. Fluorescence microscopy analysis of extracellular tachyzoites expressing GFP-TgAtg8 or its glycine mutant version, before or after induction of autophagy for 8 hours in HBSS. Autophagosomes are labelled by GFP-TgAtg8 in control parasites (arrowheads), but not by the glycine mutant version of the protein. B. Extracellular tachyzoites expressing GFP-TgAtg8 or its glycine mutant version were put to starve in HBSS medium for increased lengths of time and the proportions of cells displaying punctate or cytosolic GFP signals were assessed. Data are mean from n = 4 independent experiments ±SEM. C. Protein extracts corresponding to tachyzoites expressing GFP-TgAtg8 and glycine mutant version incubated in HBSS for up to 8 hours, were separated by urea SDS-PAGE and analysed with an anti-GFP antibody to detect GFP-TgAtg8 and lipidated GFP-TgAtg8-PE forms. Anti-ROP5 was used as a loading control.
Figure 3(A) Glucose consumption and lactate excretion in KP and KPΔNF1 clones normalized by cell count (n = 4 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs). (B) Glutamine consumption in KP and KPΔNF1 clones normalized by cell count (n = 4 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs). (C) Relative viability of KP and KPΔNF1 cells after 72 h of GPNA treatment assayed by cell-titer glo (relative luminescent units) (n = 4 biological replicates). (D) Cumulative population doublings of KP and KPΔNF1 cells cultured with 2.0 or 0.5 mM glutamine (n = 4 biological replicates). (E) Two patient-derived KRAS;NF1-mutant LUAD cell lines (PDKN1, PDKN2), as well as the control patient-derived KRAS-mutant/NF1-WT LUAD cell line (PDK), were passaged for 14 population doublings prior to collection. The heatmap displays the most significantly upregulated genes in the PDKN1 and PDKN2 cell lines (relative to the PDK control), with the degree of upregulation depicted by color as indicated in the legend. Quantification of the expression of the six key altered genes in cultured PDKN1 cells (passaged for 14 population doublings) vs. their respective original patient tumor samples using qPCR. For bar charts, data presented as means with error bars representing standard deviations (SDs). Quantification of the expression of the six key altered genes in cultured PDKN2 cells (passaged for 14 population doublings) vs. their respective original patient tumor samples using qPCR. For bar charts, data presented as means with error bars representing standard deviations (SDs). (H) Western blotting analysis of Psat1 expression in KP and KPΔNF1 cells infected with sgTom or sgPsat1 following selection. GAPDH was used as a loading control. (I) Cumulative population doublings of KP and KPΔNF1 cells after transduction with sgTom- or sgPsat1-containing vectors (n = 4 biological replicates). (J-L) Cumulative population doublings of patient-derived KRAS-mutant LUAD cell lines that are either (J, K) NF1-mutant (PDKN1 and PDKN2) or (L) NF1-WT (PDK) after selection with sgTom- or sgPsat1-containing vectors (n = 4 biological replicates).
Figure 3(A) Treatment of SARS-CoV-2 (106 PFU) infected Vero-E6 cells with Human recombinant soluble ACE 2 (hrsACE2) (200µg/ml) and remdesivir (Remd. 4µM). Viral RNA level was determined at 15 hours after virus inoculation. (B) Treatment of SARS-CoV-2 (106 PFU) infected human kidney organoids with hrsACE2 (200µg/ml) and/or remdesivir (Remd.4µM)). Viral RNA was determined by qRT-PCR 72 hours after the inoculation of 106 PFU of SARS-CoV-2. (C) Treatment of SARS-CoV-2 (106 PFU) infected Vero-E6 cells with clinical doses of hrsACE2 (5 and 10µg/ml) and remdesivir (Remd. 4µM)). (D) Treatment of SARS-CoV-2 (106 PFU) infected kidney organoids with hrsACE2 (10µg/ml) and remdesivir (4µM). (E,F) Progeny virus released from untreated Vero-E6 cells or Vero-E6 cells treated with clinical doses of hrsACE2 (5 and 10µg/ml) and remdesivir (Remd. 4µM). Progeny was determined (E) 15 hours and (F) 48 hours post-infection (hpi).
a–d, THP1 cells expressing Flag–NLRP3 (a, b) or Flag–ASC (c, d) were analysed for the co-localization of Flag–NLRP3 with the endoplasmic reticulum (ER) marker calreticulin or mitochondria (Mitotracker) using confocal microscopy.f3a-d, THP1 cells expressing Flag-NLRP3 (a, b) or Flag-ASC (c, d) were analysed for the co-localization of Flag-NLRP3 with the endoplasmic reticulum (ER) marker calreticulin or mitochondria (Mitotracker) using confocal microscopy. b, THP1 cells expressing Flag-NLRP3 were analysed for the co-localization of Flag-NLRP3 with the endoplasmic reticulum (ER) marker calreticulin using confocal microscopy. e, THP1 cells were stimulated with MSU or nigericin and then fractionated. The cytosolic, ER, MAM and mitochondrial fractions were analysed by western blotting as indicated in the text. f, THP1 cells stably expressing shRNA against NLRP3 were stimulated with nigericin and subcellular fractions analysed as in e. g, TXNIP associates with mitochondria after NLRP3 inflammasome activation in a ROS-dependent manner. THP1 cells were stimulated with MSU and nigericin, and TXNIP localization was investigated in the presence or absence of the antioxidant APDC. Data shown are representative of two or three independent experiments. Scale bars: a-g, 20 µm.
Figure 7A Top panels, western blot results of HRH1 and phospho-Ser616 DRP1 expression in vehicle control and single-drug treated tumors. Bottom panels show quantifications; significant differences correspond to one-sided t-test (*P = 0.011 and **P = 2x10-3; tumors/group n = 4). Average values are indicated with lilac-colored lines. B Western blot results showing increased HRH1 expression in combinations relative to rapamycin alone, reduced expression of S6 total in the rapamycin-loratadine combination, and raised phospho-Ser616 DRP1 expression in this combination. Bottom panels show quantifications and significant differences with one-sided t-test (HRH1, clorgyline P = 0.011, loratadine P = 0.049, rasagiline P = 0.021; total S6, loratadine P = 4x10-4; phospho-Ser616 DRP1, loratadine P = 0.037; tumors/group n = 4). Average values are indicated with lilac-colored lines. C Graph showing the percentages of MitoSOX red-positive Tsc2-deficient 105K cells treated for 24 hours with DMSO or drugs in vitro with 10% FBS complete medium (except for the condition without L-histidine). Clorgyline 1 μM, loratadine 100 nM, rasagiline 1 μM, and rapamycin 20 nM. Differences relative to rapamycin alone were determined by a two-sided t-test (DMSO P = 6x10-5, clorgyline P = 2x10-5, loratadine P = 6x10-4, rasagiline P = 4x10-5; combination with clorgyline P = 1x10-4, loratadine P = 3x10-5, and rasagiline P = 9x10-4; replicates/condition n = 5; assays n = 2). The bars indicate mean ± SD. D Results of blind histopathological evaluation of Tsc2-deficient 105K tumors treated with vehicle or drugs, in monotherapy or in combination with rapamycin. The histograms show the proportion of defined phenotypic scores: 1+, < 5% of the tumor; 2+, 5-50% of the tumor; and 3+, > 50% of the tumor. Cytological atypia was graded as mild (1+), moderate (2+), or severe (3+). Significant differences relative to rapamycin were determined using Fisher's exact test (epithelioid ***P = 9x10-4 and *P = 0.025; fibrosis, clorgyline and loratadine *P = 0.015, and rasagiline *P = 0.032; glandular, clorgyline *P = 0.030 and rasagiline *P = 0.045; and atypia, loratadine P = 0.010). E Representative images of hematoxylin-eosin-stained Tsc2-deficient 105K tumors treated with vehicle or monotherapies. Tumors treated with rapamycin tend to have a fascicular growth pattern with bundles of spindle cells and foci of fibrosis, whereas most of the tumors of the other treatment groups showed extensive epithelioid morphology. Scale bars are shown. F Representative images of hematoxylin-eosin-stained Tsc2-deficient 105K tumors treated with rapamycin alone or rapamycin combinations. With the addition of a second drug to rapamycin, tumors more frequently tended to show glandular differentiation and less atypia. Scale bars are shown.
Phosphorylation of VAMP8 suppresses fusion but not docking in mast cell secretion.Figure 4A. IgE-induced beta-hexosaminidase release in cells with siRNA knockdown of endogenous VAMP8. Wild type RBL cells in white, VAMP8 in black, VAMP8Ala (T47A, T53A, S54A) in blue and VAMP8Glu (T47E, T53E, S54E) in red. VAMP8 siRNA reduced secretion, which was rescued by VAMP8 overexpression. VAMP8Ala further enhanced secretion, whereas VAMPGlu failed to rescue the siRNA effect (n = 12). Values are mean ± SEM; ***P < 0.001. B. Single-site phosphomimetic mutant of VAMP8 failed to rescue the VAMP8 siRNA effect (n = 11). Values are mean ± SEM. C. Immunocytochemistry of VAMP8 imaged at the plasma membrane using TIRF microscopy, following 4 minutes of antigen stimulation. Endogenous VAMP8 was knocked down with siRNA. Expressed wild type VAMP8 and VAMP8Ala (T47A, T53A, S54A) quickly assumed a uniform plasma membrane stain (due to VAMP8 vesicles fusion), whereas VAMP8Glu (T47E, T53E, S54E) showed a punctate stain, representing vesicles docked at the plasma membrane without fusion. Scale bar 10 µm.D. Identification of puncta in TIRF immunocytochemistry images using MATLAB (see Method). Identified puncta are shown in white in the middle image. Scale bar 10 µm. E. Quantification of relative puncta density in TIRF immunocytochemistry images (n = 15, in total >400 cells per condition). Values are mean ± SEM; ***P < 0.001. F. PKCB forms an incoherent feed-forward loop with two parallel pathways, one suppressing fusion through VAMP8 phosphorylation and one promoting docking through putative phosphorylation substrates. G. Vesicle fusion is prevented by VAMP8 phosphorylation.
Figure 5A. Location of position 236 of TrwB/VirD4 where a FLAG tag was introduced for immuno-labelling of TrwB/VirD4 within the T4SS3-10+D4 complex. Left: structure of hexameric TrwBΔN70/VirD4ΔN70 (PDB entry code 1GKI). Only two diametrically opposed subunits are shown. The position of residue 236 is shown with a red sphere. Right: region of the T4SS3-10+D4 IMC where the location of the residue 236 of TrwB/VirD4 would be expected (indicated by a red sphere) and schematic representation of an anti-FLAG antibody bound to that location. B. Class averages of antibody-bound T4SS3-10+D4FLAG. Class averages of the aligned NS-EM images of the T4SS3-10+D4FLAG with 3-4 particles per class. Red circles indicate the position of the antibody. C. Network of spatial restraints identified by XL-MS mapped onto primary sequences of the TrwB/VirD4 subunit (blue) and the subunits interacting with it, TrwE/VirB10 (grey) and TrwK/VirB4 (yellow). Intermolecular and intramolecular crosslinks are shown in green and orange, respectively. * indicates crosslinks mapped in the structure shown in Figure EV3B.
Figure 9WT and Mic10-KO cells were transfected with a scrambled control (Ctrl.) or siRNA pools against OPA1 (OPA1-KD) for 72 h. (A,B) STED nanoscopy of OPA1 depleted WT (A) and Mic10-KO (B) cells. Cells were immunolabeled for Mic60 and ATPB. (C) TEM of OPA1 depleted Mic10-KO cells. Left: Representative TEM recordings showing CJs marked by arrows in a control cell and a similar view for an OPA1-KD cell. The CMs are colored in green and the OM and IBM together in magenta. Right: Quantification of mitochondria containing CJs or septa junctions. Sections that contained CJ-like structures were analyzed. Of these, the number of sections exhibiting septa or cristae structures (other) was quantified. (D Depletion of ATP5ME in HeLa cells. Cells were transfected with a scrambled control or siRNA pools against ATP5ME for 4 d. TEM recording (left) and quantification of the number of cristae per mitochondrial section (right). (E) Cells depleted of ATP5ME were immunolabeled for Mic60 and recorded with STED. The arrows point to crossing points of the opposite Mic60 distribution bands.