EMBO/sd-character-level-ner huggingface dataset:
Figure 5: Immunization scheme. *monitoring: diabetes symptoms observed (red bar). Blood glucose levels of InsA-peptide immunized mice (n=3) and CI (n=7) mice were assessed at indicated days post immunization. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test, all comparisons were not significant. (C, F) Urine glucose levels of InsA peptide immunized (n=3) and CI (n=3) mice were monitored at indicated days post immunization. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test, *P < 0.05. (D, E) Serum anti-Insulin-immunoglobulin titers of mice immunized with InsA-peptide (day 59, day 78: n=3, day 49: n=7) and control-immunization (CI: CpG only, n=3) measured by ELISA at indicated days (coating: Insulin). Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Mann-Whitney-U test (D) and repeated measure ANOVA test (E). *P < 0.05, **P < 0.01.
Figure 5A. Injection schema for blood-retina barrier assay in adult Tspan12-/- mice with established retinal vascular defects. B. Quantification of extravasated FITC-dextran in retinal lysates from WT and Tspan12-/- mice injected with isotype control antibody or F4L5.13. Data are presented as mean± SEM, n=6 retinas from 3 mice per group. Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test (*p<0.05 as compared to Tspan12-/- control treatment). C. Quantification of the vascularized area in adult Tspan12-/- mice injected with isotype control or F4L5.13. Data are presented as mean± SD, n=4 retinas (4 fields of view per retina) from 2 mice per group. p-value was calculated by Student's t‐test. D D. Flat-mounted retinas of adult Tspan12-/- mice injected systemically with sulfo-NHS-biotin show that F4L5.13 partially restored BRB function. PECAM was used to stain endothelial cells. Images are representative of 4 retinas per group. Scale bar: 500 µm. E. Maximum intensity projection of adult Tspan12-/- retinas after mice were injected with F4L5.13 or isotype control. Endothelial cells were stained with IB4-Alexa 647. Images are representative of 4 retinas per group. Scale bar: 100 µm.
Figure 9A. Schematic representation of Puromycin- Proximity Ligation Assay. Concentration used: 2ng/μL (Puromycin). Abbreviations: TUBB3, tubulin beta 3 class III; PLA probes, Proximity Ligation Assay probes. B. Schematic representation of the experimental paradigm. Representative images of local translation events of TUBB3 in the growth cone. Dashed white line delineates the growth cone. Each white arrow points to a puro-PLA puncta. Abbreviations: TUBB3, tubulin beta 3 class III; Puro, puromycin. Scale bars: 5 μm. Quantification of number of puncta per growth cone. Each data point corresponds to one growth cone. Total number of growth cones analyzed: 30 (PBS), 36 (Sema3A). n= 2 independent experiments. Values are median with interquartile range. D. Schematic representation of the experimental paradigm. Concentrations used: 2 μM co-MO; 2 μM MOs-3p; 200ng/mL Sema3A. Representative images of local translation events of TUBB3 in the growth cone of transfected axons. Dashed white line delineates the growth cone. White arrows represent number of puro-PLA puncta per growth cone. Abbreviations: co-MO, control morpholino. Scale bars: 5 μm. E. Quantification of number of puncta per growth cone. Each data point corresponds to one growth cone. Total number of growth cones analyzed: 32 (co-MO + PBS), 45 (co-MO + Sema3A), 27 (MOs-3p + Sema3A). n=2 independent experiments. Values are median with interquartile range. Abbreviations: co-MO, control morpholino; ns, not significant. F. Schematic representation of the experimental paradigm. Concentrations used: 4μM coMO; 2μM MOs-3p + 2μM co-MO; 2μM MOs-3p + 2μM MOs-TUBB3; 200ng/mL Sema3A. Abbreviations: co-MO, control morpholino; TUBB3, tubulin beta 3 class III. G. Percentage of collapsed growth cones in axons transfected with co-MO, MOs-3p + co-MO and MOs-3p + MOs-TUBB3. Each data point corresponds to one independent experiment. Total number of growth cones counted: 152 (co-MO), 155 (MOs-3p + coMO), 179 (MOs-3p + MOs-TUBB3). n= 3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino; TUBB3, tubulin beta 3 class III. H. Working model: Pre-miR-181a-1 is transported along RGC axons tethered to CD63-positive late endosomes / lysosomes. Under non-stimulated conditions TUBB3 undergoes basal translation in the axonal compartment. Upon Sema3A exposure, pre-miR-181a-1 is locally processed and the expression level of the concomitant newly generated miRNAs increase locally within the growth cone. miR-181a-5p, the predominant mature miRNA generated from pre-miR-181a-1 targets TUBB3, thereby silencing protein synthesis through LPS inhibition (LPS-I). Abbreviations: ns, not significant; LE/Ly, late endosomes / lysosomes; LPS, local protein synthesis; LPS-I, LPS inhibition; TUBB3, tubulin beta 3 class III.
Figure 5A Scheme of Bem1 and Cdc24 proteins indicating the relative position of the mutations in the lipid tethering motifs (black x). B Percentage of full-length Cdc24 and cdc24 ph domain mutant (cdc24 ph) associated with liposomes containing 75% PC 20% PS and 5% PI4P. C Percentage of Cdc24 and cdc24 ph mutant associated with liposomes of the composition shown in (B) in the presence of the indicated bem1 protein. Note how additive mutations in the Bem1 lipid binding sequences reduce the percentage of Cdc24 associated with the liposomes. D Percentage of Cdc24 and cdc24 ph associated with liposomes of the composition shown in (B) as a function of the indicated bem1 protein concentration. The curves denote the regression fit of the data to equation 2 in the materials and methods. E Ten-fold serial dilutions and subsequent colony formation of the indicated mutant cells at the indicated temperatures. F DIC images of the indicated bem1 and cdc24 mutants showing the increased morphological defects ensuing from loss of lipid tethering in the bem1 and cdc24 mutants. G Western blots probed with anti-HA antibody to detect Cdc24 or cdc24 ph tagged with 3XHA in the indicated mutant strains (top panel). Detection of Ade13 was used as a loading control (bottom panel).
a) Immunoblot analysis of ASC immunoprecipitates (IP; left) and total lysates (right) of differentiated THP-1 cells left untransfected or transfected for 2 h with poly(dA:dT) (1.figf5(a) Immunoblot analysis of ASC immunoprecipitates (IP; left) and total lysates (right) of differentiated THP-1 cells left untransfected or transfected for 2 h with poly(dA:dT) (1.5 μg/ml), probed for beclin-1 and ASC (below), then stripped and reprobed for p62 (above). (b) Confocal microscopy of differentiated THP-1 cells treated as in a, immunostained for p62 (green) and ASC (red); arrows indicate colocalized proteins. (c) Confocal microscopy of differentiated THP-1 cells treated for 2 h with LPS (500 ng/ml) and ATP (3mM), immunostained for p62 (green) and ASC (red): left two images, merged channel volume and surface rendering of a three-dimensional reconstruction; right four images, enlargement of images at left (yellow arrow), with arrows indicating removal of part of the signal to reveal the red signal (ASC) inside the green-stained area (p62). (d) Immunoblot analysis of lysates of cells transfected twice with siRNA with scrambled sequence (−) or siRNA specific for beclin-1 or p62; numbers below lanes indicate band intensity (as in Fig. 1b). (e) Immunoblot analysis of the inflammasome and autophagosome fractions and total lysates of differentiated THP-1 cells transfected as in d, with poly(dA:dT) included in the second transfection; lysates were prepared 4 h after the final transfection. (f) Immunoblot analysis of supernatants and lysates of differentiated THP-1 cells treated as in e; supernatants were collected after culture for 6 h in serum-free medium. Scale bars (b,c), 10 μm. Data are representative of at least two experiments.
Figure 3(A) In situ hybridization of E10.5 embryos using Pax3 probe. Pax3 expression (brown signal) was observed in limb bud and demomyotome (DM), respectively. Cells labeled with Pax3 were indicated by red arrow. Scale bars, 200 μm. (B) Gross appearance of E14.5 embryos of indicated genotype. Scale bars, 2.5 mm. (C) HE staining of forelimb, hindlimb, and paraspinal muscle of E14.5 embryos. Scale bars, 100 μm. (D) Gross appearance of E16.5 embryos of indicated genotype. Scale bars, 2.5 mm. (E) HE staining of forelimb, hindlimb, and paraspinal muscle of E16.5 embryos. n = 3, scale bars, 200 μm. (F) Anti-Ki67 antibody was used to label proliferative myoblasts (red fluorescence) with DAPI labeled nucleuses (blue fluorescence) in paraspinal muscle. Scale bar, 25 μm. (G) Bar graph showing statistical analysis of positive rate of Ki67-labeled nuclei, n = 3 with more than 150 cells analyzed per n, **P < 0.01, by chi-square test (χ2 test). WT means wild types, Hetero means heterozygotes, and Homo means homozygotes.
Figure 3Mitochondrial preparations of both Hek293T (left) and HeLa (right) cells transiently transfected with an HA-tagged constitutively active Akt T308D/S473D (Akt D/D) construct were analyzed by western blotting. GAPDH: cytosolic marker; TIM23: mitochondrial marker Resting mitochondrial calcium levels in the control (ctrl) and Akt D/D-expressing HeLa cells evaluated by ratiometric imaging of the mitochondrial-targeted GCaMP6m (n=4 independent experiments; 44-49 cells) Schematic model of the HA-tagged constitutively active Akt form targeted to the mitochondrial intermembrane space (mt-Akt D/D). Both the expression and the mitochondrial localization of the chimera were assessed by immunofluorescence. TOM20 was used as a mitochondrial marker. Merged images are indicated (merge). Scale bar 10 μm Hek293T cells were transfected with the indicated constructs. The GFP-tagged MICU1 was immunoprecipitated from the whole-cell lysate, and the precipitated proteins were immunoblotted with the HA and phosphorylated (S473) Akt (p-Akt) antibodies Hek293T cells were transfected with the indicated constructs. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blotting Resting mitochondrial calcium levels in control (ctrl) and mitochondrial-targeted Akt D/D (mt-Akt D/D)-expressing HeLa cells, evaluated through ratiometric imaging of the mitochondrial-targeted GCaMP6m (n=4 independent experiments; 43-47 cells) Resting mitochondrial calcium levels in ShRNA MICU1 HeLa stable cells transfected with the indicated constructs and evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m (n=3 independent experiments; 38-50 cells) Representative kinetics (H) and analysis (I) of aequorin-based [Ca2+]m measurements in permeabilized ShRNA MICU1 HeLa stable cells transfected with the indicated constructs and challenged with 400 nM buffered [Ca2+] (n=3 independent experiments).
Rapamycin reduces the ER Ca2+-leak rate.Figure 3A-B) Western-blot analysis for luminal Ca2+-binding proteins in HeLa cells treated with the indicated concentrations of rapamycin (Rapa) for 5 h: calreticulin (CRT) (A) and BiP/Grp78 (BiP) (B). Upper panels: representative Western blots; lower panels: quantification of the protein/GAPDH ratio (n = 4). C) Western-blot analysis for SERCA2 in HeLa cells treated with the indicated concentrations of rapamycin for 5 h. Upper panels: representative Western blots; lower panel: quantification of the SERCA2/GAPDH ratio (n = 4). D) Representative plot showing the decrease in ER 45Ca2+ content (logarithmic scale) in a Ca2+-free efflux medium without ATP as a function of time in permeabilized HeLa cells pretreated for 5 h with 1 µM rapamycin or with DMSO. The passively bound Ca2+ was determined by loading the cells with 45Ca2+ in the presence of 10 µM of the Ca2+ ionophore A23187 and then subtracted from the stored 45Ca2+. The ER Ca2+-leak rate can be estimated as the rate of decline of the ER 45Ca2+-store content as a function of time. E) Quantification of the mean 45Ca2+-store content at the beginning of the measurement (t0) (n = 5). F) Quantification of the mean slope of the curve in D after transformation to a linear scale, which is a measure of the 45Ca2+-leak rate (n = 5). * p<0.05; ** p<0.01, paired Student's t-test.
ACE2 expression is upregulated in human primary keratinocytes exposed to IFNα and IFNγ.Figure 8. ACE2 expression is upregulated in human primary keratinocytes exposed to IFNα and IFNγ.Primary keratinocytes from 3 donors were either untreated (control) or exposed to cytokines IFNα, IFNγ. 24 hrs after treatment, total RNA was extracted for subsequent microarray analysis. ACE2 expression data was retrieved from GEO microarray database (NCBI Accession: PRJNA153023; GEO: GSE36287).
Figure 8During mitotic progression, β-catenin binds to Plk1 PBD in a phosphorylation-independent manner and localizes at the midbody/contractile ring of telophase cells (β-catenin p-S60 is surrounded by actomyosin contractile ring during early telophase). The Ser60 residue of β-catenin is phosphorylated by bound Plk1, which recruits Ect2 to the midbody and facilitates the formation of the β-catenin-Plk1-Ect2 complex. The resulting complex induces RhoA activation and mediates the progression of cleavage furrow ingression and subsequent cytokinesis steps.
Therapeutic drugs ameliorate behavioral phenotypes of KO mice A.Figure 4A. Fear-conditioning test. Freezing (%) was monitored in the cued (top) and contextual (bottom) tests before (pre) and after (post) administration of clozapine (left) or haloperidol (right). Data are presented as means ± S.E.M. (n = 7-9/group). *, p < 0.05; ***, p < 0.001 (Tukey test); #, p < 0.05 (t-test, KO/saline vs. KO/clozapine). B. Home cage-activity test. Spontaneous locomotor activity of mice chronically fed chow with or without 0.24% (w/w) lithium carbonate for two months was monitored for 6 days in the home cage.
Figure 4(A) The molecular species-selectivity of PS-to-PE conversion is recapitulated in wild type BY4741. WT cells overexpressing SCT1 vs. control were pulsed for 20 min with 2H3-serine prior to lipid extraction and ESI-MS/MS analysis of 2H3-PS (*PS) and 2H3-PE (*PE). Data is presented as mean of 4 biological replicates with the individual values indicated. Underlying data for this figure can be found in Dataset EV2. (B) Acyl chain composition of WT and psd1∆ cells overexpressing SCT1 (pSCT1) vs. empty vector control (pEmpty). Data is presented as mean ± SD (n>10). **** p < 0.0001, multiple t-test. (C) Ratio of the proportions of saturated over unsaturated acyl chains (SFA/UFA) of WT, psd1∆, crd1∆ and psd2∆ overexpressing SCT1 vs. empty vector control. Data is presented as mean ± SD (n≥3). ** p<0.01, **** p < 0.0001, unpaired two-tailed t-test. (D) OLE1 transcript levels in WT and psd1∆ transformed with pSCT1 vs. pEmpty as determined by RT-qPCR. Data were normalized to endogenous ACT1 mRNA, and to the average of WT pEmpty (n=3).
Figure 6CFTR splicing assay was examined in cells expressing various TDP-43 constructs by PCR analysis. The numbers represented the relative ratios of exon 9 exclusion (-) vs. inclusion (+) (top panel). Both endogenous (endo.) and exogenous (exo.) TDP-43 protein levels in the indicated samples were examined by immunoblotting (bottom panel). CFTR splicing assay was performed in cells with knockdown of either OGT, TDP-43, or combination. ACTB levels served as loading controls (top panel). The indicated protein levels were examined by immunoblotting (bottom panel). CFTR splicing assay was examined in siTDP-43 cells overexpressing the WT or mutant OGT by PCR analysis. ACTB levels served as loading controls (top panel). Various protein levels were examined by immunoblotting (bottom panel). Representative gel images for alternative exons in Dnajc5 and Sort1 genes in Neuro 2a cells expressing the indicated Flag-TDP-43 constructs were shown (top panel). TDP-43 protein levels were examined by immunoblotting (bottom panel). The relative ratios of exclusion to inclusion (-/+) were quantified. Neuro-2a cells expressing doxycycline-inducible shRNA knockdown of TDP-43 were transfected with the indicated TDP-43 constructs, and the alternative exons in Dnajc5 and Sort1genes were examined by PCR. Representative gel images were shown (top panel). TDP-43 protein levels were examined by immunoblotting (bottom panel). The relative ratios of exclusion to inclusion (-/+) were quantified. Representative PCR analysis to examine expression levels of the spliced mRNA isoform containing exon 2a of STMN2 in SH-SY5Y cells with the indicated constructs (top panel). TDP-43 protein levels were examined by immunoblotting (bottom panel). Quantification of the relative levels of truncated STMN2. Quantification of the relative mRNA levels of STMN2. Results were normalized to expression of the gene encoding ACTB and were quantified by the change-in-threshold method (ΔΔCT).
Figure 5Rab8a (T22N) protein accumulated in BAG6-knockdown cells. HeLa cells were transfected with siRNA duplexes for BAG6 or control siRNA. At 48 h after siRNA transfection, Flag tagged-Rab8a (T22N) was expressed in the cells. At 24 h after Rab8a (T22N) transfection, the cells were chased with 50 μg/mL CHX and harvested at the indicated time after CHX addition. Actin was used as a loading control. Anti-Flag blot signals in the control or BAG6 siRNA-treated cells were quantified, and relative signal intensities after CHX addition were calculated. The value of the Flag signal at 0 h was defined as 1.0. Note that all signal intensities of the Flag-tag were normalized by that of actin, a loading control, in each sample. Polyubiquitin modification of Rab8a was abolished in BAG6-knockdown cells. Flag-Rab8a (T22N) immunoprecipitates were blotted with an anti-polyubiquitin antibody (FK2, left panel). As a negative control, siRNA#1scr was used. Anti-polyubiquitin signals co-precipitated with Rab8a (T22N) (a representative example is shown in the left panel) were quantified (right panel). Note that the intensities of the co-precipitated polyubiquitin signal were normalized both by the input ubiquitin-signal and bait Flag-signal. Defective distribution of the endosomal protein TfnR in HeLa cells with the excess accumulation of the inactive form of Rab8a. Right panel shows a merged image of TfnR staining (shown as green), Rab8a (T22N) staining (magenta), and Hoechst 33342 nuclear staining (blue).
Figure 7A HEK293T cells were transfected with plasmids expressing HA-tagged SHP2 and GFP-tagged TLR7. After 48 h, cells were left unstimulated or stimulated by IMQ (10 µg/ml) for 30 min. Cell lysates were immunoprecipitated by anti-HA or anti-GFP and probed by indicated antibodies. B PMA-differentiated THP-1 cells were unstimulated or stimulated by IMQ (10 µg/ml) for 30 min. Cell lysates were immunoprecipitated by anti-TLR7 or anti-SHP2 and probed by indicated antibodies. C BMDMs derived from wild-type mice were stimulated by IMQ (10 µg/ml) for 30 min. Cell lysates were immunoprecipitated by anti-SHP2 and probed by indicated antibodies. D Representative images of confocal microscopy performed with PMA-differentiated THP-1 cells treated with IMQ (10 µg/ml) for indicated durations. Scale bar: 20 µm. E Immunoblotting of HEK293T cells co-transfected for 48 h with GFP-TLR7, plus HA-SHP2, HA-SHP2 mutant vectors, followed by immunoprecipitation with anti-GFP beads. F PMA-differentiated THP-1 cells were infected with vector or SHP2 lentivirus and then treated with or without R848 (10 µg/ml). Subcellular fractionation was performed, and cytoplasmic and cell membrane proteins were probed by respective antibodies. G, H Immunoblot analysis of TLR7 and SHP2 in the isolated ER, Golgi and endosome from PMA-differentiated THP-1 cells with either SHP2 overexpressed (G) and SHP2 deficiency(H). I Skin sections from psoriatic patients and healthy controls were immune-stained for TLR7 together with a marker of the early endosome (EEA1) or late endosome/lysosome (LAMP1) prior to analysis by confocal microscopy, showing TLR7 localization in different organelles (arrows). Scale bar: 50 μm.
Acute inhibition of ribosome synthesis and function in vivo reduces translation of components of the translational machinery and decreases the abundance of polysome-associated, energy metabolism-related mRNAs(A) Western analysis for on-target effects for Everolimus (EV, P-RPS6) and CX-5461 (p53).Figure 1(A) Western analysis for on-target effects for Everolimus (EV, P-RPS6) and CX-5461 (p53). Each lane represents equal amounts of protein from lymph node tissue isolated from a single mouse that received drug vehicles (everolimus vehicle: 1% methylcellulose; CX-5461 vehicle: 25 mM NaH2PO­­­4) (V/V; mouse #1-6), 5 mg/kg everolimus (EV; mouse #7-12), 35 mg/kg CX-5461 (mouse #13-18) or both drugs (CX-5461+EV; mouse #19-24) for 2 hours (n=6 per treatment group). Actin was used as a loading control. (B) A schematic illustration of the polysome profiling analysis workflow: cytoplasmic lysate was layered on top of a linear 10-40% sucrose gradient, ultracentrifuged (36,000 rpm, 2¼ hours, 40C) and fractionated using the Foxy Jr Fraction Collector with constant monitoring of absorbance at 260 (A260) nm by an ISCO UA-6 Absorbance Detector. Fractions (one fraction per minute: 800 µL per tube) corresponding to polysomal mRNAs that were bound by four or more ribosomes were pooled and analyzed by RNA-seq followed by data analysis using anota2seq or limma. (C) Enrichment analysis by MetaCore® GeneGO of genes in "translation up" (red) and "translation down" (blue) categories identified by anota2seq analysis comparing lymph node cells isolated from mice in CX-5461+EV treatment group with the V/V group (n=6). Ratio" values were obtained by dividing the number of significant genes in our data assigned to a molecular process by the total number of genes in the process in MetaCore®'s database. (D) Genes implicated in "Translation: initiation" and "Translation: Elongation-Termination" processes based on Fig 1C (E) Activity levels of key biological processes involved in cellular growth, proliferation and metabolism based on single sample gene set enrichment analysis (ssGSEA) of indicated comparisons. Percentage increase (red) or decrease (blue) in the activity levels of key biological processes involved in cellular growth, proliferation and metabolism based on ssGSEA of indicated comparisons.
Figure 1Facial phenotypes of patient A at 3.5 years of age, showing coarse facial features, broad ear lobes, macroglossia and hypertrichosis. Radiograph of the hand of patient B at 21 months of age demonstrating rough trabecular structure and thin corticalis as signs of dysostosis multiplex. Ovoid vertebrae visualized by spine radiographs of both patients. T2-weighed brain MRIs of patient A (D1-3) at 28 months of age and patient B at (D4-6) at 16 months of age, showing global brain atrophy as well as periventricular and deep white matter hyperintensities. Additional MR investigations of patient A at 28 months of age. (E) T1-weighed coronal images demonstrating thinning of the posterior corpus callosum and reduced volumes of the cerebellar hemispheres and vermis. (F) Magnetic resonance spectroscopy of the white matter showing a decreased N-acetylaspartate (NAA) peak at 2.02 ppm (arrow). (G) T1-weighed MRI of the spinal cord showing contrast enhancements around the conus medullaris (arrowheads). May-Grünwald-Giemsa-stained bone marrow smear from patient A with myelopoietic cells containing dense granules (arrowheads), suggestive of a lysosomal storage disorder.