EMBO/sd-character-level-ner huggingface dataset:
LC3B and GATE‐16 act distinctively in autophagosomal biogenesis.Figure 6(A, B) Control HeLa cells and HeLa cells stably expressing GFP‐LC3B (A) or GFP‐GATE‐16 (B) were transfected with the reciprocal siRNA pools using DharmaFect reagent. After 72 h interval, the cells were incubated for 2 h in EBSS, subjected to immunostaining with anti‐Atg16 after fixation, and analysed by confocal microscopy. Quantification of cells containing Atg16 structures larger than 2 μm (A) or Atg16‐labelled puncta structures (B) from three independent experiments in comparison to control and to G to A mutants (D) is presented at the lower panel. Scale bar: 20 μm. (C) HeLa cells stably expressing silent GFP‐LC3B or GFP‐GATE‐16 were transfected with their siRNA pools using DharmaFect reagent. After 72‐h interval, the cells were incubated for 2 h in EBSS medium and subjected to immunostaining with anti‐Atg16 and anti‐p62 antibodies followed by fixation. The cells were analysed by confocal microscopy. Arrows represent cells, which do not express GFP proteins whereas arrowheads represent cells, which express GFP‐proteins. Quantification of Atg16‐labelled puncta structures from three independent experiments is presented at the right panel. Scale bar: 20 μm. (D) HeLa cells stably expressing GFP‐LC3BG120A or GFP‐GATE‐16G116Awere transfected with either pool of GABARAP/GATE‐16 siRNAs or LC3 siRNAs, respectively, and a non‐targeting siRNA. The cells were starved for 2 h, fixed, and immunostained with anti‐Atg16 and anti‐p62 antibodies. Quantification is presented in (A, B) at the lower panels. Scale bar: 20 μm. (E) HeLa cells stably expressing GFP‐LC3B (left panel) or GFP‐GATE‐16 (right panel) were transfected with the reciprocal siRNA pool or with non‐targeting siRNA and starved for 2 h in EBSS medium. The cells were fixed and their cryo sections were immuno‐labelled using anti‐GFP antibodies and analysed by TEM as described in 'Materials and methods'. Quantification of cells containing Atg16 structures larger than 2 μm (left panel) or the size of Atg16‐labelled structures (right panel) is presented.
Increases in serum placensin and minimal changes in glucagon levels during human pregnancy and in patients with gestational diabetes mellitus (GDM).Figure 5A Immunoblotting of serum placensin from patients at second trimester (1-3), term pregnancy (4-6) using specific placensin antibodies. B Serum levels of placensin and glucagon at different weeks of pregnancy measured using specific ELISA in normal and GDM patients. Numbers in parentheses represent number of patients. C Ages of patients at different gestational stages and BMI values for normal pregnant women and GDM patients.
NSMCE2 SUMO ligase activity is largely dispensable in miceScheme illustrating the Nsmce2‐mutant allele (Nsmce2SD) used in this study, which was generated through introducing the mutations on exon 6 (see ).Figure 3Scheme illustrating the Nsmce2‐mutant allele (Nsmce2SD) used in this study, which was generated through introducing the mutations on exon 6 (see ). Aminoacid and nucleotide sequences of the Nsmce2‐mutant allele. The SUMO ligase‐inactivating mutation used C185S; H187A (SD), which is the same originally described in yeast (Andrews et al, ) and which has been used as a SUMOylation‐deficient model in multiple studies. The nucleotide sequence derives directly from sequencing DNA from Nsmce2SD/SDmice. Immunoprecipitation of SMC5 (and IgG, as a control) from a 48‐h culture of B lymphocytes of wt and Nsmce2SD/SD animals. NSMCE2 and SMC6 levels on the immunoprecipitates were analyzed by WB. The image of the Ponceau staining is shown as a loading control. IF of NSMCE2 foci in MMS‐treated Nsmce2+/+ and Nsmce2SD/SD MEFs. Scale bar, 2.5 μm. Representative picture of 6‐month‐old Nsmce2+/+ and Nsmce2SD/SD littermates. Kaplan-Meyer survival curves of Nsmce2+/+ and Nsmce2SD/SD mice. The P‐value was calculated with the Mantel-Cox long‐rank test.
∆Np63 stability is regulated by USP28 via its catalytic activity.Figure 2Co-immunoprecipitation of exogenous HA-USP28 and FLAG-ΔNp63 in HEK293 cells. Either HA-USP28 or FLAG-ΔNp63 were precipitated and blotted against FLAG-ΔNp63 or HA-USP28. The input corresponds to 10% of the total protein amount used for the IP (ACTIN as loading control). Ni-NTA His-ubiquitin pulldown in control transfected or HA-USP28 overexpressing HEK293 cells, followed by immunoblot against exogenous ΔNp63. The input corresponds to 10% of the total protein amount used for the pull down. Relative ubiquitination of the representative immunoblot was calculated using undefined for normalization. Ni-NTA His-ubiquitin pulldown K48 or K63 in control and HA-USP28 overexpressing HEK293 cells, followed by immunoblot against exogenous ΔNp63. The input corresponds to 10% of the total protein amount used for the pulldown. Relative ubiquitination of the representative immunoblot was calculated using VINCULIN for normalization. Co-immunoprecipitation of exogenous FLAG-USP28 C171A and FLAG-ΔNp63 in HEK293 cells. ΔNp63 was precipitated and blotted against FLAG-USP28 or ΔNp63. The input corresponds to 10% of the total protein amount used for the IP (ACTIN as loading control). Ni-NTA His-ubiquitin pulldown in control, FLAG-USP28 or FLAG-USP28 C171A transfected HEK293 cells, followed by immunoblot against exogenous ΔNp63. The input corresponds to 10% of the total protein amount used for the pulldown. Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization. CHX chase assay (100ug/ml) of control, FLAG-USP28 or FLAG-USP28 C171A transfected HEK293 cells for indicated time points. Representative immunoblot analysis of FLAG (USP28) and ∆Np63 as well quantification of relative protein abundance (ACTIN as loading control). Immunoblot of USP28 and ∆Np63 in transfected HEK293 cells upon treatment with either DMSO or indicated concentrations of PR-619 for 24 hours. Relative protein abundance was calculated ACTIN as loading control.
Two regulatory phases in Msn2 response Msn2-GFP localization following stress (0.Figure 5Msn2-GFP localization following stress (0.4M KCl) in selected strains. Top: The percentage of nuclear cells (Y axis) over time (X axis). Bottom: count of the number of nuclear import events in live-cell time-lapse microscopy at the two relevant periods. addition of the Δmsn5 strain. addition of the Δmsn5 strain. Top: Schematic model of Msn2 localization in different genetic backgrounds and phases of the response. Bottom: Summary of our observations in terms of nuclear localization, Msn2 activity and the effect on gene expression.
Figure 6A, B. PIME cells were treated with various doses of MI-463 for 48 hours, followed by detecting Pbk expression using WB (A) and qPCR (B). qPCR data was from three independent experimets (n=3). **P = 0.0022 (25 nM), *P = 0.0479 (50 nM), *P = 0.0233 (100 nM) (two-tailed unpaired student's t-test). ns, not statistically significant difference. C, D. Menin-null PIME cells were treated with various concentrations of MI-463 for 48 hrs, followed by detecting Pbk expression with WB (C) and qPCR (D). qPCR data was from three independent experimets (n=3). ns, not statistically significant difference (two-tailed unpaired student's t-test). E, F. MI-503 and MI-463 treatment promotes growth of PIME cells (E) and INS-1 cells (F). Three independent experimets (n=3). ns, not statistically significant difference (Two-way ANOVA). G. MI-503 increases proliferation of menin-expressing PIME cells but not menin-null PIME cells. Three independent experimets (n=3). ***P < 0.0001 (Two-way ANOVA). H-J. 5 days of MI-463 treatment increases the expression of Pbk at the protein level (H) and mRNA level (I), as well as the proliferation marker ki67 mRNA levels (J) in mouse primary islets. qPCR data was from three independent experimets (n=3). *P = 0.0105 (I, 50 nM), *P = 0.0287 (I, 100 nM), *P = 0.0150 (J, 25 nM), *P = 0.0273 (J, 50 nM), *P = 0.0314 (J, 100 nM) (two-tailed unpaired student's t-test). ns, not statistically significant difference. K-M. 5 days of MI-503 treatment impacts the expression of Pbk at protein level (K) and mRNA level (L), as well as ki67 mRNA levels (M) in human primary islets (donor number is four). qPCR data was from three independent experimets (n=3). **P = 0.0036 (L, 10 nM), **P = 0.0024 (L, 50 nM), **P = 0.0073 (L, 100 nM), *P = 0.0237 (M, 50 nM) (two-tailed unpaired student's t-test). ns, not statistically significant difference.
Figure 4Heatmap of k-means clustering derived from mean-centered expression values (log2 scale) of DEGs selected from 6000 DEGs (Fig EV4A) obtained from RNA-seq (Fig 2). See also Dataset EV1. FACS analysis of the induced fusion protein (T-V5-GR-2A-H2BGFP) and Blimp1-RFP reporter. Right: Measurements of Blimp1-RFP expression in three different cell fractions are also plotted on the histogram image. Unsupervised hierarchical clustering (correlation-based distance) obtained from RNA-seq in T-induced PGCLCs (T-PGCLCs), in vivo PGCs, and FACS-sorted cell fractions as indicated in Fig 2A-C. PCA of global gene expression in the same dataset as (D). Left: A representative confocal image of a mouse embryo at E7.0 bearing T-GFP and Blimp1-RFP transgenic reporters. Scale bar: 50um. Arrowheads indicate Blimp1-positive nascent germ cells in the posterior proximal epiblast. Right: FACS analysis of dissociated mouse embryos without visceral endoderm. Expression of the indicated genes measured by qPCR in FACS-sorted cells (E) derived from embryos at different developmental stages (E7.0-7.5). Ten FACS-sorted cells in different cell fractions (E) were used for each individual qPCR analysis with two technical replicates. The ∆Ct values from the average Ct values of Arbp and Ppia are shown (n≥3, log2 scale). P values were calculated by two-tailed unpaired t-test.
Figure 8(A) BT-474 cells were treated for the indicated times with the 0.3 μM MK-2206. The cell lysates were analysed by immunoblot using the indicated antibodies. (B) BT474ccells were treated with inhibitors as indicated either as monotherapy or in combination and cell confluency measured on the Incucyte ZOOM every 4 hours for up to 4 weeks. (C) BT-474 cells were injected subcutaneously into athymic Foxn1nu nude mice. Mice were treated with either vehicle (8 mice) or MK-2206 (100mg/kg) (10 mice) or 14h (25mg/kg) (6 mice) or both, MK-2206 and 14h (10 mice) for 24 days. The tumour volume was measured twice a week. Tumourgrowth was represented as the fold change mean ± SEM. (D) All mice were weighed at the end of the treatments. Results are presented as mean ± SD. (E) Plasma concentrations of MK-2206 and 14h were analysed in samples obtained 2-3 hours after the administration of the last dose on the 24th day of treatment. Results are presented as a mean ± SD from three different samples. (F) Tumours were harvested at the end of the experiment, four hours after the last dosage and subjected to immunohistochemistry analysis using cleaved Caspase 3 antibody (clCasp3) . Apoptotic sells were counted in 25 fields per each condition (left panel) and quantified as clCasp3 positive cells/field (right panel). Representative images are shown. Bar 100 μm (G) Tumours were harvested at the end of the experiment, four hours after the last dosage and subjected to immunohistochemistry analysis with the indicated antibodies. Representative images are shown. Bar 100 μm. (H) Tumours were obtain the same way as (G) and subjected to immunoblot analysis with indicated antibodies. Six different tumours were analysed from each treatment group and each line represents one tumour sample.
Figure 3A Comparison of the precursor and polymeric structures of UMOD shows how dissociation of the EHP triggers a major conformational change of the ZP module. This involves a significant rearrangement of the interdomain linker, which not only completely dissociates from ZP-C but also changes secondary structure upon conversion of α1 in the precursor to α1β in the polymer. Molecules are depicted in cartoon representation, with only one subunit of the UMOD precursor homodimer shown; structural elements are colored as in Fig EV1A, with the N- and C-terminal halves of the ZP-N/ZP-C linker colored bright and dark red, respectively. B In the ZP-C domain of the precursor form of UMOD (left panel, teal), the polymerization-blocking EHP β-strand interacts hydrophobically with a short α-helix (αEF) encompassing the FXF motif. Hepsin-mediated cleavage of the CCS of this molecule (chain B/UMOD 2) triggers release of its EHP, which is replaced by α1β from the interdomain linker of a second UMOD subunit (chain A/UMOD 3) already incorporated at the end of a growing filament (right panel). This allows the FXF motif of molecule B to form an intermolecular β-sheet (αEFβ/βF'; upper dashed box) with the ZP-N fg loop of a third, incoming subunit (chain C/UMOD 4, magenta). Another result of the CCS cleavage is that the C-terminus of mature UMOD 2 is freed for interaction with the D8C domain of the same molecule (not shown). Elements are shown as in panel A, with disulfide bonds and glycan residues represented by thick dark grey and thin black sticks, respectively; β-strands are labelled as in the UMOD precursor (Bokhove et al, 2016a). C Hidden Markov model logos, highlighting the conservation of selected residues shown in panels B, D and E. D Hydrophobic interactions stabilize the interface between the E'FG extension of the ZP-N domain of chain A and the ZP-C domain of chain B, corresponding to the dashed box in the lower right part of panel B. E Details of the interface between the ZP-C domain of chain B and the ZP-N domain of chain C, showing a different view of the area boxed in the upper right part of panel B.
Figure 2(A) Sketch of UbLRad23-GFP-95 consisting of the UbL domain of S. cerevisiae Rad23, followed by superfolder GFP and a 95-amino-acid-long tail derived from S. cerevisiae cytochrome b2. (B) In vitro degradation of UbLRad23-GFP-95 by purified S. cerevisiae proteasome. The graph plots the amount of substrate over time, estimated by fluorescence intensity monitored by plate-reader (green) or electronic autoradiography of SDS-PAGE gel bands (red), normalized to the initial substrate amount as described in Methods. (C) Michaelis-Menten analysis of UbLRad23-GFP-95 degradation by different concentrations of purified S. cerevisiae proteasome (green, 10 nM; red, 40 nM). (D) UbLRad23 inhibits UbLRad23-GFP-95 degradation. The initial degradation rates of UbLRad23-GFP-95 at different concentrations of purified UbLRad23 domain are plotted and fitted to the equation describing competitive inhibition. (E) The UbLDsk2 domain is a competitive inhibitor of UbLRad23-GFP-95 degradation. Lineweaver-Burk plot of UbLRad23GFP95 degradation with different concentrations of purified UbLDsk2 domain. (C-E) Proteasomal degradation of UbLRad23-GFP-95 monitored by fluorescence intensity in a Tecan plate reader as described in Methods.
Computational analysis of the TCGA Provisional LIHC data set for expression of PIDDosome components reveals correlation with proliferation markers.Figure 6(A) Genes co-expressed with the PIDDosome components were examined with respect to the associated Hallmark pathways taking positive (pos) and negative (neg) correlation into account. (B) E2F target genes expression was analyzed for correlation with expression of CASP2, RAIDD and PIDD1 using Spearman's correlation coefficient. The central band represents the median, the boxes indicate the 2nd and 3rd quartile, and the whiskers are 1.5 times the interquartile range (n=200 genes). Correlation (Pearson) of CASP2 mRNA with transcript levels of Ki67 in the TCGA data set. Correlation (Pearson) of RAIDD and PIDD1 mRNA with transcript levels of Ki67 in the TCGA data set.
Reduced autophagy after PINK1 knockdown.Figure 22A) SH-SY5Y cells were starved for 2 h in HBSS with Bafilomycin (+Baf) or left untreated in RPMI+5% FCS (- Baf). The LC3-II and actin content were determined by western blotting. A representative blot is shown on the right, showing only LC3-II bands in the Bafilomycin-treated samples. The LC3-II bands of Bafilomycin treated samples were normalized to actin and the relative LC3-II content of nt cells was set as 1. Cells with stable PINK1 knockdown exhibited a reduced LC3-II/actin ratio compared to the control (nt) cells; n = 4, p<0.005. 2B) Cortical neurons from 3 different isolations (10-20 DIV) of WT and PINK1 KO mice were either non-starved or starved for 2 h in HBSS and the LC3-II and actin content was determined by western blotting. A representative blot is shown on the right. The relative LC3-II content of WT and PINK1 KO mice, respectively, was set as 1. Primary neurons showed a strong upregulation of autophagy in reaction to starvation but cells derived from PINK1 KO mice exhibited a reduced LC3-II/actin ratio compared to the WT cells; n = 5, p<0.001. 2C) HeLa cells were transiently transfected with scrambled siRNA or PINK1 siRNA. After 48 h the LC3-II and actin content was determined by western blotting. A representative blot is shown on the right. The relative LC3-II content of cells transfected with scrambled siRNA was set as 1. Transient PINK1 knockdown resulted in a reduced LC3-II/actin ratio compared to the cells transfected with scrambled siRNA; n = 6; p<0.005. 2D) HeLa cells were transiently transfected with PINK1-GFP, PINK1G309D-GFP or GFP. After 24 h cells were starved for 2 h in HBSS. Afterwards the LC3-II and actin content were determined by western blotting. A representative blot is shown on the right. The relative LC3-II content of cells transfected with GFP was set as 1. Transient PINK1 or PINK1G309D overexpression resulted in an increased LC3-II/actin ratio compared to cells transfected with GFP; n = 3, PINK1: p<0.01; PINK1G309D: p<0.05.
Linkage analysis and homozygosity mapping.Fig 2(A) A family comprising six animals and one very distantly related case were available for the initial mapping of the disease locus. We performed parametric linkage analysis for a recessive trait in the family and homozygosity analysis across the three cases. (B) The analyses yielded 25 linked genome segments (orange) and 11 homozygous genome segments (red). Only three regions on chromosomes 11, 13, and 20 showed both linkage and homozygosity and were considered the critical intervals (arrows).
Model of T6SS binary effector delivery.Figure 6The toxin, VP1390, and its co-effector, VP1388, are loaded together onto the T6SS spike and are delivered into the periplasm of a neighboring prey cell. If the prey cell expresses the cognate immunity protein, VP1389, then it can antagonize the attack (kin). Otherwise, the VP1390 toxin acts in the prey periplasm, leading to cell lysis (non-kin). IM, inner membrane; PG, peptidoglycan; OM, outer membrane; Cyto, cytoplasm.
Figure 6A. Alignment of SHP-box sequences from human UFD1, human p47, S. cerevisiae Dfm1, and S. pombe Rbd2 C-terminal SHP-box (aa 242-251). Asterisks denote residues identical in all sequences, colons mark conservative substitutions, and dots mark semiconservative substitutions. B. Recombinant proteins GST-fused Rbd2 C-terminus (Rbd2200-251), Dsc5 UBX (Dsc5323-425), Dsc2 UBA (Dsc2298-372), and GST-HA-V5 control were bound to GST magnetic beads and incubated with S. pombe cytosol fraction from wild-type cells. Equivalent amounts of unbound and bound fractions were probed for anti-Cdc48, anti-ubiquitin and anti-GSTIgG. C. GSTpulldown assay was performed as in (B) using truncated forms of Rbd2 C-terminus; Rbd2200-251, Rbd2200-225, Rbd2225-251 and Rbd2200-240. Unbound (1x) and bound (5x) fractions were analyzed by western blotting D. Single residue mutants of conserved glycine residues in Rbd2200-251 (G244R and G246R) were tested for Cdc48 binding as in (C). Western blots (B-D) were imaged using chemiluminescence.
Netrin-3 expression in cancer cell lines Exploring the expression by RNA-sequencing of netrin-1 and -3 among 675 cancer cell lines spanning 16 cancer types.Figure 1A. Expression heat map of netrin-3 and netrin-1. Receptors of netrin-1 and putative receptors of netrin-3: DCC, UNC5-A, -B, -C, -D, Neogenin, APP, CD146 are presented. The color legend (right-hand side) describes all tumor types included in the heat map. Asterisks indicate the two main pathologies in which netrin-3 expression is detectable. C. Percentage of cell lines positive for netrin-1 and/or -3 expression. Co-occurrence of expression was calculated using Fisher's exact test. P-value is indicated in the table. B. Analysis of netrin-1 and -3 expression in tumor cell lines and ranked according to netrin-1 expression
Figure 7A. Timeline for injection of mice with Stx2a and OSMI-1. Mice were pre-treated with OSMI-1 or vehicle alone beginning 1 day before injection of Stx2a. Body weight changes and mortality were monitored every day for 1 week to determine the survival rate. Blood and tissue samples were collected at day 3 after Stx2a challenge. B. Survival rate of mice challenged once with Stx2a (132.5 ng/kg) and injected daily with two doses (300 or 1000 µg/mouse) of OSMI-1 (n = 10, 5 per dose) or vehicle (n = 10). The effect of OSMI-1 (1000 µg/mouse) was compared with that of the vehicle control (a log-rank test with GraphPad Prism software). C. Chemical and hematological analyses of blood pooled from three mice at day 3 after Stx2a challenge. Data are presented as mean ± S.E.M. (n = 3 biological replicates from nine mice, two-tailed Student's t-test). The effects of OSMI-1 (1000 µg/mouse) were compared with those of the vehicle control. *P < 0.05; **P < 0.01; and ***P < 0.001 D. Schematic illustration of proposed protective mechanism from Stx-induced apoptotic and inflammatory responses through inhibition of O-GlcNAcylation.
Brain MRI study of the proband and parents.Figure 1A-F Brain MRI of the proband at 23 years. (A) Sagittal fast spin echo (FSE) T1-weighted image shows thinning of the corpus callosum, dilation of the IV ventricle and of the cisterna magna and reduced volume of vermis and brainstem; two lacunar lesions are evident in the dorsal pons. (B) T2-weighted axial fluid-attenuated inversion recovery (FLAIR) images show hyperintense periventricular white matter with several lacunar lesions also in the basal ganglia and thalami. (C) MR angiography 3D TOF (time of flight) reconstruction shows two saccular aneurisms in the right M1 (ø 4 mm) and left M2 (ø 2.5 mm) segments of middle cerebral arteries (arrows). (D) A recent ischemic hyperintense lesion is detected on diffusion tensor imaging (DTI) in the right side of the dorsal pons. Severe cavitations and lateral ventricles dilation (left > right) on FLAIR images (B, E) and diffuse microbleeds as small hypointense foci on SWI are visible (F) in the same slices of (E). R = right, L = left. G, H Brain MRI scans of the asymptomatic parents of the proband show multiple focal hyperintensities on T2-weighted images in the periventricular and subcortical cerebral white matter, expression of gliosis secondary to chronic small vessel ischemic changes, more evident in the father (G), 56 years, than in the mother (H), 54 years.
Figure 5A. Diagram of direct simultaneous measurements of the presynaptic capacitance jump (Cm) and Ca2+-currents (ICa) evoked by depolarization of a patched nerve terminal. B. Example traces of the presynaptic capacitance and Ca2+-currents at the calyx terminals induced by a 20 ms step depolarization from −80 mV to +10 mV with an extracellular solution containing 2 mM Ca2+, and an internal pipette solution containing a standard 0.05 mM BAPTA. C. Summary graphs of the depolarization-induced capacitance jump that monitors synaptic vesicle exocytosis (left), and the peak Ca2+-current density (right). D, E. Same as (B, C), except that 0.5 mM EGTA was added in addition to 0.05 mM BAPTA to the patch pipette internal solution. F, G. Same as (B, C), except that 1.0 mM EGTA was added in addition to 0.05 mM BAPTA to the patch pipette internal solution. H, I. Same as (B, C), except that 10.0 mM EGTA was added in addition to 0.05 mM BAPTA to the patch pipette internal solution. J, K. Summary plots of the relationship between the total capacitance jump (J) or between the Ca2+-current density (K) and the concentration of EGTA in the patch pipette.