EMBO/sd-character-level-ner huggingface dataset:
Normalizing endothelial cell survival and blood-brain barrier permeability by intravenous injection of AAV-BR1-NEMO in a conditional murine incontinentia pigmenti model (NemobeKO mice).Figure 8BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter. All animals were at the age of 8-12 weeks.A Representative immunostainings of cerebral microvessels. String vessels (white arrows, highlighted in white square inset) were significantly reduced in NemobeKO mice treated with AAV-BR1-NEMO compared to NemobeKO mice treated with AAV-BR1-eGFP control vector. NemoFl mice served as control animals. Scale bars represent 200 µm.B Quantification of string vessel lengths in the cerebral cortex as percentage of total vessel lengths. NemobeKO and NemoFl mice were treated with AAV-BR1-NEMO or AAV-BR1-eGFP control vector (n=9 NemoFl animals + BR1-eGFP, 10 NemobeKO animals + BR1-eGFP, 9 NemoFl animals + BR1-NEMO, and 13 NemobeKO animals + BR1-NEMO), ****p < 0.0001.C Total vessel length measured as total CD31-positive vessels. Vessels were restored in NemobeKO mice treated with AAV-BR1-NEMO compared to the AAV-BR1-eGFP injected mice. NemoFl mice served as a control (n= 9 NemoFl animals +BR1-eGFP, 10 NemobeKO animals + BR1-eGFP, 9 NemoFl animals + BR1-NEMO, and 13 NemobeKO animals + BR1-NEMO), ***p = 0.0007, **p = 0.0038 (NemoKO:eGFP vs. NemoFl:NEMO ), **p = 0.0014 (NemoKO:NEMO vs. NemoKO:eGFP). BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter. All animals were at the age of 8-12 weeks.D IgG and albuminWestern blots of brain lysates. Less leakage in the BBB was detected in NemobeKO mice treated with AAV-BR1-NEMO than in NemobeKO mice injected with AAV-BR1-eGFP (the right panel indicates the quantified gel intensity under the various treatment conditions (n= 5 animals per group). NemoFl mice served as controls, ****p < 0.0001. BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter. All animals were at the age of 8-12 weeks.E Quantitative immunoglobulin staining of coronal brain sections of NemoFl and NemobeKO mice. Ig extravasation was significantly reduced in AAV-BR1-NEMO-treated mice (n= 9 NemoFl animals + BR1-eGFP, 10 NemobeKO animals + BR1-eGFP, 9 NemoFl animals + BR1-NEMO, and 13 NemobeKO animals + BR1-NEMO), ****p < 0.0001. BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter. All animals were at the age of 8-12 weeks.F Effect of AAV-BR1 vector on BBB permeability. No vector (left) or empty AAV-BR1 vector (right) was injected i.v. to wild type mice and BBB permeability was assessed by extravasation of the fluorescent tracer sodium fluorescein (n=7 animals per group). No significant difference was detected (n.s.).
Figure 2(A) miR-124 expression is altered in DSCR1 mutants. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 animals for each condition, *P< 0.01. (B) Promoter activity of miR-124 in Neuro2 A cells with DSCR1 reduction or overexpression. Firefly luciferase reporter under the control of miR-124 promoter is measured. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05, **P< 0.01. (C) Bisulfite sequencing analyses of the miR-124 promoter. Each CpG site is indicated, and the methylation status of two different regions are shown. Open and filled circles represent unmethylated and methylated CpG sites, respectively. The percentage of methylated CpGs among total number of CpGs is also shown. (D) Neuro2A cells containing DSCR1shRNA show increased activity of miR-124 promoter measured. The miR-124 promoter was inserted in front of the luciferase reporter. Site-directed mutation of two methylation sites (31 and 58) in the promoter shows the luciferase activity similar to that of DSCR1 reduction. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05. (E) Overexpression of DSCR1 reduced the luciferase activity, while removing all methylation sites in the miR-124 promoter increases it. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05.
Figure 2Enrichment of RBP-RNA interaction sites in the SRSF3-dependent large constitutive exons (S3-LCEs). Fraction of S3-LCEs in exons having eCLIP peaks in 334 eCLIP data in ENCODE. hnRNP K-eCLIPs are indicated in red. Sashimi plots of representative SRSF3-dependent exons. C2C12 cells were treated with control siRNA (siCont), Srsf3 siRNA (siSrsf3), Hnrnpk siRNA (siHnrnpk), or both Srsf3 and Hnrnpk siRNAs (siSrsf3+siHnrnpk), and RNA-seq was performed. Lines indicate junction-spanning reads. Gene structures are shown below the Sashimi plots. Scatter plots showing PSI values of control cells (siCont), Srsf3-silenced cells (siSrsf3; left panel), and both Srsf3- and Hnrnpk-silenced cells (siSrsf3 + siHnrnpk; right panel). The PSI values of all internal exons are plotted. Yellow dotted lines indicate ΔPSI = 0.2 and -0.2. Skipped and included exons (|ΔPSI| > 0.2 and Bayes factor > 10) by siSrsf3 are indicated in red and blue, respectively. Note that exon skipping and inclusion are partly mitigated by an additional knockdown of hnRNP K (right panel). PSI values of the exons skipped by Srsf3 silencing (red circles in Fig 2C) in cells treated with the control siRNA (C), Srsf3 siRNA (S), Hnrnpk siRNA (K), or both Srsf3 and Hnrnpk siRNAs (S+K). The box plot shows the interquartile range (boxes), the median (central band) and the minimum and maximum except for the outliers at the ends of whiskers (n = 344). Relationship between exon lengths and recovery of exon skipping in Srsf3-silenced cells by additional Hnrnpk silencing. The exons skipped by Srsf3 silencing (red circles in Fig 2C) are classified into four groups according to the degree of recovery by the additional Hnrnpk silencing [ΔPSI (PSIsiSrsf3+siHnrnpk - PSIsiSrsf3)]. The box plot shows the interquartile range (boxes), the median (central band) and the minimum and maximum except for the outliers at the ends of whiskers (n = 60 for "ΔPSI ≤ 0", n = 131 for "ΔPSI 0 to 0.2", n = 95 for "ΔPSI 0.2 to 0.4", and n = 57 for "ΔPSI > 0.4"). Relationship between the number of CCC motifs in exons and recovery of exon skipping in Srsf3-silenced cells by the additional Hnrnpk silencing. The exons skipped by Srsf3 silencing (red circles in Fig 2C) are classified as in (E). The box plot shows the interquartile range (boxes), the median (central band) and the minimum and maximum except for the outliers at the ends of whiskers (n = 60 for "ΔPSI ≤ 0", n = 131 for "ΔPSI 0 to 0.2", n = 95 for "ΔPSI 0.2 to 0.4", and n = 57 for "ΔPSI > 0.4"). Distributions of hnRNP K-RNA interactions around 3′ (left) or 5′ (right) splice sites (SS) of the SRSF3-dependent exons in control cells (black) and Srsf3-silenced cells (red). The standard error of the average density of hnRNP K-tRIP reads is shown as a semi-transparent shade around the average curve.
Figure 1Transgenic ESZscan4_Emerald cells were sorted and total RNAs were extracted and subjected to RT-qPCR. The graph shows the mRNA expression level of Arg2 in ES subpopulations. (B) Transgenic ESZscan4_Emerald cells were sorted and total proteins were extracted and subjected to immunoblotting. The image is a representative of three independent biological western blotting analysis on arg2 expression in sorted ESZscan4_Emerald and control cells. Schematic illustration of Arg2 silencing and magnetic separation of the Zscan4+ cells. The modified ESZscan4_LNGFR cells were transfected with a siRNA that specifically target mouse Arg2. 96h after siRNA transfection, cells were harvested and incubated with a LNGFR magnetically labeled antibody. Positive fractions were collected through autoMacs Separator. All the analyses were performed on Zscan4+ siArg2 and Zscan4+ siCtrl cells. Transcript (left panel) and protein expression (right panel) levels of Arg2 in ESZscan4_LNGFR cells after transfection with a non-silencing siRNA (siCtrl) or siRNA against Arg2 (siArg2). Imaging flow cytometry analysis on Zscan4+ siArg2 and Zscan4+ siCtrl cells. Samples were stained by labelling histone H3 protein. The intensity of H3 fluorescent signal was measured in the nuclear region by creating a mask defined by DAPI staining. The texture feature of fluorescent signal was analysed by using the modulation method (for details see Materials and Methods). Two cell populations (R1 and R2) correspond to two different patterns of fluorescent signal: homogenous and clustered distribution, respectively.
Figure 5FDG-µPET in rodents: Axial slices (as indicated in Fig. 4A) of averaged %-FDG-µPET differences between Grn-/-or Trem2-/- mice and wt indicate decreased glucose metabolism in the cerebrum of Grn-/- and Trem2-/- (both cold color scale) when compared to wt. Scatter plot depicts single FDG-PET values deriving from a neocortical volume-of-interest. µPET results are illustrated upon an MRI template; 6-10 female mice per group (Grn-/- n=6 at 8 months, Trem2-/- n=6 at 11 months; wt n=10 at 8-11 months).
Hsc70 and DnaJB1 interaction with Hsf1 trimers lead to deprotection of segments within the trimerization domain.Figure 3A Difference plot of deuteron incorporation into Hsf1 monomers (light gray) or trimers (dark gray) in the presence of Hsc70 (left) or DnaJB1 (right) minus deuteron incorporation into Hsf1 in the absence of chaperones after 30 s incubation in D2O buffer. Shown are mean ± SD (n = 3) for peptic peptides as indicated between the panels. Left, Hsf1 domain representation. B Difference plot of deuteron incorporation into Hsf1 trimers incubated for 300 s in D2O buffer after pre-incubation in the presence of ATP, Hsc70 (70), and DnaJB1 (B1) as indicated minus deuteron incorporation into Hsf1 in the absences of additives. C Fit of two Gaussian distributions to the peak intensities for peptic peptides 159-168 (left), 169-175 (middle), and 176-189 (right). Representative plots of 3 independent experiments. D Fraction of high exchanging subpopulation for peptides 159-168, 169-175, and 176-189 under the different indicated pre-incubation conditions: ‑chaperones, +Hsc70 (70), +DnaJB1 (B1), and +ATP incubated for 0 or 30 min at 25°C. Shown are means ± SD (n=3). E Relative deuteron incorporation of the low (solid bars) and high (open bars) exchanging subpopulations.
Figure 1A Outline of the screening for HSATIII-regulated genes during thermal stress recovery. HeLa cells were transfected with a HSATIII ASO (HSATIII KD) or HSATIII sense oligonucleotide (control), exposed to thermal stress. Nuclear polyA(+) RNAs were analyzed by next-generation sequencing (NGS). NGS data have been deposited in the DDBJ Sequence Read Archive (DRA) (accession number: DRA007304). B HSATIII ASO-mediated depletion of nSBs. Thermal stress-exposed HeLa cells (42°C for 2 h and recovery for 1 h at 37°C) were visualized by HSATIII-FISH and immunofluorescence using an anti-SAFB antibody or anti-HNRNPM antibody. The nuclei were stained with DAPI. Scale bar: 10 μm. C qRT-PCR-validation of HSATIII knockdown. The graph shows the qRT-PCR level of HSATIII RNAs in control and HSATIII knockdown cells under three conditions: 37°C, 42°C for 2 h, and thermal stress followed by recovery at 37°C for 1 h (Recovery). Expression levels were calculated as ratios to GAPDH mRNA and were normalized to the levels in control cells under thermal stress conditions (42°C for 2 h). Data are shown as the mean±SD (n=3). HSATIII RNAs ratios (%) are indicated. D MA-plot (log2 fold-change over the average expression level) of all detected introns in HSATIII KD and control cells (n=3). The introns with significant changes in their expression levels (adjusted p-value < 0.01) are shown as green dots. Among these introns, up-regulated (log2 fold-change > 1) and down-regulated (log2 fold-change < -1) introns are shown with magenta and light blue dots, respectively. Genes and introns that were experimentally validated in subsequent experiments are indicated as gene symbol-i (intron) #. Total numbers of introns and genes utilized were 144769 and 32623, respectively. The numbers of introns and exons that were affected by HSATIII knockdown (fold change>2). Examples of RNA-seq read maps of HSATIII target RNAs. Affected introns and exons are indicated by blue (down-regulated upon HSATIII knockdown) G Examples of RNA-seq read maps of HSATIII target RNAs. Affected introns and exons are indicated by blue (down-regulated upon HSATIII knockdown) and magenta (up-regulated) boxes. H Cumulative frequency curves of the lengths of adjacent exons of 399 HSATIII-up-regulated internal introns. The lengths of whole annotated internal introns are shown as a reference.
Figure 4(A) WT and CRISPR-Cas9 Fam134b knockout MEFs were treated as indicated, lysed and analysed by western blot with the indicated antibodies. Western blots are representative of 4 independent experiments. (B) WT and CRISPR-Cas9 Fam134b MEFs were immunolabeled for PC1 (568, red), nuclei stained with Hoechst (blue) and analysed by scanning confocal microscopy. Scale bar = 10 µm. (C) CRISPR FAM134B MEF mock, wild type FAM134B-HA or FAM134Blir-HA transfected were immunolabeled for PC1 (568, red), LAMP1 (488, green) and HA (647, violet) and analysed by scanning confocal microscopy. Scale bar = 10 µm. Inset panels show magnification of the boxed area. Bar graph shows quantification of LAMP1 vesicles positive for PC1, expressed as % of total lysosomes (mean +/- SEM), quantification of n=10 cells per treatment; 3 independent experiments. One-way ANOVA with Dunnett's multiple comparisons test was performed. ns ≥0.05, *** P<0.0001.
Figure 5A. A deletion of daf-16 completely abolishes the increased resistance to bacterial pathogens in isp-1 mutants. p-value indicates the significance of difference between red and purple lines. Three biological replicates per strain were performed. Data information: Statistical significance was assessed using log-rank test. B,C. While daf-16 RNAi effectively decreases the expression of DAF-16 target genes (B, sod-3, dod-3, mtl-1, ftn-1, icl-1, sodh-1), it does not affect the expression of any of the innate immunity genes (C, T24B8.5, K08D8.5, F55G11.8, clec-65, clec-67, dod-22, Y9C9A.8 and C32H11.4). This indicates that DAF-16 is not required for expression of innate immune signaling pathway target genes in isp-1 mutants. daf-16 expression was knocked down using RNAi beginning at the L4 stage of the parental generation. RNA was isolated from six biological replicates at the young adult stage of the experimental generation. RNA from the six biological replicates was pooled for RNA sequencing.
miR-511 expression depends on GCs.Figure 6Adx significantly sensitizes B mice for TNF. Sham-operated mice (black, n = 12) all resisted 2 μg TNF while most Adx mice (grey, n = 12) succumbed to this dose of TNF (top panel). Effect of adrenalectomy (Adx) on relative mRNA expression in the spleen of B mice (black, n = 5) and Adx mice (grey, n = 6). Bottom left panel shows significant down-regulation of Tsc22d3 (encoding GILZ), Dusp-1 (encoding MKP-1), Mrc1 and miR-511 in Adx mice. Bottom right panel displays significant up-regulation of the protein level of TNFR1 in liver, spleen and serum of Adx mice (grey) compared to control mice (black) (all groups n = 10). Pretreatment with 50 μg DEX protects B mice against lethal SIRS induced by 30 μg TNF (−30 min, grey) compared to PBS pretreatment (250 μl, −30 min, black) (both groups n = 13) (left panel). Relative mRNA expression in the liver of B mice 6 h after i.p. injection of PBS (black, n = 10) or DEX (grey, n = 10) (right panel). Significant up-regulation of Tsc22d3, Sgk, Tat and Mrc1. Induction of miR-511 by DEX compared to PBS. Relative mRNA expression in the spleen of B mice 6 h after i.p. injection of PBS (black, n = 10) or DEX (grey, n = 10). Significant up-regulation of Tsc22d3, Sgk, Tat and Mrc1. DEX had no effect on TNFR1 mRNA levels. Significant up-regulation of miR-511 by DEX. Significant down-regulation of the protein level of TNFR1 in liver (n = 20), spleen (n = 20) and serum (n = 5) by 50 μg DEX (grey) compared to PBS (black).
Figure 5 Figure 5. EGF treatment dissociates DENND2B from ITSN A HEK-293T cells were serum starved, treated with 100 ng/mL EGF for the indicated times and cell lysates were analyzed by Western blot. B Quantification of A where P-PKD is normalized to Hsc70. Mean +/- SD. ANOVA with Dunnett post-test *p=0.015 and ** p=0.003 n=3 from 3 independent experiments. C HEK-293T cells were serum starved, treated with 100 ng/mL EGF for 5 min and cell lysates were incubated with protein G beads or protein G beads coupled to anti-Flag. Bound proteins were detected by Western blot. D Quantification of B where P-PKD substrate is normalized to Flag-DENND2B. Mean +/- SD Welch's t-test *p=0.036, n=4 from 3 independent experiments. E HEK-293T cells were serum starved, treated with 100 ng/mL EGF for 5 min and cell lysates were incubated with GST-SH3A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively. F Quantification of E where bound Flag-DENND2B is normalized to SM. Mean +/- SD, Welch's t-test *p=0.012, n=6 from 3 independent experiments. G Schematic of EGFR trafficking. ITSN promotes EGFR internalization upon activation. EGF-independent activation of EGFR: EGFR traffics on Rab13-positive recycling or late (RE/LE) endosomes to the plasma membrane where ITSN binds the DENND2B to facilitate EGFR exocytosis. EGF-dependent activation of EGFR: DENND2B is phosphorylated by PKD, binds 14-3-3 and loses its affinity for ITSN and EGFR is targeted towards lysosomal degradation.
Figure 4Serum anti-Insulin-immunoglobulin titers of mice immunized with InsA-peptide (red: 0:1, n=10 and green: 100:1, n=5) and control-immunization (CI: CpG only, grey, n=3) measured by ELISA at indicated days (coating: Insulin). Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Kruskal-Wallis test, *P < 0.05, ***P < 0.001. Ratios of IgG to IgM derived from ELISA values plotted against molar ratios of antigens for n=5/group. Mean ± SD, statistical significance was calculated by using Kruskal-Wallis test, *P < 0.05, ***P < 0.001. Western blot analysis of insulin-specific serum IgG derived from InsA-peptide immunized mice. Top panel (green): 100:1 serum, lower panel (red): 0:1 serum (sInsA:cInsA). Serum of mice was used as primary antibody and detection was done with anti-mouse-IgG-HRP. Black filled arrow: Proinsulin (12 kD), Black non-filled arrow: Insulin (6 kD), β-actin (42 kD, loading control). Representative of two independent experiments. ELISpot of InsA-peptide immunized (red, n=11) and CI mice (grey, n=8) showing insulin-specific IgG-producing splenocytes on day 14. Representative wells are shown (top lane). Mean ± SD, statistical significance was calculated by using Mann-Whitney-U test, ****P < 0.0001. Blood glucose levels of InsA-peptide immunized (red: 0:1, n=5 for E and n=10 for F; green: 100:1, n=5 and n=4 for F, d30, d32) and CI (grey, n=5 for E and n=10 for F) mice were assessed at indicated days. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Kruskal-Wallis test (E) and repeated measure ANOVA test (F), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Urine glucose levels of InsA peptide immunized (red: n=10 for d28, d32 and n=8 for d30; green: n=5 for d28, n=4 for d30, n=3 for d32) and CI (grey, n=3) mice were monitored at indicated days post immunization. Dots represent individual mice. For 100:1: representative data are shown for two independent experiments with total n=10. Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test, ***P < 0.001. Table comparing human to murine Insulin-A-chain amino acid sequences. Underlined: sequence used as peptide.
Figure 6Representative photographs of WT and DKO mice after HFD feeding for 2 months. Scale bar: 1cm. Body weight, body weight gain after HFD feeding (B) and tissue weight of iWAT and eWAT (C) of WT and DKO mice fed with HFD for 2 months (n=10-11). Concentration of serum triglyceride (TAG) (left) and non-esterified fatty acid (NEFA) (right) in WT and DKO mice after HFD feeding for 2 months (n=5). Relative mRNA expression of FoxO1, PDK4, and ATGL in GAS muscles of WT and DKO mice fed with HFD for 2 months (n=5). Western blot analysis of FoxO1, PDK4, p-PDHA1, PDHA1 and ATGL in GAS muscles of WT and DKO mice fed with HFD for 2 months. Relative mRNA expression of PGC1α, PLIN5 in GAS muscles of WT and DKO mice fed with HFD for 2 months (n=5). Relative mRNA expression of HSL in GAS muscles of WT and DKO mice fed with HFD for 2 months (n=5). Fasting blood glucose levels (left) and random blood glucose levels (right) in WT and DKO mice after HFD feeding (n=10-11). Glucose tolerance test (GTT) and area under the curve (AUC) data for GTT in WT and DKO mice after HFD feeding (n=10-11).
Efficient skipping of exon 326 in vivo-morpholino-modified antisense oligonucleotide-treated adult Ttn Ser14450fsX4 knock-in mice.Figure 7Fluorescence in situ hybridization (FISH) of heart muscle tissue from adult knock-in mice with a probe complementary to vPMO-mTtnAON. Scale bars, 250 and 50 μm (magnification). RT-PCR analysis of Ttn exon 326 transcripts from heart tissue of untreated WT and HET animals and vPMO-mScrAON- and vPMO-mTtnAON-treated mice (left). Representative direct sequencing of Ttn exon 326 transcripts from vPMO-mTtnAON-treated HET heart tissue (right). Mass spectrometry-based analysis of titin peptides in adult knock-in mice injected with vPMO-mScrAONs and vPMO-mTtnAONs. Unsupervised hierarchical clustering identified a cluster enriched in exon 326 peptides that was down-regulated in vPMO-mTtnAON-treated animals compared to vPMO-mScrAON-treated littermates. Another cluster enriched in C-terminal peptides was up-regulated in the vPMO-mTtnAON group compared to the vPMO-mScrAON group (n = 3, P = 0.02, Fisher's exact test, FDR = 0.04).
TMEM41B-deficent cells display enlarged lipid droplets A H4 Cas9 cells stably expressing NT, TMEM41B or ATG7 sgRNAs were stained with BODIPY 493, NBD cholesterol or BODIPY FL C12 probes for 2 h at 37°C and imaged live with an automated CV7000 confocal microscope.Figure 3 A H4 Cas9 cells stably expressing NT, TMEM41B or ATG7 sgRNAs were stained with BODIPY 493, NBD cholesterol or BODIPY FL C12 probes for 2 h at 37°C and imaged live with an automated CV7000 confocal microscope. Puncta area per cell was quantified using YAS and depicted as mean ± SD (n=4 technical replicates). Scale bar: 20 μm  B H4 Cas9 TMEM41B KO and NT control cells were treated overnight with 400 μM BSA-conjugated oleic acid or 0.1% BSA as vehicle control, stained for 2 h with HCS LipidTox Green Neutral Lipid Stain and imaged with an automated Operetta microscope. Scale bar: 20 μm  C Lipid droplet size, number and mean fluorescence intensity was quantified using Harmony software. Data are presented as mean ± SD (n=3 technical replicates). An average of 1500 cells was analyzed per replicate  D H4 Cas9 TMEM41B KO and NT control cells were probed by ADRP immunostaining and imaged with an automated CV7000 confocal microscope. Scale bar: 20 μm. E Size of ADRP droplets was quantified with YAS and depicted as mean ± SD (n=3 technical replicates)  F Protein level of ADRP was probed by immunoblotting in H4 Cas9 TMEM41B KO and NT control cells. G ADRP band intensities were quantified and depicted as mean ± SD (n=4 independent experiments) with paired t-test values