EMBO/sd-character-level-ner huggingface dataset:
Potent reduction of pTau proteins by AAV‐mediated TFEB expression.Figure 1A Immunofluorescence staining of cortex of 4‐month‐old rTg4510 mice either untreated (Tau) or injected with TFEB (Tau + TFEB) at P0 using phospho‐Tau antibodies AT8 (S202/T205) and PHF1 (S396/S404) or conformation‐specific antibody MC1 and counter‐stained with DAPI. AAV‐TFEB was injected at P0 and mice were analyzed at 4 months. Scale bar: 200 μm. B Quantification of staining intensities. Student's t‐test was performed to analyze the significance. n = 5 mice/genotype/treatment group. **P = 0.0011, ***P = 6.85 × 10−5, and ***P = 9.33 × 10−5 for AT8, PHF1 and MC1, respectively. Each bar represents average ± s.e.m. C Western blot analysis of detergent‐extracted brain lysates using anti‐total Tau, Tau1, AT8 or MC1 antibodies. WT: wild‐type; WT + TFEB: wide‐type mice injected with TFEB; Tau: rTg4510 transgenic mice; Tau + TFEB: rTg4510 transgenic mice injected with TFEB. Two exposures were displayed for total : the short exposure was used for quantification of Tau transgenic samples; the long exposure was used for side‐by‐side comparisons of the total levels in WT and Taumice and for quantification of WT total Tau. γ‐tubulin was used as a loading control. D Western blot analysis of TFEB expression in 4‐month‐old WT or Tau mice with (+TFEB) or without AAV‐TFEB P0 injection (the same as in A and C but from another independent batch of experiments). PHF1 and total Tau antibodies were used to blot the Tau transgenic lysates for correlation with TFEB levels. E, F Quantification of relative band intensities in WT (E) or mice (F) mice. Student's t−test shows that TFEB protein levels are significantly increased in WT + TFEB vs WT (**P = 0.0026) and in Tau + TFEB vs Tau (*P = 0.047). AT8, PHF1 and MC1 levels are significantly reduced in Tau + TFEB vs Tau (*P = 0.032, 0.041, 0.040, respectively). &amp;amp;, significant correlation between TFEB protein levels and PHF−1 was determined using Pearson Product Moment Correlation test (P = 0.0044). n = 3/group/experiment. Each bar represents average ± s.e.m.
Figure 3A Cell surface expression of CD11b on BM (left), blood (middle) and splenic (right) eosinophils (CD11b+ SSChi SiglecF+) 13 days following N. brasiliensis infection of Dusp5+/+ Rag2-/- (red) and Dusp5-/- Rag2-/- (blue) mice.B Graphs represent corresponding quantitative analysis of CD11b MFI (geometric mean of fluorescence intensity) on eosinophils from Dusp5+/+ Rag2-/- (open circles) and Dusp5-/- Rag2-/- (grey squares) BM (left), blood (middle) and splenic (right) eosinophils as described in (A). Data in (B) are graphical representation of the FACS plots in (A) and from multiple experiments. C Cell surface expression of CD11b of donor-derived (CD45.2+) bloodeosinophils during N. brasiliensis infection. Histogram of CD11b+SiglecF+eosinophils from donor-derived (CD45.2+) Dusp5+/+ (red shaded histogram) and Dusp5-/- (blue line) BM chimeric mice.D Graphical representation of MFI of CD11b cell surface expression on bloodeosinophils from N. brasiliensis infected Dusp5+/+ (open circles) and Dusp5-/- chimeric mice (grey squares) from donor-derived (CD45.2+) cells shown in (C). Data in (D) are graphical representation of the FACS plots in (C) and from multiple experiments.
Effects of FDA-approved drugs on pseudotyped SARS-CoV-2 viral entry in Huh-7 cells versus human Airway Chips.Fig. 4.(A) Graphs showing the inhibitory effects of chloroquine, arbidol, toremifene, clomiphene, amodiaquine, verapamil, and amiodarone when added at 0, 1, or 5 uM to Huh-7 cells infected with SARS-CoV-2pp for 72 h (black bars). The number of pseudoparticles in the infected cells was quantified by measuring luciferase activity; viral entry in untreated cells was set as 100%. VSVpp were tested in parallel to exclude toxic and nonspecific effects of the drugs tested (grey bars). (B) The efficacy of the same drugs in human Airway Chips infected with CoV-2pp. Chloroquine, Arbidol, toremifene, clomiphene, amodiaquine, Verapamil, and Amiodarone were delivered into apical and basal channels of the chip at their respective Cmax in human blood, and ne day later chips were infected with CoV-2pp while in the continued presence of the drugs for 2 more days. The epithelium from the chips were collected for detection of viral pol gene by qRT-PCR; viral entry in untreated chips was set as 100%. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Prediction of a peptide hormone in the C-terminal region of FBN2.Figure 1A Exon-intron structures for human FBN1 and FBN2 genes. Conserved furin cut sites are indicated. Asprosin (Asp) is encoded by exons 65-66 of the FBN1 gene whereas a conserved Asp-like sequence is found in exons 64-65 of FBN2. B Sequence comparison between C-terminal regions of human FBN1 and FBN2, encoding asprosin and placensin, respectively. * identical residues. C Conservation of FBN2 furin cleavage sites and C-terminal regions among diverse vertebrate species. Boxed area represents conserved furin cleavage site. * identical residues conserved in all species examined, : highly conserved residues.
PELP1 localizes to the granular region of the nucleolus.Figure 3(A-C) Immunofluorescence staining of endogenous PELP1, SENP3, UBF1 and NPM1 in HeLa cells. Nuclei were visualized using DAPI staining.
a) Left, immunofluorescence microscopy was performed on RPE cells following treatment with 100 nM bafilomycin A1 for 2 h.figf5(a) Left, immunofluorescence microscopy was performed on RPE cells following treatment with 100 nM bafilomycin A1 for 2 h. Immunolabelling was performed against the indicated endogenous proteins optineurin, T6BP and NDP52 (red), and LC3 (green). The insets show higher magnifications of the areas outlined in the main images. Right, from the confocal images, a Pearson's coefficient was calculated to estimate the degree of co-localization of the different autophagy adaptors with LC3. As a negative control, a Pearson's coefficient was calculated after rotation of one colour image by 90°. The results represent >20 cells from n = 2 independent experiments. (b) Left, stable cherry-LC3-expressing RPE cells were transiently transfected with GFP-myosin VI, treated with Torin1 for 3 h to induce autophagy, and subsequently processed for confocal immunofluorescence microscopy. Areas of co-localization are white in the merged three-colour images and the insets show higher magnifications of the areas outlined in the main images. Right, a Pearson's coefficient was calculated for the degree of co-localization between the three colours. As a negative control, a Pearson's coefficient was calculated after rotating one colour image by 90°. Results represent >10 cells from n = 2 independent experiments. (c) HeLa cells were either mock transfected or co-transfected with siRNA targeted against TNO. Cells were either left untreated or treated with100 nM bafilomycin A1 (BfnA1) for 4 h. Western blot analysis was performed against the indicated proteins. (d) Quantification of LC3-II intensity (±s.d.) was performed from western blot data of TNO-siRNA treated HeLa cells. The results represent n = 3independent experiments. **P0.01, ***P0.001 (e) Immunofluorescence microscopy of RFP-GFP-LC3-expressing HeLa cells transfected with TNO siRNA was performed and confocal images were evaluated for the correlation in GFP and RFP punctae signal overlap using the Image JACoP plugin. The results are calculated from >100 cells from n = 2 independent experiments represented as the Pearson's coefficient of GFP/RFP signal overlap. Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.
Fig.Figure 2Immunoblotting of His-tagged PLDα6 (arrow) expressed in E. coli as separated on an 10% SDS-PAGE and blotted to a membrane. Lipid hydrolyzing activity assayed in the presence of different phospholipids using purified PLDα6 from E. coli. Solid bars are activities assayed using purified PLDα6 whereas open bars were empty vector control that used an equal volume of eluents from bacteria containing the empty vector that was identically processed as those expressing PLDα6. Values are means ± SD (n = 3 biological replicates).
Figure 3A Treatment response in 16 hematologic malignancy (HM) and non-CNS solid PDX models. Objective responses including maintained complete response (MCR), complete response (CR) and partial response (PR) were observed in 10 of 16 models. Drugs are indicated as chemotherapy (Ch), targeted agent (T) or combination treatment (C). Event-free survival (EFS) and percentage of human CD45+ leukocytes in peripheral blood in acute lymphoblastic leukemia (ALL) orthotopic models. Event-free survival (EFS) and percentage of human CD45+ leukocytes in peripheral blood in acute lymphoblastic leukemia (ALL) orthotopic models. EFS and tumor volume in non-CNS subcutaneous PDX models which demonstrated objective response in 1 or more treatments. EFS and tumor volume in non-CNS subcutaneous PDX models which demonstrated objective response in 1 or more treatments. L EFS in a CNS orthotopic model in which drug sensitivity was observed. EFS is time of inoculation of tumor cells to event (defined by neurologic symptoms or weight loss).
Decreased PP2A activity causes Cdc25-T48 accumulation after CLU silencing .Figure 6(A) Left panel: Western blot for CLU, Cdc25C, Cdc25C-T48, Cdc25C S216 and Cdc25C S198 in PC-3 cells after siSCR or siCLU transfection, synchronization and nocodazole release at indicated time points. Vinculin was used as loading control. Right panel: Immunofluorescence microscopy for Cdc25C-T48 in PC3 cells after siSCR or siCLU transfection. DNA was counterstained with (DAPI) (blue). Scale bar represents 10 µm. (B) Western blot for CLU, Cdc25C-T48, Cdk1 and PP2A in PC3 cells after siSCR or siCLU transfection and after synchronization followed by treatment with or without okadaic acid (OA) (100 nM) for 12 hours. Proteins were extracted 15 min after nocodazole release. Vinculin was used as loading control. (C) Western blot for PP2A, CLU, Cdc25C-T48, Cdc25C, Wee1 and Cdk1 in PC3 cells transfected with siSCR or siPP2A synchronized and nocodazole released at indicated time points. Vinculin was used as loading control. (D) PP2A phosphatase activity measurement in PC3 cells after siSCR or siCLU transfection, synchronization and nocodazole release at indicated time points. Error bar represent mean ± SEM, n=3, ** p<0.01 (p=0.0025) by ANOVA followed by Bonferroni's post hoc comparisons. Cells treated with no peptide were used as a negative control.
Superposition of Nsp15 monomers:SARS-CoV-2 (teal, chain A; blue, chain B), SARS-CoV (pink), MERS-CoV (grey).Figure 2.Superposition of Nsp15 monomers:SARS-CoV-2 (teal, chain A; blue, chain B), SARS-CoV (pink), MERS-CoV (grey).
Figure 7A (left) Immunoblotting of LDHA and ATF4 in A375 cells expressing inducible (Tet-On) sh-LDHA expression vector treated with doxycycline (Dox) for 48 hr. (right) qRT-PCR (n=3) analysis of the ATF4 target genes in A375 cells treated with doxycycline (0.5 μg/ml) for 24 hr. Statistical analysis was performed by unpaired t-test. Data are shown as the mean ± SEM , ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. B Proliferation of inducible (Tet-On) sh-LDHA expression vector harboring A375 cells (n=3) treated with Dox (0.5 μg C Athymic nude mice were injected subcutaneously with A375 melanoma cells expressing inducible (Tet-On) sh-LDHA expression vector. Starting day 5, animals were treated on alternate days with 1 g/L doxycycline, 4 mg/kg rapamycin, or with a combination of the two (n=8 in each group). The tumor growth was determined by measuring the tumor volume. Statistical analysis was performed using Welch's t-test (two-tailed). Data are shown as the mean ± SEM ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. D Immunoblotting of resected tumors (4 from each group) for the indicated proteins. Representative images of H&E and cleaved caspase-3 staining from each treatment group (E). Quantification of cleaved caspase-3 (F) (n = 4, each 4 fields). Scale bars, 50 μm. Statistical analysis was performed using two-way Anova. Data are shown as the mean ± SEM , ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. G Relative mTORC1 expression in pan-tumor samples segregated based on high (LDHAhi) (n=2917) and low LDHA (LDHAlo) (n=2914) expression. The p-value of paired Wilcoxon test following the adjustment multiple hypothesis correction (FDR). Data information: In the boxplots, the top and bottom horizontal lines represent the 75th and the 25th percentile respectively, and the middle horizontal line represents the median. The size of the box represents the interquartile range and the top and bottom whiskers represent the maximum and the minimum values respectively. H Conditional essentiality of the indicated genes in LDHAlo and LDHAhi cell lines (n=17, each group). The p-value of paired Wilcoxon test following the adjustment multiple hypothesis correction (FDR). p≤0.1 is significant. Data information: In the boxplots, the top and bottom horizontal lines represent the 75th and the 25th percentile respectively, and the middle horizontal line represents the median. The size of the box represents the interquartile range and the top and bottom whiskers represent the maximum and the minimum values respectively.
Screening of epigenetic drugs for upregulation of miRNAs and downregulation of ZEB1.Figure 3Heat map showing the relative expression levels after drug treatment for 48 h in Panc1. Values measured by qRT-PCR were depicted with the software GENE-E. Only mocetinostat upregulated the miRNAs and downregulated ZEB1. Relative expression of indicated genes in Panc1 measured by qRT-PCR after treatment with different HDAC inhibitors. Note the downregulation of ZEB1 and upregulation of miR-203, miR-200, and E-cadherin by mocetinostat. n = 3, mean ± SEM; unpaired Student's t-test. For significance, see Supplementary Table S1. Immunoblot and immunofluorescence showing that mocetinostat treatment (1 μM, 48 h) reduced ZEB1 expression and induced E-cadherin in Panc1. Expression of histone deacetylases was not altered by mocetinostat, but histone acetylation was induced. Scale bar 10 μm. Chromatin immunoprecipitation analysis validated mocetinostat-induced (1 μM, 48 h) enrichment of the active histone marks H3ac, H4ac, H3K9ac, and H3K4me3 at ZEB1 target gene loci in Panc1. n = 3, mean ± SEM; unpaired Student's t-test. Mocetinostat treatment reduced expression of the anti-apoptotic miR-203 target survivin and sphere-forming capacity in Panc1 when pre-treated with mocetinostat for 48 h. n = 3, mean ± SEM; Mann-Whitney U-test.
Figure 6WT BMDMs were stimulated with LPS for 1 h. Immunoblot analysis of NLRP3, ABRO1, and BRCC3 from the cell lysates immunoprecipitated with control IgG, anti-ABRO1, anti-BRCC3 or anti-NLRP3 antibody. Brcc3+/+ and Brcc3−/− BMDMs were treated with or without LPS for 1 h. Before LPS treatment, Brcc3−/− BMDMs were pretreated with MG132 (10 μM) for 6 h to rescue the expression of ABRO1. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with anti-ABRO1 antibody. Abro1+/+ and Abro1−/− BMDMs were treated with or without LPS for 1 h. Before LPS treatment, Abro1−/− BMDMs were pretreated with MG132 (10 μM) for 6 h to rescue the expression of BRCC3. Immunoblot analysis of NLRP3 and BRCC3 proteins in cell lysates immunoprecipitated with anti-BRCC3 antibody. A constant amount of Myc-NLRP3 plasmid together with an increasing amount of Myc-ABRO1 plasmid were co-transfected in HEK-293T cells. Immunoblot analysis of NLRP3 and BRCC3 from the cell lysates immunoprecipitated with control IgG or anti-BRCC3 antibody. Brcc3+/+ and Brcc3−/− BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP were treated with LPS and nigericin. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-NLRP3 antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody Abro1+/+, Abro1−/−, BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP were treated with LPS and nigericin. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-NLRP3 antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody Brcc3+/+ and Brcc3−/− BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP CBA analysis of IL-1β in culture supernatants Abro1+/+, Abro1−/−, BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP CBA analysis of IL-1β in culture supernatants
Figure 4Differences in % deuterium uptake between the pParkin:pUb and pParkin:pUb in complex with UbcH7-Ub states plotted at the one minute time point. The width of the bars shown represent the lengths of the peptides observed and measured, while the heights represent the relative difference in hydrogen-deuterium exchange between the two species. Error bars represent standard deviation above the average for triplicate measurements. Positive bars indicate a greater deuterium uptake in the pParkin:pUb:UbcH7-Ub state compared to the pParkin:pUb state, signifying a greater exposure to solvent, while a negative change indicates the complex state is more protected. The linear domain structure is shown below the data to indicate the location of peptides. Relative HDX differences from (A) are mapped to the structure of pParkin:pUb and UbcH7-Ub complex. Regions where HDX is faster in the pParkin:pUb:UbcH7-Ub complex at the one minute time point are indicated in red while those regions in blue exchange more slowly using a similar the gradient as Fig. 1. Based on HDX and NMR experiments (C, D) the pUbl domain is shown oriented towards the RING0 domain (dashed arrow, top). The increased HDX for RING2(Rcat) and RING0 suggests the RING2(Rcat) domain has a weaker interaction with the RING0/RING1 domains upon UbcH7~Ub binding (dashed arrow, bottom). Regions shown in grey are peptides that were not mapped. 1H-15N HSQC spectra of full-length 15N-labelled pParkin: pUb (green contours) compared to that for the isolated 15N-labelled pUbl domain (grey contours). The spectrum of the full-length pParkin:pUb complex was collected using a 1H-15N CPMG, T2-filtered HSQC (Aguirre et al, 2017) to show only the more mobile regions of pParkin. In the spectrum of full-length pParkin many additional resonances arising from flexible loops in the protein are observed and are not labelled for clarity. Residues that experience small chemical shift changes are indicated by arrows. 1H-15N HSQC spectra of 15N-labelled pUbl (grey contours) and in the presence of an equimolar amount of unlabelled R0RBR:pUb (blue contours) shows little change. Upon addition of unlabelled UbcH7-Ub (red contours) small changes in chemical shifts of pSer65, D62, Q63, Q64 are observed as indicated by arrows.
Examples of translation initiation events confirmed by Ribo-Seq in Jurkat cells.Figure 2Figure 2 - Examples of translation initiation events confirmed by Ribo-Seq in Jurkat cells. A-B. Alternative translation initiation due to leaky scanning. C. Alternative splicing. D. The preferential expression of an N-terminally truncated proteoform confirmed by Ribo-Seq data. E-F. Evidence for alternative translation initiation occurring at two consecutive AUG codons.
Figure 2A) Left panel: Schematic diagram displaying relevant genomic structure of the strains used to measure inter-chromosomal DNA repair efficiency. The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated. Note that crossover and non-crossover product have different restriction fragment sizes that can be differentiated in a Southern blot assay. Arrow depicts the localization of the double-strand break. Right panel: Physical analysis of wild-type and cdc14-1 mutant strains carrying the inter-chromosomal gene conversion assay. Cells were grown overnight before adding galactose at semipermisive temperature to induce HO expression, thus producing a DSB on chromosome V. Samples were taken at different time-points, genomic DNA was extracted, digested with EcoRI and analysed by Southern blot. Blots were hybridized with a MATa-only DNA sequence and an actin probe was used as loading control. Asterisk denotes an overexposed film to visualize crossover formation. Immunoblot of Rad53 was performed as previously described. Coomassie staining is shown as a control for loading. Graphs show the mean ± S.D. of gene conversion, DSB induction and crossover vs non-crossover ratio from three independent experiments. All data was normalized using actin as loading control. Replicates were averaged and statistical significance of differences assessed by a 2-tailed unpaired Student t test. B) Left panel: Schematic representation depicting the genomic structure of the strains used to evaluate repair pathway choice. The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated. Note that the use of NHEJ or HR generates different product that can be recognized in Southern blots by the different disposition of the restriction sites for the endonucleases used. Arrow depicts the localization of the double-strand break. Right panel: Physical analysis of wild-type and cdc14-1 cells harbouring the repair pathway choice assay. Cells were grown overnight and HO induction was attained through addition of galactose for 1.5 hour at semipermisive temperature. After induction of the HO, glucose was added to repress the HO expression and samples were taken to analyse repair efficiency. DNA was extracted, digested with EcoRV, separated on agarose gels and blotted. Blots were hybridized with a probe corresponding to the MATa-only DNA sequence. A second probe to the HIS3gene was used to control the amount of genomic DNA loaded at each time-point. Graphs show quantification of mating type switching by gene conversion (HR), restoration of MATa (NHEJ) and the kinetics of the DSB induction. All data was normalized with the HIS3 gene. Graphs show the mean ± S.D. from three independent experiments. Replicates were averaged and statistical significance of differences assessed by a 2-tailed unpaired Student t test.
Figure 3Representative 3D-reconstructions of CA1 pyramidal neurons from AAV-Venus injected LM controls (left), AAV-Venus injected cDKOs (middle, red) and AAV-APPsα injected cDKOs (right, green). Schematic representation of parameters assessed. Compared to LM controls AAV-Venus injected cDKO mice show a significantly reduced basal (##p<0.01, C) and apical (##p<0.01, E) dendritic length and reduced branching in basal (##p<0.01, D) and apical (##p<0.01, F) dendrites. AAV-APPsα injection did neither affect basal (nsp>0.05, C) nor apical (nsp>0.05, E) total dendritic length. However, the total number of basal nodes differed significantly compared to AAV-Venus injected cDKO mice (*p<0.05, D). Sholl analysis of basal dendritic length reveals a significant group effect (repeated measures ANOVA: genotype F(2, 29)=5.038, p<0.05) and a significant distance effect (repeated measures ANOVA: genotype F(5, 145)=250.2, p<0.0001). Due to a significant interaction effect (repeated measures ANOVA: genotype F(10, 145)=4.466, p<0.0001), a post-hoc Sidak's multiple comparison test was performed to further evaluate effects between groups at distinct distances from soma. Compared to Venus injection, AAV-APPsα injection of cDKO significantly increased basal dendritic length at 60 µm (*p<0.05). H Sholl analysis of apical dendritic length reveals an overall significant group effect (repeated measures ANOVA: genotype F(2, 37)=4.776, p<0.05) and a significant distance effect (repeated measures ANOVA: genotype F(18, 666)=47.54, p<0.0001). Due to a significant interaction effect (repeated measures ANOVA: genotype F(36, 666)=1.640, p<0.05), a post-hoc Sidak's multiple comparison test was performed to further evaluate effects between groups at distinct distances from soma. AAV-APPsα expression increased midapical dendritic length at 300 µm (**p<0.01) and 330 µm (**p<0.01) distance from soma compared to AAV-Venus injected cDKO mice.