EMBO/sd-character-level-ner huggingface dataset:
Figure 2(a) Top: schematic representation of the STG-p62 construct. Bottom: characterization of several STG-p62 clones in comparison to parental Hap1 cells (b) Wild type or STG-p62 Hap1 cells were mock or puromycin treated for 2 hours and stained for ubiquitin. The endogenous p62 was stained with an anti-p62 antibody in the parental Hap1 cells, while the fluorescence of the GFP tag was recorded in STG-p62 cells. Arrowheads indicate co-localizing puncta (c) Purified STG-p62 was tested for cluster formation in presence of 20µM GST-4xUb. Averages and SDs of three independent replicates are shown for the +GST-4xUb sample and of two for STG-p62 alone (d,e) FCS analysis of diffusion (d) and brightness (e) of the GFP1-5 ruler and of STG-p62 in the cytoplasm of Hap1 cells. Dashed lines represent the linear regression of the GFP1-5 ruler data. Regression coefficients are indicated. For all data points averages and SDs (on both axes for STG-p62) are shown (see methods for a list of the number of measurements performed for each construct). MW: molecular weight. D: diffusion coefficient (µm2/s)
Figure 3Mice were treated as described in Fig.2A. LP cells were isolated from each mouse and were culture for 48h. Cytokine and chemokine production of LP cells were detected using multiplex cytokine assay. Color from blue to red indicates enrichment of gene expression. mRNA expressions of IL-22, β-defensin, Reg3g, and Cxcl1 in LP cells were detected by qPCR. LP cells and mLNs were isolated from each mouse and were culture for 48h. Production of IL-22 in LP cells and mLNs were detected by ELISA. LP cells were isolated from each mouse and were culture for 48h. Expression of Il-17 in LP cells were detected by qPCR. Production of IL-17 in LP cells were detected by ELISA. Protein level of p-STAT3 and STAT3 in colonic epithelial cells was detected by using Western Blot (one mouse per lane). The relative expression of p-STAT3 was calculated. WT and Dectin-3-/- mice were intraperitoneally treated with anti-IL22 antibody or anti-IgG antibody as control during AOM-DSS administration (n=5, each group). Mice were euthanized on Day 100, tumor number, tumor size, and tumor load in colons were measured.
Figure 3A-C. Muscle strength and performance of PEO patients (P1-P4) on normal diet (ND) and after modified Atkins diet (mAD) (one and six months and 2.5 years after initiation of diet). D-G. Spiroergometry results from PEO patients and control subjects on normal diet (ND), as well as one month and six months after mAD initiation. PEO patients had higher serumlactate levels, and attenuated respiratory response to exercise. These effects were further enhanced after mAD and remained after 6 months of the study. mAD increased patients' serumammonium (NH4+) levels. Data presented as mean and SD.
NAF‐1 knockdown reverses BCL‐2 lowering of ER Ca2+ stores.Figure 6(A) H1299 neo and HA‐BCL‐2b5 cells treated with either CTRL or NAF‐1 shRNA were loaded with Fura‐2AM, and ER Ca2+ stores were measured as the difference in cytoplasmic Ca2+ concentration before and after addition of TG (2 μM). Shown are representative traces of Fura‐2AM fluorescence measured at 340/380 nm excitation wavelength ratio at 510 nm wavelength emission. Arrow indicates time at which TG was added, delta values indicate TG‐sensitive ER Ca2+ stores. (B) Differences in ER Ca2+ stores are shown as the average±s.e.m. of three independent experiments as described in (A). (C) Co‐immunoprecipitation of endogenous NAF‐1 and endogenous IP3 receptor type I. Lysate from H1299 HA‐BCL‐2b5 cells was collected and immunoprecipitation was performed with anti‐IP3R1 antibody. The resulting precipitate was analysed by immunoblot.
HIV genomes form large nuclear foci in macrophages (A-C) EdU-labeled viral DNA forms large foci in ThP1 cells infected by HIV-1.Figure 1EdU-labeled viral DNA forms large foci in ThP1 cells infected by HIV-1. EdU images are shown in red, DAPI images in blue. (A) Left: nucleus of an infected cell at 48 h p.i. displays three large and bright EdU foci (colored arrows), as well as small and dim punctae (grey arrows). Center: Magnified views have colored borders matching the colors of the arrows. 3D images show that EdU foci are located within the nucleoplasm (Movie EV1, Appendix Fig. S2). Right: nucleus of an uninfected cell displays no EdU signal. (B) Graph shows EdU intensity profiles along lines shown in A, with colors matching the corresponding arrows. (C) Dual-color image of an infected cell with EdU (red) and immunolabeled lamin (grey). Curves to the right plot the intensity profile of EdU and lamin along the dashed blue line. The EdU intensity peaks do not coincide with lamin enrichment. See also Fig. EV1A,B. (D) Multi-color images of an infected cell showing EdU (red) with CA (yellow) and integrase (green). The colocalization of nuclear EdU foci with CA and IN (arrows) confirms that EdU specifically labels viral DNA and shows the presence of these proteins in vDNA foci. See also Appendix Fig. S3. (E,F) vDNA foci are much larger than the predicted size of single viral genomes. (E) Left image shows an observed, EdU-labeled vDNA focus. Right: simulations of a single linear or circular 10 Kb long chromatinized DNA polymer chain and corresponding predicted images in diffraction-limited (~300 nm resolution) microscopy. (F) Boxplots show the distribution of sizes (FWHM, full width at half maximum) of n=40 measured vDNA foci (for MOI 100) compared to the sizes predicted for linear (left) and circular (right) chains based on n=100 simulated configurations each. Blue lines in boxes define medians, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. All differences are highly significant (Wilcoxon test data vs. model: ***p ≈ 3 ⋅ 10 − 20, circular vs. linear model: ***p ≈ 10 − 17). (G) Images of vDNA foci (arrows) in ThP1 cells infected with multiplicities of infection (MOI) 10, 50 and 100. Images for MOI 10 are shown at two different contrast levels to reveal dimmer spots. Scale bars, 3 µm. (H) Boxplots compare the peak intensities inside individual vDNA foci for MOIs 10, 50 and 100. Central lines in boxes define medians, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. Intensities increase significantly with MOI (Wilcoxon tests: MOI 10 vs. MOI 50: *** p ≈ 5 ⋅ 10 − 16; MOI 50 vs. MOI 100: ***p ≈ 5 ⋅ 10 − 5; MOI 10 vs. MOI 100: ***p ≈ 2 ⋅ 10 − 23). Data from one experiment. A replicate showed similar results. (I) Histograms show the number of foci per nucleus for MOIs of 10 (blue), 50 (green) and 100 (red).
Histidine metabolic perturbations affect nerfin-1 neuron to NB reversion A) Schematic depicting the metabolic reaction between histidine and its downstream metabolites histamine and 3-methyl-L-histidine.Figure 2A) Schematic depicting the metabolic reaction between histidine and its downstream metabolites histamine and 3-methyl-L-histidine. B) Concentration of histidine and 3-methyl-L-histidine from whole Drosophila adults extracts measured by 1H NMR after 3 days of feeding on either CDD or -his diet (n=3,3). Both compounds are reduced to below detection levels (<0.3mM) upon dietary his depletion. C) Dietary supplementation of 3-methyl-L-his for 4 days significantly increased adult nerfin-1 clonal growth, and rescued nerfin-1 growth inhibition due to his dietary withdrawal (n =18,14, 7,9). D) Dietary supplementation of histamine does not significantly increase adult nerfin-1 clone size, but can rescue nerfin-1 clonal growth inhibition due to his dietary withdrawal (n =42,14,23,25). E) Larval nerfin-1 clonal growth is significantly decreased upon overexpression of HDC RNAi (n=23,36). F), Schematic depicting stepwise neurons to NBs reversion in nerfin-1 type I lineages where neurons express Elav, NBs express Dpn, and reverting neurons express both markers. G) Hdc inhibition significantly reduced the percentage of Dpn+ NBs (n=16,17), and significantly increased the percentage of Elav+ neurons per clone (n=16,17). H) HdcRi overexpression reduced cellular growth of nerfin-1 NBs (n=17,19) and neurons (n=36,19) measured as the ratio of nucleolus/nuclear volume.
Figure 8Experimental overview of the tamoxifen-inducible disruption of Il7r following generation of 2W1S-specific Th1 memory T cells. CD4creERT2 x Il7rf/f or Il7rf/f (littermate control) mice were injected i.v. with Lm-2W1S, tamoxifen administered for 4 consective days (19-21 dpi) and spleen cells harvested for ATAC-Seq 14-18 days later. Flow cytometric analysis of IL-7Rα expression by 2W1S-specific CD44hi CD4 T cells in the presence or absence of cre recombinase. Average number of 2W1S-specific cells recovered/mouse after sorting 3 replicate samples of Il7rf/f compared to CD4creERT2 x Il7rf/f . Errors bars indicate the standard deviation. Average ATAC-Seq signal at the 411 IL-2 pDHSs which contain a STAT binding motif. UCSC genome browser tracks at loci with IL-2 pDHSs showing replicate ATAC-Seq samples for CD4creERT2 x Il7rf/f memory T cells, Il7rf/f control memory T cells, replicate DNase-Seq samples for TB IL-2 and TB IL-2nil and TB IL-2 and TB IL‑2nil STAT5 ChIP-Seq samples. The grey boxes signify IL-2 pDHSs which diminish after disruption of the Il7r gene.
Figure 6Biochemical strategy employed to trap a post-translocated state of IAPV-IRES in the ribosome. General view of the IAPV-IRES in a post-translocated state on the ribosome: 60S depicted as blue ribbons, 40S as yellow ribbons, IAPV-IRES represented as solid Van Der Waals surface colored according to secondary structure elements described in figure 1A and eRF1* depicted orange. Close up view of the SL-III inserted in the experimental unsharpened cryo-EM density. On the right, refined model with residues from the 60S (blue) in interacting distance with the SL-III (green) indicated. Comparison of the final refined model for IAPV-IRES colored according to the secondary structure described in figure 1A with canonical tRNAs (PDBID: 4V5D) in the pre-translocated state (left) and after translocation (right). Left, overall top view of the inter-subunit space of the 40S for the post-translocated state. Inset, close up view of the 40S tRNA binding sites where it can be appreciated the insertion of PKI of the IAPV-IRES in the P site, projecting the VLR towards the E site. The elements of the 18S rRNA forming the "P site gate" are indicated by solid arrows. On the right, equivalent view for canonical tRNAs.
Figure 6(A) EVD, ACT2 and TAS1c capped and uncapped 5'ends from nanoPARE and Smart-seq2 libraries along 20-21 nt siRNA in WT and ddm1. (B) RIBO-seq coverage profiles from 35S:EVD in WT or rdr6. RPM: Reads per million. (C) Ribosomal footprints on shGAG in rdr6 displaying codon occupancy at P-sites to calculate codon coverage. The coverage observed at each codon position was divided by the expected mean coverage along the entire GAG coding sequence. (D) Maximal individual codon coverage over the expected coverage for all translated transcripts of Arabidopsis. Vertical lines indicate the strength of stalling sites of shGAG in the WT or rdr6 background. Percentages specify the proportion of transcripts with more pronounced stalling events than shGAG. (E) Overlay between 35S:EVD siRNAs (red) and RIBO-seq profiles (black) in the WT background. (F) Schematic representation of putative ribosome stalling-linked mRNA breakage generating 5'OH ends. Lack of 5'PO4 prevents XRN 5'->3' exonucleolytic activity (see Fig.6A), granting the RNA to be used as template by RDR6. (G) Overlap between ribosomal footprints and mapping of 5'OH ends from 35S:EVD in rdr6 cloned through RtbC ligation. Regions investigated are highlighted in grey. 5'OH ends were only successfully cloned from region #1. Alignment of sequenced clones to EVD is displayed in Appendix Fig. S9.
Loss of pkma2 and ppargc1a affects cardiomyocyte dedifferentiation and proliferation following cardiac injury.Figure 2(a Immunostaining of heart sections for PCNA and MEF2 in 5 dpci pkma2+/-; pkmb+/- and pkma2-/-; pkmb+/- animals; magnified view of area in white boxes shown below; arrowheads point to PCNA+ CMs; white dashed lines mark the wound border; percentage of PCNA+ CMs in the border zone shown on the right (n = 4-6 ventricles of each genotype). Immunostaining of heart sections for N2.261 and MEF2 in 5 dpci pkma2+/-; pkmb+/- and pkma2-/-; pkmb+/- animals; magnified view of area in white boxes shown below; arrowheads point to N2.261+ CMs; white dashed lines mark the wound border; percentage of N2.261+ in the border zone shown on the right (n = 4-6 ventricles of each genotype). Immunostaining of heart sections for PCNA and MEF2 in 5 dpci ppargc1a+/+ and ppargc1a-/- animals; magnified view of area in white boxes shown below; arrowheads point to PCNA+ CMs; white dashed lines mark the wound border; percentage of PCNA+ CMs in the border zone shown on the right (n = 4-6 ventricles of each genotype). d) Immunostaining of heart sections for N2.261 and MEF2 in 5 dpci ppargc1a+/+ and ppargc1a-/- animals; magnified view of area in white boxes shown below; arrowheads point to N2.261+ CMs; white dashed lines mark the wound border; percentage of N2.261+ CMs in the border zone shown on the right (n = 4-6 ventricles of each genotype). (e AFOG staining of heart sections from 60 dpci pkma2+/-; pkmb+/- and pkma2-/-; pkmb+/- animals; black dashed lines outline the scar area; scar area measured on the right (n = 3-5 ventricles of each genotype). f) AFOG staining of heart sections from 45 dpci ppargc1a+/+ and ppargc1a-/- animals; black dashed lines outline the scar area; scar area measured on the right (n = 3-5 ventricles of each genotype).
Assembly of microtubules and actin filaments on isolated centrosomes.Figure 3Two sets of representative images showing fluorescent microtubules and actin filaments assembled from isolated centrosomes. Centrosomes were isolated from Jurkat cells expressing centrin1-GFP. Upper and lower lines show actin filaments and microtubules radiating from two distinct centrosomes with low (top) and high (bottom) densities of actin filaments. Scale bars: 10 μm. The graph shows the number of microtubules per centrosome relative to the density of actin filaments. Inset shows actin filaments at the centrosome with a FIRE look-up table and a 20 µm-wide circle in which actin fluorescence intensity is measured. Measurements were pooled from 5 independent experiments; n= 50. Microtubules (top line) and actin filaments (bottom line) assembly from isolated centrosomes in the presence of increasing concentration of monomeric actin (from left to right). Scale bar: 20 μm. The graph shows the number of microtubules per centrosome in response to increasing concentrations of monomeric actin. Data were pooled from 2 independent experiments; 0 µM: n=21; 0.3 µM: n=17; 0.5 µM: n=17; 1.0 µM: n=17. ****:P<0.001 Mann-Whitney test. The image shows the density of actin filaments (in the presence of 1µM of actin monomers) at the centrosome color-coded with the FIRE look-up table and the definition of central, peripheral and distal regions corresponding to decreasing concentrations of actin filaments. Scale bar: 20 μm. The graph shows the measurements of microtubule growth rate in each region. Data were pooled from 3 independent experiments: central zone: n=58, peripheral: n=104, outside: n=61; n. s. means no statistical difference between the data set according to Mann-Whitney test. The graphs show the various intensities of centrosome immuno-staining with antibodies against gamma-tubulin on the same coverslip depending on the presence/absence of actin filaments (left) or on the amount of actin filaments (right). Data were pooled from 3 independent experiments. Left graph: without actin n=69, with actin n=26, right graph n=26. N.s. means no statistical difference between the data set according to Mann-Whitney test.
Figure 8A. Western blot analysis to detect phosphorylation levels of Akt (S473) on lysates from Pmp22-/- and WT mice at P20, with quantification. Results are mean ± SEM, p=0.6857, two-tailed Mann Whitney U test, representative of three independent experiments. B. Western blot analysis for Mbp and P0 on lysates from Pmp22-/- and WT mice P20 shows decreased myelin protein expression levels at both time points in Pmp22-/- nerves, results are mean ± SEM, p=0.0500 with the exception of the P0 protein level at P20, for which the difference between Pmp22-/- and WT nerves is not statistically significant, p=0.1000, one-tailed Mann Whitney U test, representative of two independent experiments. C. Representative images of semithin section analysis of WT and Pmp22-/- sciatic nerves at P30. Tomacula are abundant in Pmp22-/- nerves at this age, where both fibers with thicker myelin (in the range of <3-4 μm in diameter) and thinner myelin (with diameters greater than 4 μm, particularly in motor fasicles) can be observed.C'. Niaspan does not ameliorate the phenotype in Pmp22-/- mice, as indicated by the number of tomacula (quantification of the percentage of tomacula in the entire sciatic nerve section, results are mean ± SEM, p=0.7400, Mann Whitney U test, n= 15 animals per condition). D. Growth curve of Pmp22-/- mice treated with Niaspan administered by i.p. injection daily at 160mg/Kg and starting at P15 for 15 days. A significant time by group effect is noted between WT saline and Pmp22-/- saline (group effect p=0.9907 and time effect p=0.0057), indicating that the two groups start with similar weight values but then the WT saline group (n=6 animals) grows more in time. When Pmp22-/- saline (n=5) and Pmp22-/- Niaspan-treated (n=6) are compared, the group effect is statistically significant (p=0.0076) while the time effect is not (p=0.6677), suggesting that these two groups have a similar growth trend but Pmp22-/- Niaspan-treated group starts with a higher baseline weight. E. Niaspan does not ameliorate myelin thickness as indicated by g-ratio analysis, with axonal diameter distribution: WT 0.68 ± 0.003, 2945 fibers, n=5 animals; Pmp22-/- 0.64 ± 0.003, 1557 fibers, n= 6 animals; Pmp22-/- Niaspan 0.63 ± 0.003, 1445 fibers, n=6 animals, results are mean ± SEM. WT saline as compared to Pmp22-/- saline-treated p<0.0001; WT saline as compared to Pmp22-/- Niaspan-treated p<0.0001, repeated measures ANOVA. Note the loss of myelinated fibers in Pmp22-/- nerves for all caliber axons.
trimers are acetylated to increase binding to the second zinc finger of PARP-1 A 2D gel electrophoresis of lysate from WT cells followed by Western blot analysis with α-NEDD8 antibody indicates free NEDD8 migrates near its predicted isoelectic point (pI) of 6.Figure 62D gel electrophoresis of lysate from WT cells followed by Western blot analysis with α-NEDD8 antibody indicates free NEDD8 migrates near its predicted isoelectic point (pI) of 6.59 2D Gel electrophoresis of pulldowns from NEDP1 KO lysate with GST-Zn1+2 followed by Western blot analysis with α-NEDD8 antibody. Some of the 25-kDa NEDD8 band migrates to the same isoelectric point as unconjugated NEDD8 (denoted by white triangle), indicating the species is most likely an unanchored NEDD8 trimer. However, the majority of NEDD8 trimer migrates to multiple spots of lower pI (indicated in the brackets), which suggests that NEDD8 trimers undergo a post-translational modification that either adds a negative charge or blocks a positive charge on the NEDD8 trimer. Overexpression of HDAC1 or HDAC2 decreases PARP-1-NEDD8 trimer binding. NEDP1 KO U2OS cells were transfected with GFP or GFP fused to the first two zinc finger domains of PARP-1 (GFP-Zn1+2). Cells were also co-transfected, as indicated, with empty FLAG vector, HDAC1-FLAG, or HDAC2-FLAG. 24 hours post transfection, cells were harvested and immunoprecipitation was performed on the lysates with GFP-Trap. Immunoprecipitated proteins were resolved by SDS-PAGE followed by Western blot analysis with the indicated antibodies. HDAC inhibition increases PARP-1-NEDD8 trimer binding. NEDP1 KO U2OS cells were transfected with GFP or GFP fused to the first two zinc finger domains of PARP-1 (GFP-Zn1+2). 24 hours later, cells were left untreated or treated with the HDAC inhibitor sodium butyrate (NaB) (10 mM) for 4 hours. Cells were harvested and immunoprecipitation was performed on the lysates with GFP-Trap. Immunoprecipitated proteins were resolved by SDS-PAGE followed by Western blot analysis with the indicated antibodies. HDAC inhibition increases PARP-1-NEDD8 trimer binding. NEDP1 KO U2OS cells were left untreated or treated with the HDAC inhibitor sodium butyrate (NaB) (10 mM) for 4 hours. Cells were harvested and lysates were incubated with recombinant Zn1-GFP or Zn2-GFP (25 nM) for 1 hour followed by immunoprecipitation with GFP-Trap. Immunoprecipitated proteins were resolved by SDS-PAGE followed by Western blot analysis with the indicated antibodies. Overexpression of HDAC1 or HDAC2 in NEDP1 KO cells can rescue the induction of PAR polymer following H2O2 treatment. U2OS WT or NEDP1 KO cells were transfected with the indicated FLAG vectors. 48 post-transfection cells, were treated with H2O2 (600 μM) for the indicated amount of time and harvested directly in sample loading buffer. Lysates were resolved by SDS-PAGE and processed for Western blot analysis with the indicated antibodies.
a, b) Bottom view (a) and side view (b) of the complex structure.figf2(a, b) Bottom view (a) and side view (b) of the complex structure. The ribbon model represents Keap1-DC and the stick model shows p62-KIR. Each β-propeller blade is numbered from 1 to 6. (c) The simulated-annealing Fo − Fc omit map is contoured at 3 σ. p62-KIR (pink) was omitted from the calculation. The electron density of the peptide-bound region V348 to L355 of p62-KIR was unambiguously visible. (d) Intermolecular hydrogen bonds of Keap1-DC in complex with p62-KIR (left; PDB ID 3ADE) and in complex with the Nrf2-ETGE region (right; PDB ID 1x2r). (e) Intra-peptide hydrogen bonds of p62-KIR in the Keap1-DC complex. Hydrogen bonds (green broken lines) and their distances (Å) are displayed in d and e.
Mithramycin blocked sodium butyrate induced endoplasmic reticulum stress in colorectal cancer cells.Fig 7  HCT-116 (A, B) or HT-29 (C,D) cells were treated with 0.1μM mithramycin for 30 min followed by sodium butyrate (NaB) for 24 h. Representative Western blots showing the expression of IRE-1a, BIP, and PDI in HCT-116 (A) or HT-29 (C) cells are shown. GAPDH was used as loading control. Protein expression (IRE-1a, BIP, and PDI) was quantified by densitometry and normalized to GAPDH (ratio of protein:GAPDH). The fold change from control for each protein is expressed as mean ± SD of three independent experiments for HCT-116 (B) and HT-29 (D) cells. One-way ANOVA was used for statistical analysis. * P<0.05, ** p<0.01, compared to the respective control group.