EMBO/sd-character-level-ner huggingface dataset:
uORF sequence features are targets of selection.Figure 4(A) uORF initiation contexts display signatures of selection. Plot displays cumulative distribution of AUG context scores calculated using nucleotide scoring matrices (Grzegorski et al, 2014) across multiple classes of ORFs (translated uORFs (high confidence), untranslated uORFs, CDS ORFs, and TLS background), with a higher score indicating better initiation context. TLS background represents the distribution of scores of a randomly sampled set of 50,000 sequences from zebrafish 5' UTRs. Insets display sequence logos for CDSs, translated uORFs, and oORFs. (B) uORF initiation context influences repression of downstream translation. Plot displays cumulative distribution of translation efficiency in transcripts with single uORFs in favorable initiation contexts (top quintile of all uORFs) vs. unfavorable contexts (bottom quintile of all uORFs). Inset displays where these quintiles lie on the distribution of all uORF AUG scores. (C) AUG frequency is lower proximal to the CDS start codon -Plot displays AUG frequency (as a fraction of all codons), split by frame relative to the CDS start codon. Points show frequencies at individual codon positions and loess regression lines display the overall trend. (D) Vertebrate TLSs contain fewer uORFs than expected. Histograms show the distribution of Z-scores in zebrafish, mouse, and human TLSs, with positive z-scores indicating uORF enrichment and negative z-scores indicating uORF depletion, relative to sequence-shuffled TLSs. (E) uORFs are shorter than expected by chance - Histogram showing length distribution of all uORFs versus canonical protein coding regions, with inset providing a closer look at uORFs (bin size 10nt). Vertical dotted lines indicate the observed mean length of endogeneous uORFs and the mean length of uORFs obtained by sequence shuffling of zebrafish TLSs, which differ significantly (Two-sided p<4.5e-308).
Figure 4A. Schematic representation of experimental setup for trans-endothelial insulin transport assay. B. AKT phosphorylation in myogenic C2C12 cells treated with different doses of insulin. n=3, data represent mean ± SEM, unpaired t-test. C. Representative western blot of AKT phosphorylation in C2C12 cells upon Cav1 knockdown in mouse cardiac endothelial cells (MCECs). D. Densitometric analysis of western blot in C. Data represented as fold change over control. n=3, data represent mean ± SEM, unpaired t-test. E. Proximity ligation assay showing the association of CAV1 and INSR (insulin receptor) in MCECs upon stimulation with insulin. n=86-120 cells from 8 microscopic high power fields per time point, data represent mean ± SEM, unpaired t-test, *** means p<0.001.
ATF7IP-mediated SETDB1 nuclear localization enhances SETDB1 ubiquitination A Co-transfection with ATF7IP augments the K885 ubiquitination of SETDB1.Figure 5A Co-transfection with ATF7IP augments the K885 ubiquitination of SETDB1. HEK293T cells were transfected After the transfection, V5-SETDB1 was immune-purified with anti-V5 antibody and used for WB analysis. B ATF7IP regulates the ubiquitination of SETDB1 in mESCs. Endogenous SETDB1 was immune-purified from WT or Atf7ip KO mESCs cells and used for WB analysis. C LMB treatment increased the relative intensity of the upper ubiquitinated upper band of SETDB1 in Atf7ip KO mESCs. Cells were treated with 10 ng/mL LMB or mock for 5 h before analysis. D HEK293T cells were transfected with either 3xF- or 3xF-NLS-SETDB1. Over 50 transfected cells per sample were analysed. The representative data of two independent experiments is shown. Scale bar: 10 µm. E Nuclear localization itself promotes the ubiquitination of SETDB1. HEK293T cells were transfected with 3xFLAG- or 3xFLAG-NLS-SETDB1. After FLAG-IP, the purified SETDB1s were analyzed by WB. F, G IF analysis was performed with Atf7ip KO mESCs stably expressing 3xF- or 3xF-NLS-SETDB1. Representative images are shown in F, and the quantitative analysis is shown in G. Scale bar: 10 µm. Addition of NLS increases nuclear localization of SETDB1 in Atf7ip KO mESCs. The mean from three independent experiments is shown as a bar-graph with jittered points indicating the average % intensity of each experiment. Over 50 cells were analysed per sample per experiment. ***P < 0.001 by unpaired Student's t-test. H Nuclear localization promotes the ubiquitination of SETDB1 in Atf7ip KO mESCs. Atf7ip KO mESCs expressing 3xF- or 3xF-NLS-SETDB were used for FLAG-IP, and the purified SETDB1s were analyzed by WB.
Figure 7Nuclear HDAC1 activity in mice after 2 months of compound 5104434 treatment. The nuclear extracts of the frontal cortices and hippocampus from 8-mon-old of mice were separated from total tissue lysates and were immunoprecipitated for HDAC1, and were then conducted to HDAC1 activity assay. N = 6 mice per group. TSA: nuclear extracts that were treated with Trichostatin A (TSA, an HDAC inhibitor) as a negative control for HDAC1-transferred fluorescent activity during the HDAC1 activity assay. * p = 0.0203 (WT vs Tg-Veh) or 0.0408 (Tg-Veh vs Tg+5104434) by multiple comparison. Data information: All data are presented as mean ± SEM (% Representative data of western blot for nuclear acetylated-histone 3 (Lys 9/14), total histone H3, and quantification of protein levels. N = 6 mice per group. * p = 0.0105 or *** p = 0.0008 by multiple comparison. Data information: All data are presented as mean ± SEM (%) Representative data of western blot for nuclear HDAC1 levels and semi-quantification of the expression levels. N = 6 mice per group. ** p = 0.0022 (Tg)/ 0.0012 (Tg+5104434) by multiple comparison. Data information: All data are presented as mean ± SEM (%) Left panel: representative data of western blot for cell cycle- and DNA damage-related proteins. Right histogram: semi-quantification of protein levels. N = 6 per group. *WT+Vehicle mice vs. Tg+Vehicle mice, #Tg+5104434 mice vs. Tg+Vehicle mice. Statistical analysis by multiple t-test with FDR correction, Q = 1%. **P ≤ 0.01; ###P ≤ 0.001 and ****/####P ≤ 0.0001. Data information: All data are presented as mean ± SEM (%) IF staining of γH2AX in the frontal cortices and quantification of the immunoreactive cells from 8-mon-old of WT+Vehicle, Tg+Vehicle, Tg+5104434. n = 9 sections per mouse, N = 6 mice per group. Scale bar: 50 μm. * p = 0.0421 or **** p < 0.0001 by multiple comparison. Data information: number of cells in each view is presented. Immunohistochemistry of NeuN and the quantification of the immunoreactive cells in the hippocampus of three groups of mice, 8-mon-old WT+Vehicle, Tg+Vehicle and Tg+5104434. n = 9 sections per mouse, N = 6 mice per group. Scale bar: 50 μm.* p = 0.0414 or *** p = 0.0006 by multiple comparison. Data information: All data are presented as mean ± SEM (%) IF staining of GFAP and the quantification of the immunoreactive cells in the hippocampus of three groups of mice, 12-month-old WT+Vehicle, Tg+Vehicle and Tg+5104434. n = 9 sections per mouse, N = 6 mice per group. Scale bar: 50 μm. ** p = 0.002 and **** p < 0.0001 by multiple comparison. Data information: where number of cells in each view is presented.
Figure 3(A) Immunoprecipitation against IgG, USP7 and PP2A-C in whole cell extracts of RPE cells that were treated with DMSO (control) or 25 μM P22077 (USP7i). Levels of USP7, PP2A-A, PP2A-B and PP2A-C are shown. Images from two different exposure times are shown for the better inspection of saturated bands. (B) Western blot showing the levels of MPM-2, pAKT, AKT, pPRC1, pKAP, KAP, pMAPKAPK-2, pSTAT3, STAT3 and β-Actin in whole cell extracts of RPE cells treated with DMSO (Control), 0.5 μM Okadaic acid for 2 hrs, 25 μM P22077 for 4 hrs or 25 μM BAY 11-7082 for 2 hrs. Ponceau staining is shown as loading control. (C) Western blot showing the levels of PP2A-A, PP2A-B, PP2A-C and H2A in the nuclear fraction of RPE cells treated with DMSO (Control), 0.5 μM Okadaic acid for 2 hrs, 25 μM P22077 for 4 hrs and 25 μM BAY 11-7082 for 2 hrs. (D) Immunofluorescence of PP2A-A (green) in U2OS cells treated with DMSO (CONTROL), 50 μM P22077 (USP7i) for 4 hrs or 0.5 μM Okadaic acid (OA) for 1 hr 30 min. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. (E) Western blot showing the levels of MPM-2, pAKT, AKT, pPRC1, pMAPKAPK-2, p4e-BP1, 4e-BP1 and β-Actin, in whole cell extracts of RPE cells treated with DMSO (Control), 25 μM P22077 (USP7i) and 10 μM of the PP2A activator DT-061 for 2-4 hrs. Ponceau staining is shown as loading control.
Figure 6(A) representative electron microscopy of ER-mitochondria associations in NSC34 cells treated with either vehicle or GSK-3β inhibitors AR-A014418 (1 M) or CT99021 (100 nM) for 16 h. Arrowheads with loops show regions of association; scale bar=200 nm. Bar charts shows % of the mitochondrial surface closely apposed to ER in the different samples. Data were analysed by one-way ANOVA followed by Tukey's multiple comparison test. N=32 cells and 181-210 mitochondria. Error bars are s.e.m.; *p<0.05. (B) VAPB-PTPIP51 proximity ligation assays of NSC34 cells treated with either vehicle 1 M AR-A014418 or 100 nM CT99021 for 16 h. Cells were also stained for nuclei with DAPI. Bar chart shows relative number of proximity ligation assay signals/cell.
Figure 6A. β-galactosidase staining of cleared whole lung preparations isolated from control and injured BASC v-race animals 21 days (bleomycin, naphthalene) or 35 days (influenza) following injury. Scale bar: 5 mm (whole lungs) and 1 mm (magnification). B. Sequential microscopic imaging of epifluorescence and β-galactosidase activity in lung sections of control and injured BASC v-race mice. Clusters of BASC-derived AT2 (YFP+) and Club cells (mCherry+) are highlighted. Blue: DAPI. Scale bar: 200 µm. C. Contribution of BASCs to different cellular compartments. Bronchiolar and alveolar accumulations of lineage-traced BASC derivatives are expressed as percentage of all clusters containing ≥ 10 β-gal+ cells. Data depicted as mean±SD, n=4 (bleomycin, naphthalene) or n=5 (influenza).
Mocetinostat sensitizes to gemcitabine in vivo.Figure 5Relative tumor volume (RTV) of Panc1-derived tumors in NMRI nu/nu mice. Eleven days after implantation, mice were randomized according to tumor volume. Treatment with mocetinostat (60 or 90 mg/kg/day) and gemcitabine (25 mg/kg/day) was implemented (day 0) as depicted in the scheme. Shown are the group medians of the RTVs over time (left) and the individual RTVs on day 32 (right). n = 5 for each treatment group; nonparametric Mann-Whitney U-test. Representative immunohistochemical stainings of serial sections showing reduced ZEB1 and increased E-cadherin in tumor tissues of mice treated with mocetinostat. Scale bar 40 μm, inserts for higher magnifications 10 μm. Squares indicate magnified regions. Representative pictures of in situ hybridization for miR-203 and control probe showing gain of miR-203 and associated loss of ZEB1 detected by immunohistochemistry in serial sections of mocetinostat-treated xenograft tumors. Scale bar 40 μm, inserts for higher magnifications 5 μm. Squares indicate magnified regions. Schematic outline and results for xenografts of ex vivo treated Panc1 in Foxn1 nude mice. Panc1 cells were pre-treated with mocetinostat (1 μM) and/or gemcitabine (50 nM) for 48 h, followed by a 7-day recovery period before being injected subcutaneously (left). Equal numbers of viable cells were injected in 75 μl volume. At day 9 after injection, tumor growth was detectable in all groups (lower right). To better visualize and compare tumor growth, the tumor volume at day 9 was set to 1 and the increasing slope of the tumor volume to day 37 is depicted (upper right). The individual absolute tumor volumes on day 37 (lower left) and the group medians of the absolute tumor volumes over time (lower right) are shown. For cells pre-treated with the combination of mocetinostat and gemcitabine, tumor growth was arrested. n = 4 for each treatment group; nonparametric Mann-Whitney U-test.
Figure 2 A) Spectrophotometric activities of the respiratory chain (n=4-5/group). CS: citrate synthase; CIV: complex IV. Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by two way ANOVA with Tukey's correction ****p<0.0001 (CS: WT vs. Cox15sm/sm), ***p=0.0010 (CS: Cox15sm/sm vs. Cox15sm/sm rapamycin), ****p<0.0001 (CIV: WT vs. Cox15sm/sm), **p=0.0060 (CIV/CS: WT vs. Cox15sm/sm)  B) Western blot immunovisualization of subunits of the respiratory complexes. Note the increased protein levels in Cox15sm/sm vs. WT samples, which are reduced to normal levels upon rapamycin treatment. Additional samples were run on a separate gel (not shown)  C) BNGE in gel activity for cIV. Sc: supercomplexes. Note that the COX reaction is slightly increased in rapamycin-treated vs. untreated Cox15sm/sm samples  D) Immunoblot of 1D-BNGE using an anti-cIV antibody (COX1). Note that COX amount is slightly increased in rapamycin treated vs. untreated Cox15sm/sm muscles. SDHB was used as a loading control  E) State III (succinate-driven) oxygen consumption (n=3/group). Error bars represent SEM. The asterisks represent the significance levels calculated by unpaired, two tail Student's t test **p=0.0016 (WT vs. Cox15sm/sm); *p=0.047 (Cox15sm/sm vs. Cox15sm/sm rapamycin)  F) Analysis of mtDNA amount (n=9/group). Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey's correction: **p=0.0020; *p=0.0296
trans-acting quantitative trait loci co-regulate chromatin accessibility and gene expression in ESCs and EpiLCs.Figure 4A. PCA of total transcript abundance in ESCs and EpiLCs from parental (3 biological replicates) and 33 distinct recombinant inbred BXD strains generated from crosses between B6 and D2. PC1 captures variation between cell state (57.6%) and PC3 captures transcript variation within cell states. B. Scatterplot of genomic locations of individual chromatin accessibility (ca) QTL (x-axis, LOD > 5) versus location of ATAC peak being regulated (y-axis) for ESCs. Genetic effects that act locally (n = 3,973), i.e. in cis, fall on the diagonal line, while distal acting QTL (n = 13,767) lie off the diagonal. Several prominent distal QTL hotspots appear as vertical lines. C. Similar to B, plotting expression (e) QTL position versus gene location (LOD > 5, n = 701 local-eQTL, n = 357 distal-eQTL). D D. Number of distal caQTL in 1 Mb windows versus genomic location mapped in ESCs (maroon) and EpiLCs (teal). E. Similar to D comparing eQTL location and number between ESCs and EpiLCs. F. Distribution of caQTL hotspot targets in relation to nearest transcription start site in ESCs as percentage of total targets. G. Coverage profile for Gstp2 locus on Chr 19 showing H3K4me3, ATAC, and RNA read depth from merged replicates. Locations of Chr 7 trans caQTL targets are indicated with black bars and LOD scores listed below. H. LOD score plot from QTL scan in ESCs for chromatin accessibility at the putative enhancer at Gstp2. Genetic variation at Chr 7 QTL distally regulates chromatin accessibility on Chr 19. I, Phenotype by genotype plot of the Chr 7 caQTL from h. A B6 haplotype at Chr 7 is associated with increased chromatin accessibility on Chr 19 (horizontal line represents mean). J&K. Similar to H&I showing QTL mapping and haplotype effect for expression of Gstp2 in ESCs (horizontal line represents mean). L. Euler diagram showing overlap between genes in the neurogenesis module (m5 from Fig 2H) and Chr 12 trans eQTL (LOD >4) in EpiLCs (enrichment compared to all eQTL in neurogenesis module, Fisher's exact test - odds ratio (OR) = 7.78, p-value = 1.087e-06).
RT-PCR assays towards viral RNA extracted by different pcMNPs-based methods.Figure3RT-PCR assays towards viral RNA extracted by different pcMNPs-based methods.RT-PCR assays amplifying (A) the ORF1ab region and (B) the N gene in pseudoviral RNA extracted by a manual protocol.
TDP-43 accumulates in cytoplasmic ubiquitin-positive structures in Tsg101- and Vps24-depleted cells.Figure 8.TDP-43 accumulates in cytoplasmic ubiquitin-positive structures in Tsg101- and Vps24-depleted cells. HeLa cells transfected with control (A), Tsg101 (B), or Vps24 (C) siRNA were fixed, permeabilized, and stained with antibodies against TDP-43 (green), ubiquitin (red), and p62 (blue). Single channel images in black and white are shown. Colocalization of all proteins is indicated in white in the merged picture. Bars, 10 μm.
Figure 2A. Immunoblot analysis of indicated proteins in control and siRNA treated cells. B. BN-PAGE analysis of siRNA-mediated depletion shows an OXPHOS defect as revealed by subunit-specific antibodies against individual OXPHOS complexes.C. Quantification of the levels of individual OXPHOS complexes normalized to Complex II levels. The graph represents the relative abundance of individual complexes in cells treated with the specified siRNA versus controls. The bars represent the mean ± SEM of 3-7 independent experiments. P-values were calculated using paired two-tailed t-test (* P<0.05; ** P<0.01; ***P<0.001). D, E. Pulse-labeling mitochondrial translation experiment of the 13 mitochondria-encoded polypeptides (seven subunits of complex I [ND], three subunits of complex IV [COX], two subunits of complex V [ATP] and one subunit of complex III [cyt b]) in control and siRNA treated cells. (E) Detail of a mitochondrial pulse-labeling experiment showing a severe decrease in the translation of ATP6 and ATP8 in TRUB2 depleted cells.
Figure 3A Dose-response curves showing the effects of two independent anti-DDX3X siRNAs on the expression of AUG-NL-3xF (top) and (CGG)100 +1 NL-3xF (bottom) reporters. Plasmid-based reporters were transfected into HeLa cells 24 hours after knockdown, and reporter expression was quantified by luminescence. Nanoluciferase (NL) luminescence has been normalized to luminescence from firefly luciferase (FF), which was co-transfected, in order to control for transfection variability. Asterisks refer to comparisons between anti-DDX3X siRNAs and siRNAs against EGFP (siEGFP; two-way ANOVA with Dunnett's multiple comparisons test; n=12/condition). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD (compiled from ≥3 replicates). (CGG)n +1 NL-3xF expression (normalized to FF) with and without DDX3X knockdown across a range of CGG repeat sizes. Black asterisks refer to comparisons between siDDX3X- and siEGFP-treated cells; orange asterisks refer to comparisons between siDDX3X-treated cells expressing AUG-NL-3xF and those expressing a different reporter (two-way ANOVA with Tukey's multiple comparisons test; n=17-30/condition). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD (compiled from ≥3 replicates). (CGG)n +2 NL-3xF expression (normalized to FF) with and without DDX3X knockdown across a range of CGG repeat sizes. Black asterisks refer to comparisons between siDDX3X- and siEGFP-treated cells; orange asterisks refer to comparisons between siDDX3X-treated cells expressing AUG-NL-3xF and those expressing a different reporter (two-way ANOVA with Tukey's multiple comparisons test; n=17-30/condition). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD (compiled from ≥3 replicates). Western blots of FMRpolyG-NL-3xF products with and without DDX3X knockdown across a range of repeat sizes. Western blots of FMRpolyA-NL-3xF products with and without DDX3X knockdown across a range of repeat sizes.
SARS-CoV-2 miR-O7a.Figure 5(A, B) Volcano plots showing the log2 fold change and corresponding significance levels of ISGs upon 8 hours of IFN-α treatment in A549-ACE2 cells transfected with CoV2-miR-O7a.1 (A) or CoV2-miR-O7a.2 (B) compared to control mimic. Significantly downregulated genes are marked in red, and upregulated genes in blue. The orange horizontal line indicates two-tailed P = 0.05. n=2, dashed lines represent +1 and -1 log2 fold change. (C) Log2 fold change of ISGs at 8, 16, 24 hours of IFN-α treatment and categorized based on CoV2-miR-O7a.2 target sites 8mer, 7mer-m8, 7mer-A1, and no seed as shown in the schematic. The mean and standard error of the mean is shown. Number of ISGs with 8mer site n=8, 7mer-m8 n= 32, 7mer-A1 n=26 and no seed n= 92. The two-tailed p values were calculated using the Mann-Whitney-Wilcoxon test. ISGs were calculated as all the upregulated genes (≥ 3-fold; padj < 0.05) in two replicates of IFN-induced versus non-induced conditions in all time points.
PI(5)P Synthesized by PIKfyve Regulates Autophagosome Formation (A) Schematic representation of pathways for PI(5)P and PI(3)P synthesis/turnover and drugs targeting enzymes involved in these pathways.Figure 2Schematic representation of pathways for PI(5) P and PI(3) P synthesis/turnover and drugs targeting enzymes involved in these pathways. (B and C) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells treated with DMSO or increasing concentrations of YM-201636 for 2 hr in the presence or absence of BAF (mean ± SEM). (D and E) HeLa cells transiently transfected with Strawberry-ATG16L1 were treated with YM-201636 (100 nM, 2 hr) in HBSS, then fixed and stained for endogenous WIPI-2. Bar, 10 μm. (E) Quantification of ATG16L1 and WIPI2 structures per cell, n = 20 cells (mean ± SEM; n = 3 independent experiments; ∗∗∗p < 0.001, t test). (F) Western blot analysis of free ATG12 and ATG5-ATG12 complex levels with anti-HA antibody in HeLa cells transfected with HA-ATG12 and ATG5 and treated with YM-201636 (100 nM, 2 hr) (mean ± SEM). (G) HeLa cells transfected with GFP-PHD3X and Strawberry-ATG16L1 for 16 hr were left in HBSS for 1 hr, then fixed and imaged on Elyra superresolution microscope. Final visualization was performed as previously described. Bar, 1.13 μm. (H and I) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in cells incubated with YM-201636 (100 nM, 2 hr) and loaded with exogenous PI(5) P for the last 1 hr in the presence or absence of BAF (mean ± SEM). See also Figure S2.
a–c) Representative western blots and (d).f8(a-c) Representative western blots and (d) quantification show increased accumulation of some of the HACE1-interacting proteins in the Hace1−/− sTAC myocardium. n=3 mice per group, error bars represent s.e.m., P-value between KO and WT sTAC as indicated (one-way analysis of variance). Gapdh was used as a loading control.