EMBO/sd-character-level-ner huggingface dataset:
Figure 5(A) The interaction between Htt-Q97 and Sp1 is decreased by linear ubiquitination. HEK293T cells were co-transfected with Htt-Q97-HA and the siRNAs indicated. Htt-Q97-GFP was transfected as a control for specific immunoprecipitation of HA-Htt-Q97. Nuclear (left panel) and cytosolic fractions (middle panel) were subjected to an anti-HA immunoprecipitation followed by Western blotting using an anti-Sp1 antibody. Right panel: Input controls (whole cell lysates). (B) Quantification of the interaction between Htt-Q97 and Sp1 in nuclear fractions. Shown is the relative amount of Sp1 co-purifying with Htt-Q97. Signal intensities were normalized to nuclear Sp1. Data are displayed as mean ± SD and were analyzed by 2 independent Mann-Whitney U-tests, n = 4. *p ≤ 0.05, ***p ≤ 0.001. (C, D) Impairment of Sp1-mediated transcription by Htt-Q97 is modified by HOIP. (C) Shown is the relative luciferase activity in HEK293T cells expressing Htt-Q97-HA, a Sp1 luciferase reporter construct and HOIP cDNA. Data are displayed as mean ± SD and were analyzed by Kruskal-Wallis test followed by Dunn's Multiple Comparison Test, n = 9-11. (D) Shown is the relative luciferase activity in HEK293T cells expressing Htt-Q97-HA, a Sp1 luciferase reporter construct and HOIP siRNA. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 9. *p ≤ 0.05, ***p ≤ 0.001. (E) Sp1 silencing decreases HOIP and SHARPIN protein expression. SH-SY5Y cells were transfected with control or Sp1-specific siRNAs and analyzed for protein expression 2 days after transfection by immunoblotting (left panel). Quantification of normalized Sp1, HOIP and SHARPIN protein expression levels (right panel). Data are displayed as mean ± SEM with n=5 from unpaired two-tailed Student's t-tests. *p ≤ 0.05, ***p ≤ 0.001. (F) HOIP-, HOIL-1L- and SHARPIN-specific mRNA is decreased in R6/2 striatum. Quantification of mRNA from the striatum of 9-12 weeks old R6/2 mice and non-transgenic littermates. Data are displayed as mean ± SD with n = 6-9 biological replicates from unpaired two-tailed Student's t-tests. *p ≤ 0.05, ***p ≤ 0.001. (G) HOIP protein levels are decreased in human HD brain. Total protein lysates from human brain (anterior cingulate cortex) analyzed by immunoblotting using antibodies against HOIP and βIII-tubulin. HOIP-positive signals were normalized to βIII-tubulin. To verify the specificity of the HOIP antibody, WT and HOIP KO HAP1 cells were analyzed in parallel on a different gel. Data are displayed as mean ± SEM with n=5 for control and n=7 for HD patients from unpaired two-tailed Student's t-tests. *p ≤ 0.05, ***p ≤ 0.001.
Figure 9A. HeLa cells were treated with non-target siRNAs (siNT) or those targeting NSUN3 (siNSUN3_1 or siNSUN3_2) or ABH1 (siABH1_1 or siABH1_2) for 72 h before labelling of mitochondrial translation products with [35S]methionine. Protein samples were separated by SDS-PAGE then transferred to a membrane. Labelled proteins were detected using a phosphorimager and the levels of tubulin were determined by western blotting using an antibody against the endogenous protein for normalisation.B. Mitochondrially-translated proteins that could be clearly detected were quantified in three independent experiments and the results are shown graphically as mean ± SD. C. Overview of the modification pathway of C34 in mt-tRNAMet. NSUN3 introduces an m5C methylation on C34 using S-adenosyl methionine as methyl group donor and this can be further oxidised by ABH1 in the presence of O2, Fe(II) (Fe2+) and alpha-ketoglutarate (αKG) to produce f5C34.
Figure 1The two steps of GENDULF: (Step 1) Mine transcriptomics of healthy disease-relevant tissues to identify PMs, which are genes that are differentially under-expressed when the GCD is lowly expressed. GENDULF does not compute a correlation across the whole range of expression values, but specifically searches only for a significant association at the lower level range, as shown in the region boxed in the left scatter plot, where the dots are in blue and the y-axis is labeled 'PM' for `potential modifier'. The scatter plot on the upper right depicts an example of relationship between expression of the GCD and another gene in which GENDULF is not expected to find the other gene as a modifier, and hence the y-axis is labeled non-PM expression. (Step 2) Evaluate the expression of PMs identified in step 1 in transcriptomic data sets containing both diseased and control samples to find a subset of PMs that we label as DPMs (left graphs)- PM genes that are not down regulated in the disease tissues.
Mithramycin reprograms rhabdoid tumor promoters to trigger a change in cellular state and favor a differentiation phenotype.Figure 6A: Heatmaps depicting ATAC-seq global chromatin structure following 8-hours (middle) and 18-hours (right) 100nM MMA treatment. A 2kb window is centered on the TSS. Chromatin accessibility clusters at the TSS and does not change globally relative to solvent. Quantification of genes that gain accessibility or reduce accessibility on the right. B: CTCF is the top down-regulated motif from the ATAC-seq analysis (P = 1e-58). C: Schematic for the 18 state chromHMM model built for MRTs and collapsed into 6 super states. Chromatin states were called if the state was present in at least 50% of samples. ATAC-seq and H3K27ac ChIP-seq peaks were queried against the 6 super states. P-values derived from the homer motif analysis package. D: Donut plots representing the percentage of each chromatin super state across treatment time (from 8h to 18h) that increased 1.5-fold (left) or decreased 1.5-fold (right). Below, Motif analysis of the top up-regulated motifs from the H3K27ac ChIP-seq gene lists that pass a 1.5 logFC. P-values derived from the homer motif analysis package using a binomial algorithm. E: IGV tracks of rhabdoid tumor genes previously identified to be occupied by non-canonical SWI/SNF (Michel et al., 2018). ID3 and JUND3 decrease in H3K27ac occupancy and chromatin accessibility following exposure to mithramycin for 8-hours or 18-hours. F: IGV tracks of rhabdoid tumor genes previously identified to gain H3K27me3 upon SWI/SNF loss (Erkek et al., 2019). CDK6 and CDK2 decrease in H3K27ac occupancy and chromatin accessibility following exposure to 8-hours and 18-hours mithramycin treatment. G: IGV tracks of genes identified to have an increase in accessibility, H3K27ac and gene expression following mithramycin exposure. BMP1 and ADIPOR1 play crucial roles in bone and adipogenic differentiation, respectively.
Figure 3(A) Staining for Pax6 and p-Vim in E15 WT and hFOXM1-cKI mice. The right A and B are high-magnification images. Arrows indicate double positive of Pax6+ p-Vim+ cells with cells with a basal process (arrowheads). Scale bars, 50μm (left); 5μm (right). (B) Graph shows the percentage of Pax6+ cells in enlarged SVZ (n = 6 each group; two-tailed unpaired t-test, P=0.0002). (C) Graph shows the number of Pax6+ p-Vim+ cells in 4x10^4 μm2 in enlarged SVZ (n = 6 each group; two-tailed unpaired t-test, P=0.0003). (D (D) Staining for Sox2 in E15 WT and hFOXM1-cKI mice. Scale bar, 50μm. (E) Graph shows the number of Sox2+ cells in 4x104 μm2 in enlarged SVZ (WT n=3; hFOXM1-cKI n=4; two-tailed unpaired t-test, P=0.0292). ( (F) Staining for Tbr2 in E15 WT and hFOXM1-cKI mice. Scale bar, 50μm. (G) Graph shows the number of Tbr2+ cells in 4x104 μm2 in enlarged SVZ (WT n=6; hFOXM1-cKI n=9; two-tailed unpaired t-test, P=0.0003). ( (H) Staining for HOPX in E15 WT and hFOXM1-cKI mice. Scale bar, 50μm. (I) Graph shows the number of HOPX+ cells in 4×104 μm2 in enlarged SVZ (n = 4 each group; two-tailed unpaired t-test, P= 0.0044). ( (J) Staining for Ki67 in E15 WT and hFOXM1-cKI mice. Scale bar, 50μm. (K) Graph shows the percentage of Ki67+ cells in enlarged SVZ (n = 4 biological replicates; two-tailed unpaired t-test, P=0.0038). (L) The experimental scheme and staining for BrdU and EdU in E15 WT and hFOXM1-cKI mice. White arrows represent BrdU and EdU double-positive cells. Scale bars, 50μm(left);20μm(right). (M) Graph shows the percentage of BrdU and EdU double-positive cells in E15 WT and hFOXM1-cKI mice (n = 6 each group; two-tailed unpaired t-test, P=0.0413). D
A.Figure 9A. Confluent monolayers of fibroblasts were infected either with extra copy-expressing imyc-sTgAtg3 parental cell line in the presence of ATc (a), imyc-sTgAtg3-ΔAtg3 mutant cell line without ATc (b), mutant cell line in the presence of ATc (c), or mutant cell line pre-incubated with ATc before start of the experiment and maintained in the presence of ATc. Plaques (arrowheads) resulting from the lysis of host cells due to the multiplication of the parasites are only visible when TgAtg3 is still expressed. B. Mean plaque area comparisons between fibroblast layers infected with controls and TgAtg3-depleted parasites (a, b, c: legend as in A). Plaques observed with the mutant in the presence of ATc were significantly smaller than with control cell lines (* p<0.005, Student's T test). Data are mean from n = 3 independent experiments ±SEM. C. Numbers of parasites per vacuole are significantly lower in TgAtg3-depleted cell line (pre-incubated for 4 days with ATc) compared with controls 24 or 48 hours post invasion. Data are mean from n = 3 independent experiments ±SEM.
Figure 3A. Scheme illustrating replication in egg extracts of p2xDPCLeads, a plasmid containing two M.HpaII crosslinked on opposite strands. Under these conditions, the vast majority of replication forks encounter the DPC on their leading strand template B. p2xDPCLeads or p2xDPCSUMOLeads were replicated in egg extracts in the presence [α-32P]dATP. Where indicated, 50 μM of SUMOi was added to the extracts. Reaction samples were analyzed by native agarose gel electrophoresis. RI, replication intermediates; OC, open circular; SC, supercoiled. Red arrowheads indicate OC molecules that have not yet undergone repair.D. Scheme illustrating sequential SUMOylation and ubiquitylation of DPC-containing plasmids in non-replicating egg extracts. First, p4xDPCSUMO is incubated in NPE for 60-90 min to achieve poly-SUMOylation of the DPCs. NPE is then supplemented with recombinant RNF4 to a final concentration of 7 ng/μL (E) or an equal volume of CSF-arrested whole egg extract (F,G) to trigger DPC ubiquitylation. E. p4xDPCSUMO was incubated in NPE for 60 min and supplemented with buffer or recombinant RNF4 (WT or C130A). At the indicated time points following RNF4 addition, the DPC plasmid was recovered by DPC pull-down and immunoblotted against M.HpaII. F. pDPCSUMO was subjected to sequential extract addition as depicted in (D). Recombinant UBC9DN was added to NPE where indicated to block de novo SUMOylation. At the indicated time points following addition of CSF-arrested egg extract, the plasmid was recovered via DPC pull-down and immunoblotted against M.HpaII. G. pDPCSUMO was subjected to sequential extract addition as depicted in (D). Ninety min after addition of CSF-arrested extract, samples were recovered via DPC pull-down and treated with the SUMO protease Ulp1 or the ubiquitin protease Usp2 as indicated. Samples were then immunoblotted against M.HpaII. H. CSF-arrested extracts were either mock- or RNF4-depleted and recombinant His-RNF4 was supplemented to RNF4-depleted extracts to a final concentration of 7 ng/μL where indicated. Protein samples were immunoblotted with the indicated antibodies. I. p4xDPCSUMO was polySUMOylated in NPE, recovered via DPC pull-down and incubated in fresh CSF-arrested extract that was either mock- or RNF4-depleted. At indicated time points following CSF extract addition, the plasmid was recovered and immunoblotted against M.HpaII. J. As in (I), but using RNF4-depleted CSF-arrested extracts reconstituted with recombinant RNF4 WT or C130A from (H). K. CSF-arrested extracts were either mock- or PSMA1-depleted. Protein samples were immunoblotted with the indicated antibodies. L. p4xDPCSUMO was subjected to sequential addition of NPE and CSF-arrested extract as in (I). CSF-arrested extracts from (K) were supplemented with recombinant RNF4, and the DPC plasmid was recovered by DPC pull-down at the indicated time points and immunoblotted against M.HpaII.
Figure 2(A) Schematic representation of the sams genes in C. elegans. Boxes indicate exons. Coding regions of productive mRNAs are in orange. A truncated coding region with a premature termination codon in a pseudogene sams-2 is colored cyan. Double arrows illustrate regions deleted in mutant alleles indicated. (B) Alternative splicing of sams-3, sams-4 and sams-5 in unfed and fed worms without or with a protein synthesis inhibitor emetine. Synchronized L1 larvae of an NMD-deficient strain KH1668: smg-2 (yb979) (lanes 1-3) and a wild-type strain N2 (lanes 4-6) were incubated in S-complete medium alone (lanes 1, 4), with a standard E. coli strain OP50 (OD600=10.0) (lanes 2, 5) or OP50 supplemented with 10 mg/ml emetine (lanes 3, 6) for 3 hours at 20°C. Total RNAs were extracted from whole animals and subjected to semi-quantitative RT-PCR, whose products were analyzed by capillary electrophoresis. Representative gel-like presentation is indicated (n=3). Schematic structure of each PCR product is indicated on the right. Open reading frames (ORFs) for full-length and truncated proteins are in orange and cyan, respectively. rpl-12 was used as an unaffected control.
dTor loss impairs NPC insertion during nuclear growth in developing fat body cells A) Interphase NPC insertion occurs via inside-out extrusion of the INM, followed by INM/ONM fission and fusion (Otsuka & Ellenberg, 2018).Figure 5Interphase NPC insertion occurs via inside-out extrusion of the INM, followed by INM/ONM fission and fusion (Otsuka & Ellenberg, 2018). Nucleoporins are not depicted. The mature NPC, highlighting proteins examined in fat body cells. Green, cytosolic filaments; yellow, outer ring subcomplex; pale blue, inner ring subcomplex; brown, transmembrane nucleoporins; red, phenylalanine-glycine repeats (FG-Nups) that form the permeability barrier; white/blue, nuclear basket Nups. Fat body nuclei increase in size during development. Graph shows mean ± SD for each day during development of larvae. DO, day-old. Control (w-) and dTorKO fat body nuclei labeled with anti-LaminDm0, Megator/Tpr, mAb414, or expressing UAS Nup107GFP or UAS Nup358GFP with the Cg-Gal4 driver. Representative TEM of the NE of control and dTorKO 5DO fat body cells. Red arrows show nuclear pores. Nuc, nucleus; Cyto, cytosol. The percentage of NE in individual 5DO fat body cells that has normal INM, ONM, and NPC morphology. Black lines show the mean of each group. Representative TEM showing the NE of 5DO dLap1-/- fat body cells. Red arrows in (Q) show nuclear pores. Dashed yellow line highlights unusual circular membrane profiles.
Figure 7ZR-75-1 cells were treated for 1 hour (1h) or 5 days with 1 μM MK-2206, 1 μM GDC0941 or 3 μM 14h inhibitors, alone or in combination, as indicated. (A) The cell lysates were analysed by immunoblot using the indicated antibodies. ZR-75-1 cells were treated for 1 hour (1h) or 5 days with 1 μM MK-2206, 1 μM GDC0941 or 3 μM 14h inhibitors, alone or in combination, as indicated. (B) SGK3 (upper panel), Akt1 (middle panel) and S6K1 (lower panel) were immunoprecipitated from the same cell lysates and subjected to in vitro kinase assay by measuring phosphorylation of the Crosstide substrate peptide for kinases in the presence of 0.1 mM 32PATP in a 30 min, 30oC reaction. Kinase reactions are presented as means ± SD for triplicate reaction. Immunoprecipitates (IP) were also analysed by immunoblot with the indicated antibodies. (C) SGK3 was knocked down in ZR-75-1 cells by using shRNA probe B and compared to a control shRNA probe, named sh scramble. After infection, the cells were kept for 2 days in puromycin selection media and then seeded for the experiment. The cells were treated with 1 µM MK-2206 for 1 hour (1h) or 5 days. The cell lysates were subjected to immunoblot analysis with the indicated antibodies.
Figure 5A) Serum TSH and T4 levels in 3-month-old Tubb1-/- and wild-type mice. Numbers of animals tested were 10 wild-type males and 14 Tubb1-/- males for TSH and for 14 wild-type and 22 Tubb1-/-males for T4. Tubb1-/- mice had hypothyroidism with elevated TSH and decreased T4 versus wild-type mice. Student"s test, ** p< B) Nkx2-1 (in brown) immunostaining of adult thyroid tissue. Note the disorganisation of the thyroid tissue in Tubb1-/- versus wild-type mice C) Ultrastructural alterations shown by electron microscopy in wild-type and Tubb1-/- thyroid tissues. In Tubb1-/- tissues, note the disorganisation of secretion vesicles (white asterisks) and rods of identical density to secretion vesicles (white arrow). The ER is considerably dilated in Tubb1-/- thyrocytes. Representative views. Scale bars at the bottom left for each view. Co: colloi D) ER stress in Tubb1-/- thyroid tissue. Top: Chop and XBPs expression by quantitative PCR in adults, normalized to peptidylprolyl isomerase A. Experiments with at least three tissues per stage for each genotype; wt in black and Tubb1-/- in grey. Bottom: Chop and Actin protein expression by western blotting in three representative wild-type and Tubb1-/- mice. The Chop band quantification normalised for Actin confirms the increased Chop expression demonstrated by quantitative PCR in Tubb1-/- versus wild-type thyroids. All lanes are from the same blot, which was cut where indicated. Student"s test, *p<0.05 and ***p< E) Co-immunostaining of endoplasmic reticulum (ER) marker (KDEL, in red), thyroglobulin (Tg in green), and both merged (from left to right) in adult thyroid tissue. Note the thyrocyte disorganisation in Tubb1-/- mice
Figure 8Representative western blot analysis of MsrB2, LC3I/II and parkin using Parkinson's disease (PD) platelets. Quantification analysis of individual band intensity. (MsrB2; *p=0.0255, LC3II; *p=0.0101 vs. HC (n=5) and PD (n=12)). GAPDH served as the loading control. Platelet apoptosis (Annexin V) were measured by flow cytometry analysis in HC (n=9) and PD (n=13) platelets. (**p<0.01 vs. HC). The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.
Figure 3A. Protein sequence alignment of C-terminal regions of UPF3 proteins from different mammalian species (labeled on the left). B. Immunoblot showing levels of EJC and UPF proteins (on the right) in input or FLAG-IP samples from 3DKO#2 cells stably expressing different FLAG-tagged proteins indicated above each lane. D. Immunoblot showing EJC/UPF proteins (on the right) in input or FLAG-IP samples from 3DKO#2 cells stably expressing FLAG-tagged proteins given above each lane (hUPF3B = human UPF3B; hUPF3B-h3AC = human UPF3B protein with C-terminal domain from human UPF3A; hUPF3B-m3AC = human UPF3B protein with C-terminal domain from mouse UPF3A). C. Bar plots showing isoform specific RT-qPCR-based measurement of relative levels of PTC+ and PTC- isoforms of genes indicated below from wild-type (WT) or 3DKO#2 cells stably expressing the specified proteins. Relative levels from each replicate are shown by white circles. Error bars indicate standard errors of means. The asterisk (*) represents statistically significant difference (p<0.05, t-test) of indicated samples from EGFP-transfected control cells, which has a value of 1 (n=3 biological replicates). E. Bar plots of isoform specific RT-qPCR-based analysis of PTC+ and PTC- transcript levels from wild-type (WT) or 3DKO#2 cells stably expressing the proteins specified in the legend. Relative levels from each replicate are shown by white circles. The asterisk (*) represents statistically significant difference (p<0.05, t-test) of indicated samples from EGFP-transfected control cells, which has a value of 1 (n=3 biological replicates).
Figure 2(A). Serum AKG concentration-time profile obtained from male C57BL/6 mice (10 weeks) fed with normal chow before or after AKG gavage (10 mg/kg). The serum AKG level were tested at 0, 1, 2, 4 and 6 hrs after gavage (n = 8 per group). (B-D). Water intake (B), cumulative food intake (C) and body weight gain (D) of male C57BL/6 mice. At 12 weeks of age, mice were switched to HFD and received tap water or water supplemented with 2% AKG for 11 weeks (n = 9 per group). Fat and lean mass (E) of body composition from male mice after 11 weeks of AKG supplementation (n = 9 per group). representative images (F) of body composition from male mice after 11 weeks of AKG supplementation (n = 9 per group). (G-I). Weight index of BAT (G), gWAT (H) and iWAT (I) from male mice after 11 weeks of AKG supplementation (n = 9 per group). (J). Serum AKG level of male mice after 11 weeks of AKG supplementation (n = 9 per group). (K-L). Representative images (K) and quantification (L) of gWAT and iWAT HE staining from male mice after 11 weeks of AKG supplementation (n = 9 per group). (M-O). Water intake (M), cumulative food intake (N) and body weight gain (O) of female C57BL/6 mice. At 12 weeks of age, mice were switched to HFD and received tap water or water supplemented with 2% AKG for 11 weeks (n = 9 per group). (P-Q). Fat and lean mass index (P) and representative image (Q) of body composition from female mice after 11 weeks of AKG supplementation (n = 9 per group). (R-T). Weight index of BAT (R), gWAT (S) and iWAT (T) from female mice after 11 weeks of AKG supplementation (n = 9 per group). (U-V). Representative images (U) and quantification (V) of gWAT and iWAT HE staining from female mice after 11 weeks of AKG supplementation (n = 9 per group).
p53 inactivation enables NFATc1 to drive dedifferentiated PDAC A Kaplan–Meier curves showing survival of KNC, KPNC, and KP∆NC mice under the control of p48‐Cre pancreas‐specific promoters (P  0.Figure 3A Kaplan-Meier curves showing survival of KNC, KPNC, and KPΔNC mice under the control of p48‐Cre pancreas‐specific promoters (P 0.0001 for KPNC or KPΔNC cohorts versus KNC, log‐rank test, for pairwise combination). B Pathological features of KPNC and KP∆NC mice before tumor extraction. Pancreatic tissue is encircled. C, D HE and corresponding immunohistochemistry stainings of (C) KPNC and (D) KP∆NCmouse tumors. Scale bars, 100 μm. E Tumor grading of PDAC from KPNC mice (n = 10).
Figure 2(A, B) U2OS Flp-In/T-Rex parental (WT) and RAP80-/- cells were transfected with siRNA pools targeting BRCA1, RNF8, RNF168 or BARD1 or with a non-targeting control siRNA (CTRL). 48 h post-transfection cells were irradiated (2 Gy) and processed for immunofluorescence 1 h post-IR treatment using antibodies against BRCA1 and γH2AX. Quantitation of the percentage of cells with > 5 BRCA1 foci that colocalize with γH2AX is shown in (A). A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D. (n=3 biological replicates). Representative micrographs are shown in (B). (C, D) Parental (WT) RPE1-hTERT p53-/- Cas9, RNF8-/-, and RNF168-/- cells were treated with either a non-targeting siRNA pool (CTRL) or a pool targeting RAP80. 48 h post-transfection cells were irradiated (2 Gy) and processed for immunofluorescence 1 h post-IR treatment using antibodies against BRCA1 and γH2AX. Quantitation of the percentage of cells with > 10 BRCA1 foci that colocalize with γH2AX is shown in (C). A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D. (n=3 biological replicates). Representative micrographs are shown in (D). All scale bars are 5 μm.