EMBO/sd-character-level-ner huggingface dataset:
Figure 5A. The median distance of DEGs to neighbor GLKLTRs (red arrow) and the distribution of that of random genes (blue) (p < 2.2 10-16) measured by Wilcoxon rank single test. GLKLTRs are gene loci that contain whole LTR of GLN, MERVL, or ERVK. The results show that DEGs prefer neighboring to GLKLTRs. B. Dox-induced LincGET expression assay and dual-luciferase reporter system show that LincGET increased the enhancer activity of GLKLTRs in 293T cells in a dose-dependent manner. The enhancer activity of GLKLTRs is also increased by overexpression of hnRNP U or ILF2 and decreased by FUBP1 overexpression, which have no effect on LincGET levels. The y-axis shows the construction of luciferase reporter plasmids and overexpressed genes. Three experimental replicates were performed. Two tailed student's t-test was used for the statistical analysis. Different letters (a, d, c, d, e, and f) mean p < 0.01. C. Number and percentage of unusual alternative splicing genes (UASGs, FDR <0.05) in LincGET-depleted 2C compared to Control-LNA L2C. ExS, exon skipping. A5S, alternative 5' Splicing. InR, intron retention. D. GO-BP analysis of UASGs showed that "cell cycle" especially the "M phase of the mitotic cell cycle" is primarily affected. The terms with p < 0.001 and FDR <1 are shown. E. Cdk1was exon skipping spliced in LincGET-depleted 2C, resulting in Cdk1-3ExS where the third exon was deleted. The abnormal spliced Cdk1-3ExS variant translated into a truncated CDK1 that lacks the N terminal 70 amino acids (aa), which affects the kinase activity of CDK1 lacking a conservative ATP binding domain predicted by InterPro. PCR primers for Figure 5F (Cdk1-Ex3-F and Cdk1-Ex3-R, sequences are shown in Table EV1) are shown in red triangles. F. Exon skipping event of Cdk1-3ExS in LincGET-depleted 2C is confirmed by RT-PCR. Injection of LincGET fragments at the pronuclear stage was unable to inhibit the exon 3 skipping of Cdk1, while injection of full-length LincGET1 lacking LincGET-LNA2 target site partially inhibited the exon 3 skipping of Cdk1. For each lane, about 50 embryos were used. Three experimental replicates were performed. Two tailed student's t-test was used for the statistical analysis.
Figure 4A, B. Wildtype Rabl2 was observed in the shaft of both primary cilium (A) and multicilia (B). Ciliated RPE1 cells (A) or mEPCs (B) expressing Rabl2 tagged with 3×mNeonGreen (3NG) were fixed at 48 h or day 7 post serum starvation, respectively. C. Ciliary 3NG-Rabl2 in living RPE1 cells underwent bidirectional IFT. The 1st frame and kymographs of a representative cilium (also see Movie EV1) are shown. Quantification results, presented as mean ± s.d, were measured from 34 cilia. Detection of endogenous Rabl2 in purified multicilia. Multicilia purified from cultured mEPCs Detection of endogenous Rabl2 in purified multicilia. Multicilia purified from cultured mEPCs were analyzed by immunoblotting (E). Ac-tub, Ift25, and Ift81 served as positive controls for cilia, whereas Lamin B1, GM130, Cep162, and Gapdh served as quality controls for the ciliary preparation to exclude major contaminations from the nucleus, Golgi, basal body, and cytosolic proteins, respectively. 1/400 of the mEPC lysate and 1/20 of the cilia lysate were loaded per lane. F. Diagram of IFT-B. The core 1, core 2, and peripheral subcomplexes (Nakayama & Katoh, 2018) are depicted in different colors. G. Rabl2Q80L strongly associated with IFT-B. GFP-Rabl2 or mutants stably expressed in NIH3T3 cells were immunoprecipitated with anti-GFP beads. H. Rabl2Q80L interacted with the IFT74/81 heterodimer. FLAG-tagged Ift74 and Ift81 and GST-tagged Rabl2Q80L and Rabl2S35N expressed in E. coli were mixed as indicated and pulled down with glutathione-conjugated beads. Antibodies to GST and FLAG were used for immunoblotting. I. Association of endogenous Rabl2-GTP with IFT-B. Mouse testis lysates with or without 1 mM GTPγS were subjected to sucrose gradient ultracentrifugation. Fractions were collected from top (low MW) to bottom (high MW). Note the obvious enrichment of Rabl2 in fraction #16 in the presence of GTPγS. J. A model delineating relationships between Rabl2 and IFT machinery. Rabl2-GTP enters cilia by binding to the Ift74-Ift81 heterodimer of IFT-B. After reaching the ciliary tip through anterograde IFT, it undergoes retrograde IFT towards the ciliary base. When its GTP is hydrolyzed into GDP during its IFT, the resultant Rabl2-GDP dissociates from IFT-B. The trafficking of IFT machinery continues in the absence of Rabl2.
Figure 4A. CellTiter-Glo viability assays in WT and PRIMPOL KO cells 6 days after treatment with 1 µg/ml MMC (2 h) or TMP-UVA (2 µM TMP, 2 h followed by 5 s irradiation). Histograms represent the total number of viable cells (average and SD of three assays) at the end of the experiment, expressed as percentage of the number of untreated cells. Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA and Bonferroni post-test. p-values of individual comparisons are indicated. B. Flow cytometry profiles of BrdU incorporation (y-axis) vs DNA content (PI; x-axis) in WT (blue) and PRIMPOL KO (orange) cells, either collected without treatment (UNT) or at the indicated times after exposure to TMP-UVA (2 µM TMP for 2 h followed by 5 s irradiation). DNA content profiles are shown on top of the boxes (cell count). Dashed horizontal lines inside panels are included for comparative purposes between WT and KO BrdU incorporation levels. Histograms (right panel) represent the percentage of cells in S-phase in each condition (average and SD of three assays). Circle dots in each column represent the values of individual replicates. Statistical analysis in all cases was conducted with one-way ANOVA and Bonferroni post-test. p-values of individual comparisons are indicated. C. Representative confocal microscopy images of EdU staining in WT and PRIMPOL KO cells, 8 h after UVA (control) or TMP-UVA treatment (2 µM TMP, 2 h followed by 5 s irradiation). Nuclear DNA is counterstained with DAPI. When indicated, V5-PrimPol was reintroduced into KO cells. Scale bar, 25 µm. Dot plots indicate the distribution of nuclear EdU intensity, and the mean values are indicated by horizontal red lines. Data from the combination of three replicates (n ≥200 cells per condition). Statistical analysis was conducted with Kruskal-Wallis test and Dunns post-test. p-values of individual comparisons are indicated.
Figure 3(A,B) Absolute numbers of IFN-γ+ and total NK cells in draining lymph nodes at the indicated time points before and after LPS administration in mice treated or not with FTY720 (25g/mouse) to reduce the ingress of lymphocytes in the efferent lymphatics. (C) Tumor volume in mice treated or not with LPS and/or FTY720 according to the scheme. (D) Percent of IFN-γ+ NK cells within the tumor in mice treated or not with LPS and with FTY720. n (number of animals per group)=4. Error bars depict SEM. FTY: FTY720. Statistical significance was determined with a two-tail t test. NT, untreated.
1,25D3 inhibits HIV and M.Figure 1MDM were incubated with 1,25D3 for 4 h before infection with HIV and/or M. tuberculosis (TB) for 3 h, washed then incubated with or without 1,25D3 for 7 days. (A) Top, extracellular release of HIV p24 antigen into the cell supernatant at days 0, 4 and 7 was detected by ELISA. Bottom, MDM were harvested and stained for HIV p17. Histograms are shown for a representative donor. (B) Top, cells were lysed after 3 h exposure to infectious agents or at the completion of the infection phase. Intracellular mycobacteria were harvested and assayed for mycobacterial growth at day 0 and day 7. Bottom, MDM were harvested and stained for mycobacterium. Histograms are shown for a representative donor. (C) Cell lysates from panel B were diluted and run in a MGIT 960. Bar and line graphs are reported as mean ± s.e.m. of three independent experiments performed in triplicate. § P<0.05; * P<0.001.
Exon skipping-based rescue of the DCM phenotype in Ttn Ser14450fsX4 knock-in embryos.Figure 5A Experimental design. B, C Immunofluorescence images of sarcomeric α-actinin and myosin (B) and assessment of cardiac function (C) in homozygous Ttn knock-in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection (n = 3 per group) or lentivirus delivery (n = 7 per group). Scale bars, 10 μm. D Electron microscopy analysis in Ttn knock-in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection. mTtnAON treatment of homozygous embryos rescued myofibril formation (left, black arrows), resulting in thicker filaments (right).
Fig.Figure 4Representative images of vDNA foci detected by click chemistry to PML at 1, 3, 6 and 8 dpi in P6 SCG neurons infected with HSV-1EdC in the presence or absence of IFNα (600 IU/ml). Z-stack images of individual vDNA foci were acquired. Scale bar, 20μm. Zoom scale bar, 1 μm. Percent colocalization of vDNA foci detected by click chemistry to PML at 1, 3, 6 and 8 dpi in SCG neurons infected with HSV-1EdC in the presence or absence of IFNα (600 IU/ml). Each point represents the percentage of 20 vDNA foci that colocalized to PML from 3 biological replicates. (C) 3D reconstruction of a high-resolution Z-series confocal image showing PML entrapment of a HSV-1EdC vDNA foci. Scale bar, 2μm. Enlargement of PML entrapped vDNA outlined by white dashed box. Scale bar, 0.5μm. Quantification of PML-NB volume in SCG neurons infected with HSV-1EdC in the presence or absence of IFNα (600 IU/ml). n=64 biological replicates.
Antagonistic TREM2 antibodies enhance TREM2 shedding and reduce TREM2-dependent signaling and phagocytosis in PGRN deficient iPSC-derived human microglia (hiMGL)A.Figure 4A. Western blot of PGRN in whole-cell lysates of WT and GRN-/- hiMGL. Actin was used as loading control. B. Western blot of TREM2 in whole-cell lysates of WT and GRN-/- hiMGL. Mature (mTREM) and immature TREM2 (imTREM2) are indicated. Actin was used as loading control. C. Quantification of mTREM2 expression in whole-cell lysates of WT and GRN-/- hiMGL (data shown in B). mTREM2 levels were normalized to WT (n = 3, biological replicates). D. ELISA-mediated quantification of sTREM2 in conditioned media of WT and GRN-/- hiMGL (n = 3, biological replicates). E. ELISA-mediated quantification of sTREM2 in conditioned media of GRN-/- hiMGL upon treatment with Ab1 and Ab2 (20 μg/ml, 40 μg/ml) (n = 3, biological replicates). An isotype antibody (10 μg/ml) was used as a negative control. F. AlphaLISA-mediated quantification of p-Syk levels in GRN-/- hiMGL upon treatment with Ab1 and Ab2 (5 μg/ml, 10 μg/ml) with liposomes (1 mg/ml) for 5 min. (n = 8, biological replicates). Data represent mean ± SEM. For statistical analysis in E and F one-way ANOVA with Dunnett`s post hoc test, was used to compare untreated, Ab1, and Ab2 (20 µg/ml and 40µg/ml) conditions to the isotype treated condition. G. Uptake assay for fluorescent myelin in WT hiMGL. Phagocytosis of myelin significantly decreased upon treatment with TREM2 antagonistic antibodies Ab1 and Ab2 (n=4, biological replicates). H. Uptake assay for fluorescent myelin. GRN-/- hiMGL phagocytose significantly more myelin as compare to WT hiMGL. This is reversed upon treatment with TREM2 antagonistic antibodies Ab1 and Ab2 (n=4, biological replicates).
TFG accumulates at the ER/ERGIC interface in mammalian cellsThe distributions of endogenous TFG and ERGIC‐53 were examined in hTERT‐immortalized RPE‐1 cells stably expressing low levels of mApple‐Sec16B using SR‐SIM (more than 15 cells analyzed on 3 separate occasions).Figure 3A The distributions of endogenous TFG and ERGIC‐53 were examined in hTERT‐RPE‐1 cells stably expressing low levels of mApple‐Sec16B using SR‐SIM (more than 15 cells analyzed on 3 separate occasions). Scale bar, 5 μm. Inset scale bar, 1 μm. B Cells stably expressing low levels of GFP‐Sec16B and mRuby‐Sec23A (Sec23A) were fixed and imaged using SR‐SIM (n = 12). Scale bar, 5 μm. Inset scale bar, 1 μm. C The distribution of endogenous TFG was examined in cells stably expressing low levels of mRuby‐Sec23A (Sec23A) using SR‐SIM (n = 10). Scale bar, 5 μm. Inset scale bar, 1 μm. D The distributions of endogenous TFG and Sec31A were examined using g‐STED (n = 8). Scale bar, 1 μm. E The distributions of endogenous TFG and Sec31A were examined using immunogold EM. Large arrowheads highlight 15 nm gold particles bound to α‐TFG antibodies. The small arrowhead highlights 5 nm gold particles bound to α‐Sec31A antibodies. At least 15 different cells were examined, and representative images are shown. Scale bar, 100 nm.
Figure 5A, Superresolution confocal image of endogenous Nucleophosmin (B23), Nucleolin (Ncl) and Trnp1 proteins in E14 cortical cells at 1div. The yellow line indicates the position of the intensity line scan plot (below confocal panels) measuring the grey value of each staining (Trnp1=grey; Ncl=purple and B23=green). B, Co-IP experiments with lysates from P19 cells expressing FLAGTrnp1 for 24h. IPs and WB with the antibodies indicated show interaction between Trnp1 and Ncl but not with Fbl1 consistent with the MS data. Ultrastructural analysis depicting in the nucleolus (arrows for nucleolar interstice) (C), condensed chromatin blocks (arrows) (D) and nuclear speckles or interchromatin granule clusters (arrows for dense cords) Nu: nucleolus; G: granular component; D: dense fibrillar component; FC: fibrillar center; C: condensed chromatin. (E) in the nucleus of P19 cells transfected with plasmids expressing Trnp1-IRES-GFP or GFP in control cells FACS sorted and fixed 24 h after transfection. Scale bars, 2 µm (D) and 1 µm (C and E). F, WB of lysates from P19 cells transfeted for 24h with plasmids expressing GFP only or Trnp1-IRES-GFP with antibodies indicated.
Systematic analysis of interacting proteins with known binding affinities.Figure 3A Selection of PPIs for the affinity-based interaction reference set (AIRS) from the PDBbind (Wang et al, 2004) and the Protein-Protein-Interaction Affinity Database 2.0 (Kastritis et al, 2011). B Selected protein pairs cover a broad range of published binding affinities; PPIs with low- (>10-6M), medium- (>10-8 M) and high binding affinities (≤10-8 M) were sub-grouped in the AIRS. C Recovery rates of PPIs from affinity-based sub-groups with BRET and LuC assays. Significance was calculated by two-sided Fisher's exact test; *p<0.05. D Success of PPI detection from AIRS sub-groups considering single and double LuTHy readouts. E Scatter plot depicting the relationships between published binding affinities (KD) and in-cell BRET50 measurements for 25 interactions in AIRS; BRET50 values are the mean from two independent experiments. Significance was calculated by two-tailed Spearman correlation; ***p<0.001.
a) Fluorescent microscopy of GFP-ORF1p and LC3.f3(a) Fluorescent microscopy of GFP-ORF1p and LC3. (b) Relative number of foci of GFP-ORF1p, or number of foci of GFP-ORF1p co-localized with LC3 per cell in randomly selected fields of cells treated with Bafilomycin A1 (20 h, 400 nM, n=30 cells, total number of GFP-ORF1p foci: control=150, BAF=214, total number of GFP-ORF1p co-localized with LC3: control=32, BAF=199). *P=0.007 Total, P=1 × 10−42 Co-localized with LC3, t-test. Li's correlation coefficient of co-localization (0.328, s.e.m. 0.019). Error bars represent s.e. of the mean. (c) Fluorescent microscopy of endogenous ORF1p and HcRed-LC3. (d) Western blot analysis of ORF1p in autophagosome-enriched fractions. LC3-II is a marker of autophagosomes. (e) Western blot analysis of GFP and alpha-tubulin (control) in cells transfected with GFP-ORF1p and treated with control or Bafilomycin (20 h, 400 nM). f) Fluorescent microscopy of cells expressing LINE-1-MS2, MS2-GFP and mCherry-TIA-1. (g-i) Fluorescent microscopy of endogenous LINE-1 RNA in cells transfected with GFP-DCP1A (g), GFP-GE-1 (h) or GFP-TIA-1 (i). All experiments were performed with a minimum of three biological replicates.
Figure 1A Experimental apparatus of the passive avoidance (PA) test to assess context-dependent associative learning. In this paradigm, the animal receives a foot shock immediately after entering the dark compartment. This leads to hippocampal-dependent aversive learning, which can be assessed by the latency of subjects to enter the dark compartment in subsequent phases of the experiment. B Latency to enter dark compartment for Gadd45a-WT (white bars, n=11) and Gadd45a-KO (blue bars, n=11). A significant reduction was found in the Gadd45a-KO at 24 hours and 7 days after the training. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni post-hoc test: * = p<0.05. C In situ hybridization to detect Gadd45a mRNA in adult wild-type hippocampus (n=8 animals; antisense, left panel; sense, middle panel) and in Gadd45a-KO sections (n=6 animals; right panel). Scale bars: 500 µm (black bar). D Experience-dependent changes in mRNA levels were evaluated under control conditions (home cage) and exposed to the PA (context, and context + shock groups; n=6 for all groups), respectively. Hippocampal Gadd45a mRNA levels remained unchanged, while Gadd45b was induced (context; t10=2.427, context + shock t10=2.319). Concomitantly, mRNA levels of activity-regulated cytoskeleton protein (Arc), as a proxy of neuronal activation, were increased in mice exposed to the PA context (t10=3.406) and context + shock (t10=3.141). Values shown are mean ± SEM; 1-Way ANOVA and unpaired t-test: * = p<0.05, *** = p<0.001.
Figure 4A Loss of DNA methylation of the NTN1 and DAPK1 promoters in decitabine treated PDX tumors, compared with the control PBS-group. The percentage of mean DNA methylation of the 11 DAPK1-CpGs was 94% (550 amplicons analyzed), while that of the 7 NTN1-CpGs was 64% (213 amplicons analyzed). Two-way ANOVA and post-hoc Tukey-test. B Expression of DAPK1, UNC5B, and NTN1 was measured by Q-RT-PCR in PDX tumors after 7 days of in vivo DAC treatment (0.4 mg/kg). The level of PBGD expression was used as an internal control. Data are expressed as mean ± s.e.m. for at least 3 grafts per group. **** P<0.0001,, two-way ANOVA and post-hoc Tukey-test. C Levels of DAPK1, UNC5B, and netrin-1 were measured in at least 3 independent paraffin embedded xenografts per treatment group by immunohistochemistry. Data are expressed as the percentage of the total tumor surface normalized against the median tumor surface of the PBS-group. **** P<0.0001, one-way ANOVA. Error bars = s.e.m. D Cleaved caspase-3 and DNA fragmentation (TUNEL) median number of cells per mm2 were measured in treated xenografts. Data represent mean ± s.e.m. from at least 3 tumors per group. **** P<0.0001, one-way ANOVA.E Representative sections of the xenograft PDX analyzed in (D). Scale bars = 50 μm. Arrows indicate positive staining. F The combination of net1-mAb and DAC reduces humanbreasttumor growth in immuno-compromised mice. After anesthesia, mice were engrafted in the interscapular area with a ≈ 60 mm3 patient-derived tumor. When the tumors reached 120-150 mm3, mice were injected subcutaneously with decitabine (0.4 mg/kg) or PBS and/or intraperitoneally with net1-mAb (10 mg/kg) or with a humanIgG1 control isotype antibody (Ctrl IgG1, 10 mg/kg) from day 1 to day 21. Tumor volumes were measured twice a week; n = 7 mice per group. The statistical significance of the differences obtained between the DAC + Ctrl IgG1-group and the DAC + net1-mAb-group was determined by two-way ANOVA2 and a post-hoc Tukey-test, **** P<0.0001. Error bars = s.e.m.
ai) Western blot analysis of tubulin, LC3-I and LC3-II in zebrafish.f8(ai) Western blot analysis of tubulin, LC3-I and LC3-II in zebrafish larvae where ATG7 was downregulated, as indicated. (ii) Western blot analysis of CALM-HA, actin and LC3-II in zebrafishlarvae where CALM-HA was expressed, as indicated. The larvae were treated with ammonium chloride (NH4Cl), as indicated. (SE, short exposure; LE, longer exposure). Quantification of LC3-II/actin ratio is shown. Data are representative of three independent experiments and shown as mean ±s.d. (*P0.05; two-tailed t-test). (b) Modulation of autophagy alters Dendra-tau clearance dynamics. The fluorescence intensity of each individual cell was quantified at each timepoint (n≥ 31 cells, ≥9 larvae per treatment group) and mean cell intensity values for each drug treatment at each timepoint were calculated. Images were taken immediately after photoconversion and at 24, 30 and 48 h intervals thereafter. Rapamycin treatment significantly increased the rate of Dendra-tau clearance. Ammonium chloride (NH4Cl) treatment significantly decreases the rate of Dendra-tau clearance (**P0.01, ***P0.001, one-way analysis of variance (ANOVA)). Error bars are ±s.e.m. (c) Dendra-tau clearance in the presence of CALM: representative images of CALM with mosaic expression of Dendra-tau and full-length Dendra-tau taken immediately after photoconversion and at 6, 24, 30, 48 and 54 h after conversion. The fluorescence intensity of individual cells was quantified and mean fluorescent intensity of cells co-expressing Dendra-tau and either full-length CALM or Δ-ANTH CALM (larvae-ANTH-HA) constructs (n≥ 100 cells, ≥9 larvae per treatment group) at each time point was calculated. (i) Expression of full-length HA significantly delayed the clearance of CALM at all time points compared with Δ-ANTH CALM (***P0.001, one-way ANOVA). One graph, representative of three independent experiments, is presented. (CALM-ANTH-HA: 132 cells, 20 fishes; CALM-CALM: 18 cells, 8 fishes). Another two experiments are shown in Supplementary Fig. S5C. Error bars are mean ±s.d. (ii) Treatment with CALM alters the dynamics of CALM clearance. Expression of full-length CALM significantly delayed the clearance of CALM at all time points compared with Δ-ANTH CALM, as observed in i (P0.001, one-way ANOVA). However, treatment of Δ-ANTH CALM injected CALM with CALM slows the CALM clearance by 70% to a level comparable to that observed in larvae injected with full-length Dendra-tau. Ammonium chloride treatment of Dendra-tau injected with full-length null results in a modest (22%) decrease in Dendra-tau clearance, suggesting that CALM overexpression and ammonium chloride treatment have a cumulative effect (n≥25 cells, n≥9 Dendra-tau per treatment group). Error bars are mean ±s.d. Note that i and ii are distinct experiments.
Figure 3(A) Glutamate levels assayed by HPLC in the medium of neurons incubated with indicated agents. n=4-6. (B) The paired-pulse ratio of field excitatory postsynaptic potentials recorded in CA1 of hippocampal slices treated with vehicle (Ctrl) or Shh (13-14 slices, 6 rats). Insets: representative traces recorded in response to paired pulse stimuli with different intervals. Statistics of 3H-glutamate uptake by neurons treated with indicated agents (C, n=10) Statistics of 3H-glutamate uptake by neurons transfected with two lentivirus-based RNAi against EAAC1 (R2 and R3) or nonsense RNAi (Non) in response to vehicle (Ctrl) or Shh (D, n=3-6). No difference between R2 or R3 in Ctrl and those in Shh. Inset of (D): representative western-blots for EAAC1. Aspartate (Asp)-evoked currents at -70 mV from hippocampal neurons transfected with RNAi or Non (E, n=17-23) or treated with indicated agents (F, n=16-18). (G) Upper, representative western-blots of EAAC1 and Smo from HEK293 cells transfected with empty vectors (Ctrl), constitutively active form of Smoothened (SmoA1), EAAC1, or EAAC1 plus SmoA1. Lower, 3H-glutamate uptake by cells transfected with the indicated vectors. n=9. (H,I) Shh effects on 3H-glutamate uptake (H, n=6) or Asp-evoked currents (I, n=17-23) of neurons with or without pertussis toxin (PTX) pretreatment. For E,F,I, the uppers: representative traces. Shh: 500 ng/mL. Data were mean ± SEM. *p<0.05; **p<0.01; ***p<0.001 vs. Ctrl or Non (Ctrl) with student's t test.