EMBO/sd-character-level-ner huggingface dataset:
ATP8A1 regulates endosomal membrane traffic at REs A COS‐1 cells co‐transfected with GFP‐ATP8A1 and myc‐CDC50A were stained for myc‐tag and TfnR, TGN46, GM130, VPS26, CD63, or LAMP2.Figure 1A COS‐1 cells co‐transfected with GFP‐ATP8A1 and myc‐CDC50A were stained for myc‐tag and TfnR, TGN46, GM130, VPS26, CD63, or LAMP2. mCherry‐Rab11 was expressed in a cell in the top row. Nuclei were stained with DAPI (in blue). Pearson's coefficient between GFP‐ATP8A1 and each organelle marker is shown in the merged image (mean ± SD, n > 20 cells from two experiments). Scale bars, 10 μm. B COS‐1 cells were treated with ATP8A1 siRNAs for 72 h. Cell lysates were prepared and then immunoblotted with anti‐ATP8A1 antibody. α‐tubulin was used as a loading control. C, D COS‐1 cells were treated with ATP8A1 siRNA1 for 72 h. siRNA‐resistant GFP‐ATP8A1 (WT or E191Q) and myc‐CDC50A were transfected 48 h after siRNA transfection. Cells were pulsed with Alexa 594‐Tfn and chased in medium with unlabeled Tfn. During the chase, cells were imaged every 6 min for 54 min. The fluorescence intensity of Alexa 594‐Tfn is shown as percentage of that at 0 min (mean ± SEM, n = 4-6; ***P 0.001; Student's t‐test). See Supplementary Fig S2 for the representative images. E, F COS‐1 cells were treated with ATP8A1 siRNA1 for 72 h. Cells were pulsed with Alexa 594‐CTxB for 5 min and chased for 15 or 60 min. Cells were then fixed and stained for GM130. In (E), representative images are shown. In (F), the Pearson's coefficient between CTxB and GM130 after 60‐min chase is shown. Data represent mean ± SD (n > 26 cells from three independent experiments). ***P 0.001; two‐tailed Student's t‐test. Scale bars, 10 μm.
Figure 2A) Hippocampal neurons in primary culture immunolabeled against three synaptic proteins (the postsynaptic scaffold molecule Dlg4/PSD-95, the presynaptic active zone molecule RIM and the presynaptic vesicle protein SV2A) following 10 hour incubations in carrier solution (control, upper panels) or Lactacystin (10 μM; bottom panels). Scale bar, 10μm B) Average synaptic immunofluorescence following 10 hour incubations in Lactacystin or carrier solution. Numbers within bars represent the number of fields of view analyzed for each condition (~170 synapses/field of view, on average). Error bars, SEM; P values - two tailed unpaired t-test. Note that a statistically significant accumulation was observed only for RIM, whereas immunolabeling of all other proteins was either unchanged or reduced. C) Hippocampal neurons in primary culture were grown in the presence of Lactacystin (10 μM), fixed at two hour intervals and immunolabeled against Shank3/ProSAP2. Two representative fields of view at t=0 and t=24h. Scale bar, 10μm. D) Average synaptic Shank3/ProSAP2 immunofluorescence at increasing times in the presence of Lactacystin. Fluorescence was normalized to mean fluorescence at t=0. Numbers under data points reflect the numbers of fields of view analyzed at each time point. Error bars, SEM.
Figure 2(A) Complementation constructs introduced into SERA6-mTAP:loxP parasites for episomal expression of wild-type (WT) or catalytically-dead (Cys644Ala) SERA6. The papain-like domain (hatched) and myc3 epitope tag are indicated. SERA6 expression from these constructs was under the control of its native promoter. (B) Western blot analysis of egress time-course, comparing maturation of the different SERA6 variants from DMSO- and RAP-treated SERA6-WT-myc3 [SERA6-mTAP:loxP] and SERA6-Cys644Ala-myc3 [SERA6-mTAP:loxP] parasites (representative of 2 independent experiments). Schizonts were sampled at the indicated times following washout of C2, or arrested with E64-d (50 μM), a membrane-permeable derivative of E64 which is a more potent inhibitor of SERA6 maturation (Appendix Fig S1). Samples were probed with anti-myc to detect the myc3-tag, anti-HA to detect the mTAP tag, or anti-SERA6 (S6C1) to detect a different region of SERA6 respectively (see Fig 1A for antibody epitope positions). One of the blots was stripped and re-probed with anti-AMA1 as a loading control. Nomenclature for each protein species was based on apparent molecular mass or predicted composition. (C) Giemsa-stained thin films and western blot of DMSO- and RAP-treated SERA6-WT-myc3 [SERA6-mTAP:loxP] and SERA6-Cys644Ala-myc3 [SERA6-mTAP:loxP] parasites showing that cleavage of host RBC β-spectrin (red arrowhead) does not occur unless a functional copy of SERA6 is present, and is tightly associated with egress (reproducible in 2 independent experiments). Schizonts were sampled at the indicated times following washout of C2. Scale bar, 5 μm. Also see Fig EV2.
Different molecular signatures of mutant and non-mutant epidermal stem cells (EpSC) A.Figure 5Differentially expressed genes (DEGs) from the four subpopulations (bulge HF-SCGFP; bulge HF-SCTom; b-KCGFP; and b-KCTom) from psoriatic DKO*-mT/mG mice in comparison with their control counterparts (Co). The graphs represent the number of genes differentially up-regulated or down-regulated specifically in each group of interest obtained by Venn diagram analysis Heat map for specific genes relative to biological process enriched based on Gene Ontology IDs for DEGs that are: i) exclusively up-regulated in HF-SCGFP; ii) exclusively up-regulated in HF-SCTom; iii) commonly up-regulated in HF-SCGFP and HF-SCTom. Prediction of secreted proteins enriched in GFP+ subpopulations by the analysis of DEG commonly up-regulated in HF-SCGFP and b-KCGFP by ProteINSIDE analysis of RNA-seq. Gene expression of TSLP in different epidermal cell subpopulations sorted at mid-term (D7) or late-term (D30) of psoriasis-like progression in DKO* mice. protein expression of TSLP in different epidermal cell subpopulations sorted at mid-term (D7) or late-term (D30) of psoriasis-like progression in DKO* mice. n=2-3. Quantification of TSLP production in sera of control, DKO* and DKO*K15 mice at day 30 of psoriasis-like disease by ELISA. n = 4 per group.
Knockdown or overexpression of aluRNA impact size of nucleoli and rDNA transcriptionCLSM images showing NPM (immunofluorescence, red) in HeLa cells transfected with control ASO, aluRNA ASO, or treated with transfection reagent only (mock).Figure 4CLSMimages showing NPM (immunofluorescence, red) in HeLacells transfected with control ASO, aluRNAASO, or treated with transfection reagent only (mock). Nuclei were counterstained with DAPI (blue). A fraction of 80 ± 8% of the cells treated with aluRNAASO (n = 143) showed irregular nucleoli or dispersed nucleoli (53% of the cells). Only 26 ± 9% of the cells treated with control ASO presented abnormal nucleoli, with none of them showing a dispersion of nucleolar domains (n = 123), P‐value 0.01, t‐test. CLSM images showing NCL localization (immunofluorescence), aluRNA (RNA FISH) and DNA (DAPI). Transfection of HeLa cells was done as in (A). HeLa cells were transfected as in (A), and nascent RNAs were visualized by pulse labeling with ethynyl uridine (EU) for 30 min. Nuclei were counterstained with DAPI (DNA). Levels of 47Spre‐rRNA in mock‐, control ASO‐ and aluRNAASO‐treated HeLacells. Top: Northern blot. Center: Agarose gel electrophoretic analysis of RNA. Bottom panel: Quantification from RT-qPCR levels of 47Spre‐rRNA normalized to 18SrRNA. Error bars represent the standard deviation (n = 6). **P‐value 0.01, t‐test.
Figure 4Carbonate-washed total membrane fractions from wild type and SPPL2c-/- testis (n=5) were subjected to a label-free quantitative proteome analysis. For each quantified protein, a log2 intensity ratio between SPPL2c-deficient and wild type samples was calculated. Negative values indicate a depletion and positive values an enrichment in SPPL2c-/- samples. In the depicted volcano plot the negative log10 of the p value (y axis) is plotted versus the log2 intensity ratio of SPPL2c-/- versus wild type samples (x axis). All proteins above the significance level of p<0.01 (unpaired Student's t test) are coloured in red. The hyperbolic curve indicates the threshold for a permutation-based FDR correction for multiple hypotheses with p = 0.05 and s0 = 0.1. Quantification of SPPL2c, C11orf94, Syntaxin 8 (Stx8) and Phospholamban (PLN) in the proteomic dataset. HeLa cells were transiently transfected with N-terminally HA-tagged PLN (HA-PLN) and inactive (D/A) SPPL2c-myc The expressed proteins were visualised by indirect immunofluorescence via their HA and Myc epitopes. HEK293 cells were transiently transfected with HA-PLN alone or in combination with active or inactive (D/A) murine SPPL2c Isoform A (D) or Isoform B (E). Substrate processing was analysed by Western blotting with anti-HA. SPPL2c expression was confirmed with the antiserum against the C-terminal epitope. Actin was detected to confirm equal protein loading. Scheme of mutants generated for analysis of determinants within the PLN transmembrane domain (TMD) required for SPPL2c-mediated intramembrane proteolysis. Subcellular localisation of the PLN mutants was analysed by indirect immunofluorescence using anti-HA in combination with anti-protein disulphide isomerase A6 (PDIA6) to stain the ER. The 37-41A and 47-51 PLN mutants were not expressed in relevant amounts. HEK293 cells were transiently transfected with wild type or mutated HA-PLN alone or together with SPPL2c isoform A. Quantification of n=5 experiments as shown in (H). For each PLN variant, the residual PLN in SPPL2c co-expressing cells is depicted as % of PLN in absence of the protease.
Possible intermediate structures of autophagosome.Figure 9Possible intermediate structures of autophagosome. A, A membrane sac under construction. Arrows show gold particles. B and C, Isolation membranes. B, Isolation membrane is detected along the arrows. C, The small arrow shows a semicircular isolation membrane and a large arrow marks its open area. D, Apg8p-residing structures gathered in the area close to the vacuole. TK116 cells were incubated in SD(−N) medium for one hour (A and C), or two hours (B and D). The localization of 3 × HA-Apg8p was detected with anti-HA antibody, followed by the incubation with 5-nm gold- (A) or 10-nm gold- (B-D) conjugated goat anti-mouse IgG. V, vacuole.
a) Weights of mice expressing mutant huntingtin (Tg) and nontransgenic (NT) littermates treated with CCI-779 or with a placebo.figf7(a) Weights of mice expressing mutant huntingtin (Tg) and nontransgenic (NT) littermates treated with CCI-779 or with a placebo. (b) Brain weights of mice expressing mutant huntingtin treated with CCI-779 (black bars) or with a placebo (white bars) and nontransgenic untreated littermates (gray bars) at 8 weeks (CCI-779, n = 3; placebo, n = 3; nontransgenic, n = 7; CCI-779 versus placebo, P = 0.023), 12 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 5; CCI-779 versus placebo, P = 0.90) and 16 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 2; CCI-779 versus placebo, P = 0.22) of age. (c) Ratio of brain weight to body weight of mice expressing mutant huntingtin treated with CCI-779 (black bars) or with a placebo (white bars) and nontransgenic untreated littermates (gray bars) at 8 weeks (CCI-779, n = 3; placebo, n = 3; nontransgenic, n = 7; CCI-779 versus placebo, P = 0.06), 12 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 5; CCI-779 versus placebo, P = 0.38) and 16 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 2; CCI-779 versus placebo, P = 0.56) of age.
Regulatory function of RsaI is not impaired by binding of RsaG.Figure 5(A) Ternary complex formation between RsaI, various mRNAs (glcU_2, HG001_00942 and HG001_1242), and RsaG. The 5'-end labeled RsaI was incubated with increasing concentrations of the target mRNA alone or in the presence of 50 nM of RsaG. The various complexes are notified on the sides of the autoradiographies. (B) RsaG does not form stable complexes with glcU_2 and HG001_01242. Binding assays were done in the presence of 5' end-labeled RsaG and either cold RsaI (300 nM) or increasing concentrations of glcU_2 and HG001_01242. (C) Measurements of the half-lives of RsaI and RsaG in HG001-∆rsaG or HG001-∆rsaI mutant strains using Northern blot experiments. The cells were treated with rifampicin at 4h of growth and total RNAs were extracted after 2, 4, 8, 15, 30 and 60 min at 37°C in BHI. 5S rRNA was probed to quantify the yield of RNAs in each lane using the same samples, which were however run on two different gels. Calculated half-lives are shown beneath the autoradiographies and are the average of 2 experiments. The data were normalized to 5S rRNA.
NAV3 regulates microtubule dynamics and stability.Figure 4The indicated MDA-MB-231 derivatives were transfected with a GFP-EB1 plasmid. Time-lapse images were taken at 2-s intervals, and MT ends were tracked using plusTipTracker. Overlays of four MT subpopulations and representative cells are shown (scale bar, 10 μm), along with the relative proportions of subpopulations (right panel). Data (mean ± SD) were pooled from three experiments. P-values are shown for two groups.Cells from (A) were used to derive representative overlays of MT dynamics maps, depicting displacement and speed (scale bar, 10 μm). Scatter plots of mean displacement and speed of MT tracks are shown. Nine representative cells (three of each experiment) were selected for analysis of MT's mean displacement and speed. The indicated derivatives of MDA-MB-231 cells were photographed, either directly or following immunofluorescence analysis using the indicated antibodies. Nuclei were stained with DAPI (scale bars, 20 μm and 10 μm, left and right columns, respectively). The indicated stable derivatives of MDA-MB-231 cells were analyzed using RT-PCR for the levels of NAV3 expression (right panel). Shown are means ± SD from three independent analyses. Whole-cell extracts were prepared and subjected to immunoblotting with anti-NAV3, anti-acetylated tubulin and anti-α-tubulin antibodies (left panel).
Figure 2A. Schematic representation of TALEN design, its injection into one cell zebrafish embryos and screening at 2 dpf for any phenotypic changes. B. List of sequences showing indels at the veal2 locus at somatic level in F0 zebrafish embryos injected with TALEN arms. C. Schematic representation of out-cross of veal2 heterozygous mutant veal2gib005Δ8/+ and the chromatograms representing the two different types of alleles identified on genotyping of 20 embryos randomly. D. Bar graph representing the number of animals which displayed the hemorrhage phenotype across the progeny derived from breeding of two control gib004Tg(fli1a:EGFP;gata1a:DsRed) zebrafish and progeny derived from an outcross of veal2gib005Δ8/+ zebrafish. Data from three different experiments plotted as mean percentage values ± standard deviation. E. Bar graph representing the number of animals which displayed the hemorrhage phenotype across the progeny derived from an outcross of veal2gib005Δ8/+ zebrafish injected with veal2 RNA and vehicle control separately. Data from three different experiments plotted as mean percentage fold change values ± standard deviation. F-T. Representative images of 2 dpf zebrafish which displayed the rescue of the vascular integrity defects in the progeny of an outcross of veal2gib005Δ8/+ zebrafish upon complementing with the wild type (WT) veal2 RNA. (F-J) Control zebrafish embryos. (K-O) veal2gib005Δ8/+ embryos injected with vehicle control. (P-T) veal2gib005Δ8/+ embryos complemented with veal2 RNA. (F,K,P) bright field. (G,L,Q) mRFP. (H,M,R) Animals stained with O-dianisidine stain. (I,N,S) eGFP. (J,O,T) merged eGFP and mRFP filters. Arrowheads show the presence of hemorrhage due to the vascular integrity defects. (F-H,K-M,P-R) magnification-5X, scale bar-100μm (I-J,N-O,S-T) magnification-20X, Scale bar-50μm.
Sept2 depletion affects mitochondrial morphology.Figure 1(A) Mitochondrial morphology in HeLa cells depleted of Sept2, Sept7 and Sept9. Mitochondria were labeled with mitotracker (green). Scale bar: 10 µm. The inset represents a twofold enlargement. (B) Quantification of mean mitochondrial length in mock treated cells and in cells depleted of Sept2, Sept7 and Sept9. n>250 individual mitochondria from three independent experiments. (C) Mitochondrial morphology in mock treated cells or HeLa cells depleted of Sept2 and then transfected with empty vector, siRNA-resistant HA-tagged Sept2 (Sept2-rescue) or Sept7 (Sept7-rescue). Mitochondria were labeled with mitotracker (shown in green), asterisks mark transfected cells. Scale bar: 10 µm, insets are twofold enlargements. (D) Quantification of mean mitochondrial length in mock treated cells, in cells depleted of Sept2 or in cells depleted of Sept2 and transfected with siRNA-resistant Sept2 (Sept2-rescue) or Sept7 (Sept7-rescue). n>200 individual mitochondria from two independent experiments. (E) Mock treated or Sept2 depleted U2OS cells stained for Sept2 (green) and the Golgi apparatus (GM130, red), the ER (Sec61b-GFP, displayed in red) or peroxisomes (PMP70, red). (F) Quantification of the mean Golgi area (n=40 cells, three independent experiments) (G) Quantification showing the percentage of ER traversing well-resolved mitochondrial tubules (n=15 cells from three independent experiments). (H) Quantification of average peroxisome counts per cell (n=40 cells from three independent experiments). Scale bar: 10 µm, insets are enlarged twofold.
Mannitol-induced transcriptional changes of the selected TFs in proliferating, expanding and mature leaf tissue.Figure 1 The expression of the 20 genes encoding TFs was measured 24 h after mannitol treatment during the proliferating (n = 192 plants), expanding (n = 16 plants) and mature (n = 16) leaf developmental stage. Expression levels in wild-type plants transferred to mannitol-induced stress were compared to those transferred to control conditions at the same developmental stage. Data information: Data are presented as mean ± SEM, n = 4 independent experiments. FC = Fold change. * = FDR < 0.05, Unpaired two-sided Student's t-test.
Figure 5(A) Time course experiment of HuR binding of miR-122 replaced from Ago2miRNPs. A schematic representation of in vitro miRNA binding assay of HuR (left panel). Equal amounts of recombinant HuR (rHuR), after indicated time of the miRNP interaction on FLAG-beads, were immunoprecipitated and HuR associated miR-122 levels were detected by qRT-PCR (right top panel). Ago2western blot detects its presence in the FLAG beads used in the assay and its absence in HuRimmunoprecipitated materials. HuRwestern blot was used to confirm its presence and quantification of its levels in the immunoprecipitated materials, at different time points (right bottom panel). (B) A schematic diagram of in vitro miRNA binding assay of HuR, using wild type full length and delta RRMIII deletion mutant of HuR. Relative amount of miR-122 bound to full length and delta RRMIII after 60 minutes of their incubation with miR-122 containing miRNPs. (C) Coomassie stained gel picture denotes the purity of the recombinant HuR used in the assay. The same proteins were western blotted for HuR to show the specificity of the antibody used for immunoprecipitation. The recombinant P protein and N protein of Chandipura virus was used as negative control to confirm the specificity of 3A2 anti-HuR antibody. (D) HuR western blot in middle right panel confirmed the presence of HuR and the truncated version of it in the immunoprecipitated materials. A representative domain picture of HuR and HuRΔIII are shown in the upper panel. Relative levels of HuR bound miR-122 were determined by qRT-PCR (mean+/- s.e.m., n=3) (middle left panel). Amount of miR-122 recovered from the immunoprecipitated materials were normalized against the amount of HuR or HuRΔIII present in the Immunoprecipitated materials. Ago2 western blot confirmed its presence in the FLAG beads used in the assay while Ago2 was not detected in HuR immunoprecipitated materials (bottom panel). (E) RNA electrophoretic mobility shift assay done with 32P-labeled synthetic miR-122 and recombinant HuR proteins. Radiolabelled miR-122 (100 fmol) was incubated with increasing concentration of FL-HuR (5-100nM) or HuRΔIII (100 nM) and gel shifting of miR-122 were marked by arrowhead. Position of the free probes is marked by an arrow. In the right panel, 1pmol of unlabelled TNF-alpha AU-rich element encoding synthetic RNA was used to compete with miR-122 binding of full length HuR. Amount of HuR or HuRΔIII used for gel shift assay were premeasured by densitometric estimation of coomassie stained gel bands from SDS page. (F) Association of miRNAs with recombinant HuR in vitro. Small RNA pool of Huh7 cell isolated by miRVana miRNA isolation kit were incubated with recombinant HuR in an in vitro binding reaction and HuR associated RNA, recovered from immunoprecipitated HuR after the binding reaction, were quantified along with the input RNA used for binding. The mean Ct values of RNA from samples from three independent binding reactions along with input RNA were measured and plotted (mean+/- s.e.m., n=3). The Input recombinant HuR used in the binding assay and in the immunoprecipitated materials were detected by western blot.
FBXO7 ubiquitinates PSMA2 and regulates proteasome assembly.Figure 5A. Lysates from HEK293T cells, transfected with empty control vector, PSMA2 shRNA A, PSMA2 shRNA B or non-functional PSMA2 shRNA, were subjected to chymotrypsin-like proteasome activity assay. Three independent experiments were included in the analysis (ANOVA, ** p<0.01, *** p<0.001, mean + s.e.m.). B. Lysates from HEK293T cells, transfected with control vector, FBXO7 shRNA or non-functional FBXO7 shRNA, were subjected to a chymotrypsin-like proteasome activity assay. Three independent experiments were included in the analysis (ANOVA, **p<0.01, ***p<0.001, mean + s.e.m.). C. Brain lysates from wild type or homozygous FBXO7 mice were analyzed for chymotrypsin-like proteasome activity. Three independent experiments were included in the analysis (paired t-test, * p<0.05, ** p<0.01, mean + s.e.m.). D. Proteasomes purified from FBXO7+/+ or FBXO7-/- brains were analyzed by native PAGE, followed by in-gel chymotrypsin-like activity assay and immunoblotting with the PSMA2 antibody. E. and F. Lysates from HEK293T cells, transfected with empty control vector, FBXO7 shRNA or non-functional FBXO7 shRNA, were analyzed by native PAGE followed by in-gel chymotrypsin-like proteasome activity assay (LLVY-AMC), in the presence of SDS, and followed by immunoblotting with the PSMA2 (E) antibody. For 5E, ratio of free CPs to RP1+RP2-CP was calculated for each lane and normalized to control average. Quantification includes four (activity) and three (IB) independent experiments (ANOVA, *p<0.05, **p<0.01, mean+ s.e.m.). E. and F. Lysates from HEK293T cells, transfected with empty control vector, FBXO7 shRNA or non-functional FBXO7 shRNA, were analyzed by native PAGE followed by in-gel chymotrypsin-like proteasome activity assay (LLVY-AMC), in the presence of SDS, and followed by immunoblotting with the Rpt6 (F) antibody. For 5E, ratio of free CPs to RP1+RP2-CP was calculated for each lane and normalized to control average. Quantification includes four (activity) and three (IB) independent experiments (ANOVA, *p<0.05, **p<0.01, mean+ s.e.m.). G. HEK293T cells were transfected with empty control vector or the FBXO7 shRNA plasmid and subjected to fractionation using a 10-40% linear glycerol gradient and fractions were analyzed for chymotrypsin-like proteasome activity H. Brain lysates from FBXO7+/+ and FBXO7-/- mice were analyzed on native PAGE followed by in-gel chymotrypsin-like proteasome activity assay and by IB with the PSMA2 antibody. Quantification includes 6 wild type and 7 FBXO7-/- brains (activity) and 5 wild type and 5 FBXO7-/- brains (IB), (unpaired t-test, * p<0.05, mean + s.e.m.).