EMBO/sd-character-level-ner huggingface dataset:
a) Histogram showing relative phospholipid levels in CHO cells treated with 0.f4(a) Histogram showing relative phospholipid levels in CHO cells treated with 0.3% ethanol for 30 min before cell harvesting. Total PLD activity is measured as phosphatidylethanol (PEtOH) levels under the following conditions: normal medium (Nm), serum-free medium (SFM), serum-free medium with 100 nM wortmannin (SFM+W), nutrient deprivation (0.5 h St; 1.5 h St; 3.0 h St), 30 min of nutrient deprivation in presence of 100nM wortmannin (0.5h St+W). Other phospholipids were used as controls: phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC). Values denote means±s.e.m. (n=9, except for SFM+W and 0.5 h St+W, where n=3 and 5, respectively). **P0.01; *, P0.05. (b) Western blot analysis of HA-PLD1 and endogenous LC3 levels in post-nuclear supernatant (PNS, upper panel), as well as in cytosolic/soluble (S) and particulate (P) fractions prepared from PNS after high-speed centrifugation (middle panel). GAPDH was used as loading control in PNS fractions. HeLa cells were transfected with expression vectors encoding HA-PLD1 for 24 h and subjected to 2 h of nutrient deprivation in the presence or absence of the PI3K inhibitor wortmannin (100 nM). Lower panel: quantitative analysis of HA-PLD1 immunoreactivity expressed as P/S ratio. Conditions were as follows: normal medium (Nm); normal medium with wortmannin (Nm+W); starvation condition (St); starvation condition+wortmannin (St+W). Values denote means±s.e.m. (n=3). (c) Confocal analysis of the co-localization between HA-PLD1 and Lamp1 in HeLa cells subjected to nutrient deprivation for 2 h in the presence (ST+W) or absence (St) of wortmannin 100 nM. Insets show high magnification of the selected areas. Scale bar, 5 μm.
Figure 2To screen for ESCRT proteins that are involved in the endolysosomal repair process, cells were incubated with 250 μM LLOMe for 30 min and processed for immunofluorescence. TSG101, a component of the ESCRT-I complex, and EAP30, an ESCRT-II protein, are clearly recruited to the sites of damage compared to the DMSO control (Ctrl). As shown, the ESCRT-III complex together with ALIX is recruited to damaged endolysosomes. In contrast, HRS, a component of the ESCRT-0 complex, and HD-PTP show no recruitment to damaged endomembranes. Number of foci per cell was quantified from >85 cells per condition from three independent experiments and are presented as mean ± SD. Statistical significance for number of foci per cell in Ctrl versus LLOMe treatment was determined using Student's t‐test, the p-values for which are: HRS p=0.9257, GAL3 p=0.0050; TSG101 p=0.0243, GAL3 p=0.0489 ; EAP30 p=0.0006, GAL3 p=0.0307; CHMP2A p=0.0284, GAL3 p=0.0260; CHMP4B p=0.0028, GAL3 p=0.0414; VPS4A p=0.0309, GAL3 p=0.0461; ALIX p=0.0249, GAL3 p=0.0471; HD-PTP p=0.4898, GAL3 p=0.0105. Data information: Scale bars: 5 μm and 1 μm (inset).
Figure 3Representative area of the 3D cryoEM potential map for ex vivo S-Ena, at 3.2 Å resolution. An octameric peptide with sequence FCMTIRY was deduced de novo from the cryoEM potential map (shown in sticks) and used for a BLAST search of the B. cereus NVH 0075-95 genome. The red letters indicate amino acids that differ between KMP91697.1 and KMP91698.1 (see B). Multiple sequence alignment of 3 ORF's (KMP91697.1, KMP91698.1 and KMP91699.1) corresponding to DUF3992 containing proteins. Sequence motifs corresponding or similar to that deduced from the EM potential map are shaded in cyan. Secondary structure and structural elements as determined from the built model (see Fig 2) are shown schematically above the sequences (Ntc: N-terminal connecter; arrows correspond to β-strands, labelled as in Fig 2). Contiguous areas of >50% similarity are boxed blue. SDS-PAGE of recEna1B, treated with β-mercaptoethanol or TEV protease (to remove N-terminal 6xHis tag) as indicated. Bands with apparent MW of ~13 and ~15 kDa correspond recEna1B monomer with (Ena1B6His) and without (Ena1BC) the 6xHis tag and TEV recognition site, respectively. In the uncleaved sample, a band running at ~ 30 kDa (labeled *) corresponds to a non-physiological disulfide bound S-Ena1B dimer. Lower intensity in the cleaved recEna1B results from a loss of monomers to SDS-resistant high molecular weight complexes stuck in the stacking gel. Negative stain TEM images of rec1Ena1B oligomers formed after refolding, but prior to TEV removal of the N-terminal 6xHis tag. Close-up view that shows recEna1B oligomers form open crescents similar in dimensions and shape to single helical turns or arcs found in the S-Ena fiber (model - right). Steric hindrance by the 6xHis is thought to arrest recEna1B polymerization into single helical arcs. Negative stain image and 2D classification of S-Ena-like fibers formed after TEV digestion of recEna1B. Upon removal of the N-terminal 6xHis tag, recEna1B readily assembles into fibers with helical properties closely resembling those found for ex vivo S-Enas.
Overexpression of Mammalian NOS Isoforms Inhibits Autophagosome Synthesis.Figure 5(A-D) Immunoblot analyses with anti-LC3, along with anti-nNOS, anti-iNOS, and anti-eNOS antibodies, show decreased LC3-II levels in stable HEK293 cell lines overexpressing nNOS (A and D), iNOS-GFP (B and D), or eNOS (C and D) compared to HEK293 control cells in the presence or absence of bafilomycin A1. (E and F) mCherry-LC3 vesicles were reduced in mCherry-LC3-transfected stable HEK293 NOS cell lines compared to HEK293 control cells cultured in HBSS, an effect that was restored in the NOS cells by L-NAME (F). Images were acquired by a confocal microscope (E). (G and H) Confocal microscopy images (G) and immunofluorescence analysis with anti-Atg16 antibody show that stable HEK293 NOS cell lines had fewer Atg16-positive structures under starvation, compared to HEK293 control cells (H). Graphical data denote mean ± SEM.
Figure 3A-B) On the left, number of proteins that differ significantly (p-value < 0.05 in A; q-value < 0.05 in B) in abundance by AD status within each cohort. On the right, number of proteins thereof that have a consistent directionality of either elevated or reduced abundance in AD CSF in pairwise comparisons of cohorts. Correlation of protein AD/non-AD fold changes in pairwise combinations of two cohorts each. Combinations are Sweden vs Magdeburg/Kiel Proteins included differ significantly (p < 0.05) and consistently in abundance by AD status in both cohorts each. Correlation of protein AD/non-AD fold changes in pairwise combinations of two cohorts each. Combinations are Sweden vs Berlin (D), and Magdeburg/Kiel vs Berlin (E). Proteins included differ significantly (p < 0.05) and consistently in abundance by AD status in both cohorts each. Proteins that differ significantly (p-value < 0.05 in E; q-value < 0.05 in F) in abundance by AD status across all three cohorts. Z-scored abundances of proteins in the AD and non-AD groups of all cohorts shown by the heat map Hierarchical clustering separates AD from non-AD groups. Pyruvat kinase PKM (PKM) was quantified in two isoforms and UniProt IDs are given in parenthesis. Black frames highlight proteins with consistent AD/non-AD fold changes across cohorts. Proteins that differ significantly (p-value < 0.05 in E; q-value < 0.05 in F) in abundance by AD status across all three cohorts. Z-scored abundances of proteins in the AD and non-AD groups of all cohorts shown by the heat map Hierarchical clustering separates AD from non-AD groups. Pyruvat kinase PKM (PKM) was quantified in two isoforms and UniProt IDs are given in parenthesis. Black frames highlight proteins with consistent AD/non-AD fold changes across cohorts.
Figure 3A) Schematic diagram of full length TBC1D14, indicating the C-terminal TBC domain (shaded), and truncation mutants used in subsequent analyses. B) HEK 293A cell lysates (from approximately 8 × 106 cells per construct) expressing the indicated GFP fusions were subjected to IP and immunoblotting for GFP, TRAPPC12 and TRAPPC4. Data are representative of 3 independent experiments.
Exon skipping-based rescue of SRF target gene expression and Nbr1/p62/SQSTM1/MURF2 subcellular distribution in TTN Ser14450fsX4 iPSC-derived cardiomyocytes.Figure 4qRT-PCR analysis of SRF target genes (MYH6, MYH7 and ACTC1) in CTR and DCM single cardiomyocytes under basal condition (no infection, NI) and after infection with control U7snRNA-ScrAONs-IRES-GFP (Scr-AON) and the U7snRNA-TTNAONs-IRES-GFP (TTN-AON) lentiviruses. Statistical difference was tested using the two-sided Student's t-test (**P = 0.009, CTR Scr-AON versus DCM Scr-AON; *P = 0.04, DCM Scr-AON versus DCM TTN-AON for MYH6; **P = 0.002, CTR Scr-AON versus DCM Scr-AON; **P = 0.002, DCM Scr-AON versus DCM TTN-AON for MYH7; **P = 0.004, CTR Scr-AON versus DCM Scr-AON; *P = 0.02, DCM Scr-AON versus DCM TTN-AON for ACTC1). No significant differences were observed comparing the CTR NI, CTR Scr-AON and CTR TTN-AON groups and comparing the DCM NI and DCM Scr-AON groups. Expression values were relative to CTR Scr-AON, normalized to GAPDH, and presented as mean ± SEM, n = 3. Immunofluorescence images showing normal (a, b, e, f, i, l, o, p) and altered (c, d, g, h, m, n, q, r) intracellular distribution of SRF (a and b, nuclear; c and d, cytoplasmic), MURF2 (e and f, sarcomeric; g and h, nuclear), Nbr1 (i and l, sarcomeric; m and n, diffused), and SQSTM1/p62 (o and p, sarcomeric; q and r, diffused) in representative single cardiomyocytes (left). Sarcomeres are marked by α-actinin. On the right, percentage of CTR and DCM cardiomyocytes showing cytoplasmic expression of SRF, nuclear accumulation of MURF2, and diffused expression of Nbr1 and of SQSTM1/p62 after infection with control U7snRNA-ScrambleAONs-IRES-GFP (Scr-AON) and the U7snRNA-TTNAONs-IRES-GFP (TTN-AON) lentiviruses (right). Data represent mean values ± SEM from two control and two DCM clones. Statistical difference was tested using the two-sided chi-squared test (CTR Scr-AON: n = 874, n = 874, n = 882 and n = 890, CTR TTN-AON: n = 880, n = 990, n = 878 and n = 890, DCM Scr-AON: n = 890, n = 887, n = 884 and n = 886, DCM TTN-AON: n = 900, n = 875, n = 899 and n = 891 for SRF, MURF2, Nbr1 and SQSTM1/p62, respectively; *P = 0.01, CTR Scr-AON versus DCM Scr-AON; *P = 0.04, DCM Scr-AON versus DCM TTN-AON for SRF; *P = 0.02, CTR Scr-AON versus DCM Scr-AON; *P = 0.04, DCM Scr-AON versus DCM TTN-AON for MURF2; *P = 0.03, CTR Scr-AON versus DCM Scr-AON; *P = 0.03, DCM Scr-AON versus DCM TTN-AON for Nbr1; **P = 0.009, CTR Scr-AON versus DCM Scr-AON; *P = 0.01, DCM Scr-AON versus DCM TTN-AON for SQSTM1/p62). No significant differences were observed comparing CTR Scr-AON and CTR TTN-AON groups. Scale bars, 50 μm.
Figure 6USP36 promotes the binding of Nop58 to box C/D snoRNAs. H1299 cells transfected with Flag-Nop58 and V5-USP36 individually or together were assayed by RNA-IP with anti-Flag or control mouse IgG, followed by RT-qPCR detection of the indicated box C/D snoRNAs (A). Shown is one representative experiment of three independent experiments. Data were presented as mean ± SD, n=3 technical replicates. *P<0.05, compared to Nop58 alone (Student's t-test). USP36 promotes the binding of Nop58 to box C/D snoRNAs. H1299 cells transfected with Flag-Nop58 and V5-USP36 individually or together were assayed by RNA-IP with anti-Flag or control mouse IgG, followed by RT-qPCR detection of the indicated box C/D snoRNAs (A). The expression of Nop58 and USP36 proteins is shown in (B). Shown is one representative experiment of three independent experiments. Data were presented as mean ± SD, n=3 technical replicates. *P<0.05, compared to Nop58 alone (Student's t-test). USP36 promotes the binding of Nhp2 to snoRNAs. H1299 cells transfected with Flag-Nhp2 and V5-USP36 individually or together were assayed by RNA-IP with anti-Flag or control mouse IgG, followed by RT-qPCR detection of the indicated box H/ACA snoRNAs (C). Shown is one representative experiment of three independent experiments. Data were presented as mean ± SD, n=3 technical replicates. *P<0.05; **P<0.01, compared to Nhp2 alone (Student's t-test). USP36 promotes the binding of Nhp2 to snoRNAs. H1299 cells transfected with Flag-Nhp2 and V5-USP36 individually or together were assayed by RNA-IP with anti-Flag or control mouse IgG, followed by RT-qPCR detection of the indicated box H/ACA snoRNAs (C). The expression of Nhp2 and USP36 proteins is shown in (D). Shown is one representative experiment of three independent experiments. Data were presented as mean ± SD, n=3 technical replicates. *P<0.05; **P<0.01, compared to Nhp2 alone (Student's t-test).
Figure 3A, B WT and lincRNA-EPS-/- iBMMs were infected with VSV-GFP (MOI 0.1) for 6 h, and the lincRNA-EPS transcripts were measured by RT-qPCR (A), and the fluorescence of GFP were checked by microscope (B). C Cell supernatant from VSV-infected WT and lincRNA-EPS-/- iBMMs (MOI 0.1, 8 h) were collected and the viral titer were measured by plaque assay. D, E WT and lincRNA-EPS-/- iBMMs were infected with SeV (MOI 1) for 8 h (D) or HSV-1 (MOI 5) for 12 h (E). The viral RNA was measured by RT-qPCR. F Peritoneal macrophages isolated from WT and lincRNA-EPS-/- mice were infected with VSV (MOI 1) for 10 h, and the viral titer were measured by TCID50 assay. G Eight weeks female lincRNA-EPS-/- mice (n=12) and WT littermates (n=12) were injected (i.v.) with VSV (lethal dose, 1×108 pfu/g), and the survival situation was monitored for 120 h. Eight weeks female lincRNA-EPS-/- mice (n=5) and WT littermates (n=5) were injected (i.v.) with VSV (sub-lethal dose, 6×107 pfu/g) for 12 h, and negative control groups were injected with PBS (n=3). Pathological section of liver and lung were harvested by H&E staining (H). Serum, liver, and lung from the VSV-infected mice were collected. The viral load of serum and tissue homogenate were measured by TCID50 assay (I), and the serum IFN-β protein level were checked by ELISA (J).
Figure 3A In silico identification of miR-34a binding sites in the Pdgfra 3′-UTR B Immunoblot detection of PDGFRα levels in MLg cells after treatment with scrambled microRNA (SCR) or a miR-34a (MIM34a) mimic (n = 3 separate cell cultures for each group). C Quantitative RT-PCR detection of miR-34a/b/c-5p levels in MLg cells in vitro, maintained under 21% O2 or 85% O2 (n = 3 separate cell cultures for each group). D Immunoblot detection of PDGFRα levels in the lungs of mouse pups (n = 6 animals for each group) at post-natal day (P) 5, during normal (21% O2) and aberrant (85% O2) alveolarization. E Immunoblot detection of PDGFRα levels in MLg cells in vitro, maintained under 21% O2 or 85% O2, where cells had been transfected wither with a scrambled (SCR) antimiR, or an antimiR directed against miR-34a (A34a) (n = 3 separate cell cultures for each group). F Quantitative RT-PCR detection of miR-34a-5p levels in PDGFRα+ cells, sorted by FACS from the lungs of mouse pups (n = 4 animals for each group; data from an independent repetition are provided in Appendix Fig S5) at P5, maintained under 21% O2 or 85% O2 from birth. G Schematic illustration of the generation of a conditional, inducible deletion-ready mouse strain, where administration of tamoxifen (Tmxfn) abrogated miR-34a expression in Pdgfra‑expressing cells (denoted miR‑34ai∆PC/i∆PC). H Quantitative RT-PCR detection of miR-34a-5p levels in PDGFRα+ cells, sorted by FACS from the lungs of either wild-type (34awt/wt) mouse pups, or mouse pups in which miR-34a expression in Pdgfra‑expressing cells (34ai∆PC/i∆PC) at P5 (n = 4 animals for each group). I Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from 34awt/wt or 34ai∆PC/i∆PC mouse pups at P14 during aberrant (85% O2) alveolarization, compared with 34ai∆PC/i∆PC during normal (21% O2) alveolarization (scale bar, 50 µm). Data are representative of observations made in four other experiments. J Quantification of total number of alveoli by design-based stereology in 34awt/wt or 34ai∆PC/i∆PC mouse pups at P14, during normal and aberrant alveolarization (n = 5 animals for each group). K Quantification of mean septal thickness by design-based stereology in 34awt/wt or 34ai∆PC/i∆PC mouse pups at P14, during normal and aberrant alveolarization (n = 5 animals for each group).
Hdac3 and Dax1 are required for PrE lineage restriction in response to FGF, LIF, and RA.Figure 2Nanog>GFP and Gata6::mCherry fluorescence intensities of WT and Hdac3-/- Jak(i) blocks LIF, LDN19 BMP4 and PD03 FGF signaling. Average and SD of at least two independent clones. Nanog>GFP and Gata6::mCherry fluorescence intensities of WT and Hdac3-/- Fraction of Gata6::mCherry positive cells in indicated genotypes and conditions after 3d Jak(i) blocks LIF, LDN19 BMP4 and PD03 FGF signaling. Average and SD of at least two independent clones. Nanog>GFP and Gata6::mCherry fluorescence intensities of WT and Hdac3-/- Fraction of Gata6::mCherry positive cells in indicated genotypes and conditions after 4d Jak(i) blocks LIF, LDN19 BMP4 and PD03 FGF signaling. Average and SD of at least two independent clones. Nanog>GFP and Gata6::mCherry fluorescence intensities of WT and Dax1-/- (D) cells after 3d in indicated conditions. Fraction of Gata6::mCherry positive cells in indicated genotypes and conditions after 3d Nanog>GFP and Gata6::mCherry fluorescence intensities of WT and Dax1-/- (D) cells after 3d in indicated conditions. Fraction of Gata6::mCherry positive cells in indicated genotypes and conditions after 3d Average and SD of at least two independent clones. Quantification of spatial lineage segregation in spheroids. Representative spheroid segmentations of indicated genotypes Scale bar: 25 μm. Z-score-normalized fluorescence distributions in mutants compared to WT spheroids (G). Positive Z-scores indicate peripheral, outside, and negative Z-scores central, inside, enrichment. Scale bar: 25 μm.
sinhcaf is required for female sexual differentiation in zebrafish.Figure 1(A) RT-PCR analysis for temporal expression of sinhcaf mRNA during embryogenesis and early larval developmental stages. NC indicates the no template control. (B) Time-course expression of sinhcaf revealed by whole-mount in situ hybridization. Scale bars, 100 μ (C) RT-PCR analysis for spatial expression of sinhcaf mRNA in adult tissues. (D) The location of the TALEN-binding sites (underlined) on zebrafish sinhcaf gene and mutant line of TALEN-targeted sinhcaf alleles. (E) Relative mRNA level of sinhcaf in bud stage WT and MZsinhcaf -/- mutant embryos as measured by RT-qPCR. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using unpaired two-tailed Student's t-test. (F) Fluorescence microscopy of 10hpf Tg(CMV:EGFP;CMV:sinhcaf) zebrafish embryos. Scale bar, 400 μm. (G) Relative mRNA level of sinhcaf in 10hpf or 24hpf WT and Tg(CMV:EGFP;CMV:sinhcaf) embryos as measured by RT-qPCR. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using 2-way ANOVA followed by Holm-Sidak post-hoc test. (H) Percentage of male zebrafish in WT (n = 274), MZsinhcaf -/- (n = 166), Tg(CMV:EGFP;CMV:sinhcaf) (n = 158) and MZsinhcaf -/-;Tg(CMV:EGFP;CMV:sinhcaf) (n = 135) adult zebrafish. Data shown are mean ± SEM (n = the number of fish analyzed). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. The different letters (a, b, c) indicate a significant difference between the means, p<0.05.
Figure 6 A) anti-TFEB immunofluorescence on rapamycin- and rilmenidine-treated WT and Cox15sm/sm muscles vs. untreated (UT) samples. CD11C (blue signal) indicates inflammatory cells. Right panel: quantification of n=3 animals/group. The scale bars correspond to 30µm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test B) Quantification of the TFEB-positive nuclei (n=3/group). Quantification was performed using Imaris spots surface excluding the CD11C positive nuclei. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test *p=0.0199 (WT vs. WT+rapamycin), *p=0.0150 (Cox15sm/sm vs. Cox15sm/sm+rapamycin), ***p=0.0001
Figure 6A. Schematic representation of TurboID-tagged (T.I.) mCherry (negative control) and TurboID-tagged BRD9. V5 indicates the location of a V5-tag. Numbers represent amino-acid residues of the final constructs. Both constructs were stably expressed in A549 cells. B. Overview of the proximity labeling workflow: interactomes of T.I.-mCherry or T.I.-BRD9 were determined in the presence/absence of a 60min stimulation with 1000 IU/mL of IFN-α2 and 500μM biotin for 30min. C. Schematic of the non-canonical BAF (ncBAF) complex, with factors identified with high confidence (SaintExpress Score ≥ 0.99) by proximity labeling to be specific interactors of BRD9 (as compared to mCherry) colored in red. D. GO term analysis of the 145 interactors identified to be specific to BRD9. E. Comparative BRD9 interactome analysis in the presence/absence of a 60min stimulation with 1000 IU/mL of IFN-α2. Note that FDR for STAT2 was 0, but has been set to 0.001 for visualization purposes. F. Western blot analysis of samples from independent small-scale streptavidin purifications (termed 'Eluates') following the proximity labeling approach outlined in (B). The indicated proteins were detected with specific antibodies. M indicates the marker lane. Data are representative of at least two biological replicates.
Figure 7(A) Correlation of ZEB1 mRNA expression with the expression of YAP, FOSL1 and JUN across all breast cancer cell lines included in the cancer cell line encyclopedia (CCLE). rs: Spearman's rank correlation coefficient. (B) Heatmap of RNA-seq expression data of all CCLE breast cancer cell lines.
Poly-GA containing neurons display delayed cell-death profile Primary rat neurons were co-transfected with Td-tomato and eGFP-GAn plasmids or eGFP-alone.Figure 3Primary rat neurons were co-transfected with Td-tomato and eGFP-GAn plasmids or eGFP-alone. The same neurons were imaged at 24-hour intervals to assess viability. A) Representative fields (right) of eGFP and eGFP-GA50 cortical neurons at days 1, 7, and 13 post-transfection follow the survival of individual cells over time (white arrows). 20x magnification, scale bar indicates 25μm. B) Kaplan-Meier survival analysis of cortical neurons followed for 13 days post-transfection. C) Kaplan-Meier survival analysis (left) of motor neurons followed for 5 days post-transfection.