EMBO/sd-character-level-ner huggingface dataset:
Schematic of RNAi screens. SCP-1 cells expressing FUS-DDIT3 or EV were transduced with Module 1 of the DECIPHER Pooled Lentiviral Human Genome‑Wide shRNA Library. Half of the cells were harvested on day 3 (baseline sample) and day 12 (drop-out sample), respectively, and shRNA abundance was determined by next-generation sequencing (NGS).
(A, B) Depletion of plasma membrane PI(4,5) P2 with the FRB/FKBP system prevented the ANO8-mediated increase in STIM1-Orai1 current measured in pipette solutions containing 3 mM EGTA (A) or 10 mM BAPTA (B). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.
The ERMIT assay relies on the signaling properties of IRE1, one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER. The assay can be applied to heterodimerization or homodimerization events. A) Upper panel: NMR structure of the non-covalent dimer of AGR2 (PDB ID: 2LNS). The dimer domain is highlighted with a yellow circle (residue 54-70 of each monomer). Monomer A is colored green and monomer B in pink. The figure was generated in Chimera. Lower panel: a close-up illustration of the dimer domain, showing that the dimer is stabilized through two salt-bridges between E60 and K64 of each monomer.
g, h, Western blotting analysis of protein levels of indicated proteins in MEFs with indicated genotypes in normal medium or subjected to 24 hserum starvation (SS). Quantified OFD1 level was normalized with alpha-tubulin. Similar results were observed in three independent experiments.
E Real-time RT-PCR analysis of mRNA levels of GATA-1/heme-activated genes in b-estradiol-treated WT1 or double-mutant clone 1 expressing shRNA targeting Bach1. P values were calculated with respect to b-estradiol-treated control double-mutant clone 1 (n = 4, mean +/- SE).
A. Cx43 was immunoprecipitated (IP) from HEK293Cx43+ cells, after which interaction with hnRNPA2B1 and hnRNPQ was analyzed by WB.
(A) Venn diagrams summarize global changes in gene expression caused by the inactivation of Foxn1 or Aff4 and detected by RNA-seq in murine skin at P6. The upregulated or downregulated gene populations of each mutant - and the extents to which they overlap - are indicated.
(C) Schema of the construct used for overexpression of the candidate genes (under the control of the beta-actin promoter), and expression of GFP in the heart (as an internal control for genomic integration).
(A) Upon induction of autophagy, a membrane sac called the isolation membrane (IM) forms and engulfs portions of the cytoplasm. Sealing of its edges gives rise to the double-membrane bound autophagosome. Fusion of the outer membrane with a lysosome results in formation of an autolysosome, in which the inner autophagosomal membrane and its contents are degraded.
(A) LETM1 expression was suppressed in HeLa cells by transfection with targeted siRNAs (siR1, siR2 or siR3). A non-target dsRNA (NT) served as control. Cells were fixed and immunolabeled with anti-DNA antibody (green). A higher magnification of selected mtDNA foci is shown beside each picture. (B) Quantification of cells in (A) displaying mtDNA abnormalities. At least 50 cells per siRNA were counted from 4 (siR2) and 5 (siR1 or siR3) independent experiments. Data are expressed as means ± SEM. ***P<0.001 (One-way ANOVA)
H RT-qPCR analysis of viral RNA levels in U937, THP1, HT1080, 2fTGH and A549 cells infected with VSV (MOI=1.0, 24 hrs) immediately after addition of NaCl (+34 mM). I RAW264.7 cells were infected with VSV/H1N1/SeV (MOI=1.0, 24 hrs) immediately after addition of NaCl (+34 mM), or were transfected with HBV-replicon or HCV-replicon and then treated with additional NaCl (+34 mM) for 12 hrs. Viral RNA levels were analyzed by RT-qPCR. Data information: Data show the mean and SD of four or three biological replicates. For all statistical testing, p values were calculated using two-tailed unpaired Student's t-test. NS, not significant (p > 0.05). *p < 0.05, **p < 0.01 and ***p < 0.001.
C) Stable HEK293T lines were infected with SARS-CoV-2 (MOI 1) for 24 h and analyzed for percent infection by flow cytometry. Representative example plots are shown for each cell line.
a) Schematic representation of the different variants of UnaG used in the four indicated hypoxia-reporter constructs. dUnaG is identical to Fig. 1a, oUnaG is destabilized by in frame fusion with the oxygen-dependent degradation domain sequence (ODDD) from HIF1a (aa338-608), doUnaG is destabilized by both degradation motifs.
qPCR analysis of p-Stat5-associated growth genes in the livers of 9-week-old mice (n=6 per group, 2 independent repeats). Ribosomal L32, loading control for qPCR analysis.
A) 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<
N-glycosylation of endogenous SPPL2c is similar to the overexpressed protein. Total lysates from testis of wild type or SPPL2c-/- mice were treated
G. Comparison of cell growth after DMSO, 20 µg/mL 4D7, IgG or 100 nM ruxolitinib treatment over 4 days of ruxolitinib-resistant TF-1 TpoR CALRdel61. Cells counted using Trypan Blue exclusion (n=3 biological replicates with 3 technical replicates). H. Number of colonies observed from cells plated in MethoCult following 72 hours of treatment with DMSO, 20 µg/mL 4D7, IgG or 100nM ruxolitinib performed in ruxolitinib-resistant TF-1 TpoR CALRdel61 cells (n=3 biological replicates with 3 technical replicates). Data information: Error bars on represent standard deviation. All P values are unpaired Student's unpaired t-test.
Representative histopathological analysis for lung sections of WT (B) mice that were infected with M.tb and subsequent left untreated (Mock) or treated with EVs from uninfected BMMs (EVs_Control), EVs from M.tb-infected BMMs (EVs), moxifloxacin (MXF), or combination of EVs and MXF (EVs+MXF). Data shown is representative of two independent experiments. The results are the mean ± SD (n = 4 mice per group). Scale bar, 100 μM
6-week-old male CD36 wild-type (WT) or knockout mice (CD36KO) were administered with AAV-GFP (Ctrl) or AAV-KLF10 (KLF10OE) through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (G) Serum AST and ALT levels. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA
Invasion of H1299control and H1299RASSF1A cells through three-dimensional collagen matrix coated inserts, over 24 hours (n=3). Scale bars 100µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test 2-(tailed) of n=3 experiments and error bars represent the mean ± S.E.M.
Active EAE was induced by subcutaneous immunization with CFA/MOG35-55. Cells were isolated from the CNS of EAE animals at different stages of the disease (pre-onset [day 8-9], peak [day 14-17], chronic [day 22-27]; data from at least two independent experiments). Representative samples are shown, which were analyzed by flow cytometry for the expression of the markers CD11c, CD11b and Ly6C/G (pre-gated on lympho-monocytic cells by FSC/SSC; IAb+ expression was confirmed for CD11c+ cells in separate stainings).(A) In the CNS, there was a strong increase of CD11c+ proportions over the different phases of the disease. The composition of CD11c+ subpopulations changed over time, starting with a preponderance of CD11c+Ly6C/G- conventional DCs before onset of EAE. In the peak and chronic phase there was a continuous increase in the proportion of monocyte-derived CD11c+ cells.(B) For the CNS, the quantification of the absolute numbers shows a strong increase in CD11c+ cells during the course of EAE.(C) In the CNS, the proportions of the subpopulations show an increasing proportion of monocyte-derived CD11c+ cells.
(C) The different number of 4T1 cells were injected into the BALB/c mice. n = 10. Data information: Two-tailed Paired Student's t test The data represent mean ± SD.
NICD interaction with FBXW7 at endogenous levels in HEK293 cells. 500 µg of HEK293 cell lysates treated with DMSO or MLN4924 were subjected to immunoprecipitation using FBXW7 antibody, or IgG antibody as negative control, and precipitated material was analysed by western blot using NICD antibody. Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency. 10% of cell lysate before immunoprecipitation was used as input control and β-Actin served as loading control.   Quantification of the density of western blot bands in (A) performed by ImageJ software. Data are expressed as percentage changes compared to MLN4924. All data represent the mean ± SEM from three independent experiments. Student t-test was used to determine p values, with **p≤0.01.
(C) Maximal intensity projection of a confocal z-stack of 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA, GFP-Buc mRNA + RFP mRNA + Control MO and GFP-Buc mRNA + RFP mRNA + kif26ab MO. White arrowheads point to cleavage furrows of 4-cell embryos.
Mean fluorescence intensity (MFI) of extracellular TLR2, TLR4 and CD14 expression on naïve Gata6-WT and Gata6-KOmye pMɸ. n = 4 to 7 individual mice per group.
D Cells were treated with ATP8A1 siRNA1 for 72 h. ATP8A2‐1D4 (WT or mutants) and myc‐CDC50A were transfected 48 h after siRNA transfection. The cells were then stained for EHD1 and 1D4‐tag. Scale bars, 10 μm.
Western blotting of endogenous Egg immunopurified from OSCs. Anti-Egg antibodies were used for immunoprecipitation (IP). Anti-Egg and anti-Ub antibodies were used for western blotting (WB). Input: OSC total lysates used for IP. n.i.: nonimmune IgG used as an IP negative control.
Neuronal activity was induced with Chr2 stimulation by performing trains of LED light pulses at various frequencies: No stim = 0 Hz; Low = 1 Hz; Med = 10 Hz; High = 100 Hz. Trains included 30 s of light pulses followed by 25 s of recovery for desensitized ChR2; trains were performed 20 times.
Immunofluorescent staining of hCOs of day30. RGCC and PAX6 were co-stained. Scale bars: 100 μm.
(B) WT and FAK−/− PEMs were pretreated with IFN-γ (50 ng/ml), rapamycin (4 µm), or left untreated before incubation with ΔinvG Salmonella for 3 hours. Bacterial survival was then assayed as described in (A). Values are means ± SEM, N = 6, *p<0.05.
a) HeLa cells stably expressing GFP-VPS35 WT and D620N were treated with bafilomycin A1 or DMSO vehicle control. Endogenous LC3-II and tubulin levels were examined by western blot. A representative experiment of six experiments is shown. (b) Quantification of the representative experiment in triplicate shown in a, in which endogenous LC3-II levels are normalized to tubulin and expressed as a ratio of levels in WT. ***P=7.86 × 10−6 (DMSO) and 3.81 × 10−5 (Baf) by 2-tailed Student's t-test.
H-K GSEA analysis showing the significant down-regulation of genes in GO biological processes: chemokine signaling pathway (H), T cell receptor signaling pathway (I), innate immune response (J) and cytokine receptor interaction (K). The x-axis represents that the mouse genes are ranked from the most up-regulated (left end) to the most down-regulated (right end) in comparison of syngeneic tumor from Hepa1-6 tumor-bearing KO to WT mice (n=4). The y-axis is an enrichment score (ES), which goes up (>0) and down (<0) representing the enrichment of up-regulated genes and down-regulated genes, respectively. NES is the normalized enrichment score. p-value was calculated by permutation.
C. Induction of the ATP hydrolysis activity of MavQ by PtdIns is not affected by wortmannin. Recombinant MavQ was incubated with increasing amounts of wortmannin for 30 min at room temperature prior to the addition of PtdIns and ATP. The wortamannin-sensitive PI3K p110α was used as a control. The ADP-Glo assay was used to measure the kinase activity.
(e) Flag-Beclin1 Wt or T119A transfected HeLa cells were incubated in glucose-rich or nutrient-free (HBSS) media, in the presence or absence of ROCK1 inhibitor Y27632. Total cell extracts were used for IP using Flag agarose. Eluted protein was analysed by WB against phospho-T119 (Beclin) antibody and Beclin1. Input for Beclin1 was run on 7.5% gel and immunoblotted.
Cellular H2O2 concentration (HyPer) 48 h after transient silencing of TMX1 and / or NOX4 in WM3734 cells or after inhibiting NOX4 with GKT137831 (140 nM) (O) Representative ratiometric images (F505 nm / F420 nm) and (P) quantification. In (P), data are presented as mean ± SEM (n values: control=837, TMX1 kd=888, TMX1 kd + NOX4 kd=793, TMX1 kd + GKT=844).
g, Samples from drpr mutant animals (+/w; +/UAS-Atg16A; drprDgr;5/drprDgr;5), n = 10, and those with salivary gland-specific expression of Atg16A (fkh-Gal4/w; UAS-Atg16A/+; drprDgr;5/drprDgr;5), n = 16, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation. h, Quantification of data from g.
F Expression of NMT1 in NMT1-Myc overexpression plants. EF1α was used as a loading control.
(G) Waffle plot showing the impact of different signaling mechanisms on the stress phosphoproteome. Each box corresponds to 1% of the entire set. Orange boxes represent the percentage of phosphosites regulated by PP2ACdc55, whereas grey boxes represent phosphosites regulated by Hog1 and downstream of the MAPK. Light purple boxes indicate the phosphosites that are neither regulated by Hog1 nor PP2ACdc55. Dependency was defined as log2 SILAC ratio below -1 or above 1. Rck2-dependency was extracted from (Hollenstein et al., 2020).
8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on HFD-feeding for 12 weeks. n=5 per group. (I) Scheme for the experimental strategy.
The Dmax distributions (the maximum distance within a particle) derived from the EOM analysis of the measured SAXS profile (pool - the white histogram, the selected structures - the black histogram). The distribution of the selected structures shows a bimodal behaviour with an average value of 166.9 Å.
A. Representative I-V curves of the PC2_AA and PC1-CTF/PC2_AA channels in divalent ion-free bath solutions containing 100 mM of indicated ions, showing the differences in reversal potential.
(C-D) Autophagy levels were determined in Beclin 1-depleted H1299 cells with reconstituted expression of Beclin 1 WT, AA mutant or DD mutant in normal medium or after H2O2 (500μM) treatment for 3h in the presence (C) or absence (D) of CHK2.
Fluorescence complementation analysis of the effect of MB on the interaction between PD-1 with SHP2. Jurkat cells co-expressing PD1-C-Luc or PD1Y248F-C-Luc and SHP2-N-luc were seeded in a 96-well precoated with 10 μg/ml of aCD3/aCD28 with media supplemented with 10 μg/ml human PD-L1 protein in the presenc of MB at indicated concentrations for 6 hours followed by analysis of luciferase activity Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ****P < 0.0001
(b,c) Dynein mutation reduces motor coordination and muscle function in a mouse model of Huntington disease. HdhHD/+ Dnchc1Loa/+, HdhHD/+ Dnchc1+/+ and Hdh+/+ Dnchc1Loa/+ mice are shown. An average of six mice were used at every time point, with at least three mice per group after 13 weeks. (b) Grip strength. Overall effect for all time points between the three genotypes: P = 0.0024. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and HdhHD/+ Dnchc1+/+: P = 0.001. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and Hdh+/+ Dnchc1Loa/+: P = 0.0157. Effect at each time point between the three genotypes: 7 weeks, P = 0.023; 9 weeks, P = 0.001; 11 weeks, P 0.0001; 13 weeks, P = 0.004; 15 weeks, P = 0.336 (most of the double mutants were dead by 15 weeks). (c) Accelerating rotarod. Overall effect for all time points between the three genotypes: P = 0.0367. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and HdhHD/+ Dnchc1+/+: P = 0.0245. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and Hdh+/+ Dnchc1Loa/+: P = 0.0553. Effect at each time point between the three genotypes: 8 weeks, P 0.001; 10 weeks, P = 0.005; 12 weeks, P = 0.001; 14 weeks, P = 0.009; 16 weeks, P = 0.039.
E. Scatterplot showing changes in the abundances of transposon-repressing piRNAs and non-transposon-repressing piRNAs in the presence and absence of the Hel-C domain. Orange and blue dots indicate the number of normalized piRNA reads mapped uniquely to transposons in the reverse orientation and coding genes in the sense orientation, respectively.
I) HeLa cells stably expressing GFP-ATG13 together with BFP-TFGwtLIR or BFP-TFGmutLIR3 were co-transfected with a siRNA targeting endogenous TFG (siTFG 3'UTR) and mCherry-SEC16, then cultured for 40' with starvation medium. Cells were visualised with cellSens microscope for live-cell imaging. Analysis of the percentage of GFP-ATG13 lifetime colocalizing with mCherry-SEC16 in the presence of TFGwtLIR or TFGmutLIR3 is reported in the cumulative relative frequency plot. Statistical analysis was performed by Kolmogorov-Smirnov test. P <0.0001. (n=3 independent experiments, n≥995 number of colocalization events analysed).
J, R211 and PDAC8661 cells were treated with siRNA scramble, siRNA targeting p110α or a combination of siRNA targeting each class I PI3K isoform (pools) and subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. n = 3 in each group.
A NDUFC2 mRNA transcript levels in fibroblast cell lines of controls (white), Subject 1 (grey) and Subject 3 (black). Quantification of NDUFC2 mRNA transcript levels (normalised to ACTB) showed diminished transcript levels in Subject 1 while transcript levels were unaffected in Subject 3. Bars correspond to standard deviations of two independent experiments, performed in duplicate and analysed by Student's t-test; *p<0.05.
B Heatmaps of genes involved in the relevant pathways Colour code: red represents upregulation, white, unchanged, and blue downregulation of expression.
Polarized intracellular trafficking of native and virally expressed Kcnq1 was found specifically in the marginal cellsKcnq1 and cellnuclei were, respectively, labeled green and purple. The SV consists of three layers of cells; marginal cells are the first layer of cells on the side of the endolymphatic space.Cryosection through the SV of WT mice. The large arrow points to the native Kcnq1 (labeled green) in WT mice. Smaller arrows show nuclei of the marginal cells.Cryosection through the SV of a Kcnq1−/−mouse injected with AAV expressing Kcnq1. Labeled in green (bigger arrow) is the AAV1-expressed Kcnq1, found only in the apical membrane of the marginal cells. Smaller arrows show nuclei of the marginal cells. Arrowheads show Kcnq1immunolabeling in fibrocytes outside the SV.Cryosection through the lateral wall of Kcnq1−/−mice showing intracellular distribution of Kcnq1immunolabeling (green) in fibrocytes.Cryosection through the spiral ganglia of cochlea of treated Kcnq1−/−mice showing immunolabeling (green) in the cells of spiral ganglia (arrows).Data information: Purple staining in all panels is counterstaining with DAPI showing the locations of cellnuclei. Scale bars represent appropriately 50 μm.
(I) Survival of control and Ntras-silenced mice under basal conditions (black, n = 21-23 mice per group) and in hind limb ischemia (HLI; red, n = 8 mice per group). Number of survivors is indicated; experimental outline on the left. Data information: (I) Mantel-Cox-test.
A Changes in exon usage between shNTC and shH2A.B L1236 HL cells. Highlighted in red are RP genes. The significance (-log10 (P value)) is plotted against the fold change of exon usage (log2FC).
ChIP-qPCR analyses of H3 in WT and mutants as indicated (n = 3 independent experiments). Data information data are represented as mean ± SE
(B) The occurrence of GFP-ATG8 puncta in promastigotes after incubation in nutrient-deprived (PBS, ND) and nutrient-rich (HOMEM medium, NR) conditions for 2 h at 26°C. Scale bar, 10 µm
Piliation per bacterium as a function of the proportion of piliated bacteria normalized to pilESB values as measured by flow cytometry using the 20D9 monoclonal antibody. Black dots indicate all the individual de novo generated PilE mutants that were characterized. Blue dots indicate mutants of the piliation machinery and green dots mutants in the α1N region. Solid lines indicate 95% confidence interval. Quantification of aggregation normalized to pilESB for a subset of outliers and corresponding pilT double mutants. Mean values ± SEM are indicated for each strain. N≥3 independent experiments. Paired t-test. p<0.05 (*), p<0.01 (**).
(A) Western blot analysis of immunoprecipitated Bur1-HA or Bur1-∆C-HA used in (B).
STED images of heteromeric fibrils formed by co-assembly of mCHERRY-M451-90 and YPet-RIPK3387‑518
FLAG-DHHC5 immunoprecipitations were performed using lysates from Hela cells co-expressing either wild-type (WT) or palmitoylation deficient mutant (m) Golga7b. The interaction of DHHC5 with its substrate Flot2 was measured by immunoblotting and is significantly reduced in the presence of palmitoylation deficient mutant Golga7b, quantification normalised to input, *=p<0.05, ratio paired t-test, n=3.
G Volcano plot illustrates differences in expression patterns of target genes of neurons from the qPCR single-cell analyses. The dotted lines represent differentially expressed genes between the groups at P < 0.05 (unpaired Student's t-test).
Muscles were harvested 7 days after implantation of V Low, V Med, and V High clones or control cells (Ctrl).Gene expression of Pdgfb,Ang1,Ang2,Tgfb1, and Sema3a was quantified by qRT-PCR and expressed as fold-change versus control muscles. Data represent individual values, with mean ± SEM (n = 3 or 4, as indicated); *P < 0.05 and **P < 0.01 by one-way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic transformation. TGF-β1: V Low versus V Med P = 0.0047; V Low versus V High P = 0.0379; Sema3A: V Low versus V High P = 0.0042; V Med versus V High P = 0.0179.
(C) Schematic diagram of disease-relevant mutants of Parkin used in this study. IBR, in between RING; Ubl, ubiquitin like.
C RIP experiments performed using anti-myc antibodies and extracts from cells as in (A) followed by qRT-PCR analysis of G-rich and polyA+ tiTERRA. Values correspond to fraction of input RNA expressed as fold increase over an untagged (unt) control tiTEL strain (CAF545). Bars and error bars are averages and SD from three independent experiments. * P < 0.05, ** P < 0.01 (relative to unt; two-tailed Student's t test).
C. and D. Lysates of HEK293T cells, transfected as in B, were first subjected to a denaturing protocol, then to IP with the GFP antibody, followed by IB with the K63-specific ubiquitin antibody (C) or the K48-specific antibody (D). Inputs were immunoblotted with the GFP or myc antibody (lower panel). Three independent blots were quantified and band intensity normalized to control average. (ANOVA, *p<0.05, mean + s.e.m).
Expression of p21CIP1 and RAP1 after 15 dpi of cells transduced with shControl, shp21CIP1 or shp21CIP1 + shRAP1.
(D) The extent of mPTP opening in nasopharyngeal carcinoma cells (scale bar, 20 μm). calcimycin (calcium ionophore) served as a positive control. Images were analyzed using Image J software Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, ** p < 0.01).
H, Dnmt3a/b-dependent loss of DNA methylation at neuronal gene candidates was associated with reduced transcription in neurons. Depicted are mean CpG methylation levels at specified genomic regions as determined by RRBS (left) and expression fold changes in KO versus WT neurons as determined by qRT-PCR (right; n = 4 cultures per genotype; p-values from Mann-Whitney test; depicted are data points for every sample with genotype means ± SEM).