EMBO/sd-character-level-ner huggingface dataset:
Figure 7(A) Immunoblot of LC3 and p62. MEFs were transfected with PERK-ΔN or PERK shRNA. Endogenous LC3 and p62 levels were measured by immunoblotting. Data represent the ratios of LC3-II to LC3-I and p62 to actin normalized LRRK+/+ MEFs. Error bars represent ±SD from four independent experiments. Data information: For graph A, the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05. (B) Peak values of Ca2+ transients in MEFs transfected with PERK-ΔN or PERK shRNA. Error bars represent ±SD from six independent experiments. (C) Schematic of the signaling pathway consisting of LRRK2, E3 ubiquitin ligase, PERK, MAM components, and the IP3R-VDAC1 complex. Un-phosphorylated LRRK2 binds to E3 ubiquitin ligases and blocks PERKphosphorylation and activation of ligase, resulting in accumulation of MAM components. Subsequently, the accumulated MAM components strengthen the ER-mitochondrial interaction, as evidenced by increased mitochondrial Ca2+ transfer. By contrast, when LRRK2 is autophosphorylated, it detaches from E3 ubiquitin ligases and promotes PERKphosphorylation and activation of ligase, resulting in increased ubiquitination/proteosomal degradation of MAM components and reduced ER-mitochondrial interaction.
Figure 4(A) SDS gel and Western Blot of the native pull-out from S. cerevisiae TAP-tagged eEF3. eEF3* labels the TEV cleavage product of eEF3-TAP, which still carries the calmodulin binding peptide of the TAP-tag and which is recognized by the antibody. RP, ribosomal proteins. (B) Cryo-EM reconstruction of the native eEF3-80S complex with segmented densities for the 60S (grey), the 40S (tan) and eEF3 (pastel green). (C) Fitted eEF3 molecular model colored according to its domain architecture, HEAT (blue), 4HB (yellow), ABC1 (red), ABC2 (green) and CD (magenta). (D, E) (D) Overview on the eEF3-80S molecular model and (E) zoom illustrating eEF3 and its ribosomal interaction partners. ES39S (dark grey), eS19 (pale yellow), uS13 (pastel violet), uL5 (coral), uL18 (light blue), 5S rRNA (light grey).
appearance during axon specificationA.Figure 1A. Schematic illustration of centrosomal MTOC function in neurodevelopmental stages 1, 2 and 3 in human iPSC-derived NSCs/neurons. B. Typical examples of stage 1 (day 0), stage 2 (day 7) and stage 3 (day 12) human iPSC-derived NSCs/neurons immunostained for γ-Tubulin and Centrin, marked by arrows. Cells were co-immunostained with Ki67 and DAPI (day 0) or Trim46 and MAP2 (day 7 and 12) to define their neuronal stages. Arrowheads mark the AIS. Scale bar = 10µm in overview, 2µm in zooms. C. Quantifications of normalized γ-Tubulin fluorescent intensities at centrosomes in stage 1, 2 and 3 human iPSC-derived neurons. n = 46-51 cells in 2 independent experiments. Data information: Data represent mean ± SEM. One-way ANOVA including post-hoc analysis with Bonferroni correction (C). *** p<0.001, ns p>0.05 D. 3D STED imaging of αTubulin in stage 1 (day 0), stage 2 (day 7) and stage 3 (day 13) hiPSC-derived neurons. Scale bar = 5µm in overview, 1µm in zoom. E. Human iPSC-derived NSCs (day 0) immunostained for Trim46, γ-Tubulin and Centrin. Zoom represents centrosome structure. Scale bar = 5µm in overview, 2µm in zooms. F. Typical examples of centrosomes of human iPSC-derived NSCs (day 0) with STED imaging of Trim46 and Centrin immunostaining, and confocal imaging of γ-Tubulin immunostaining. Scale bar = 1µm. G. Typical examples of stage 2 and stage 3 human iPSC-derived neurons immunostained for Trim46 and MAP2. Inserts represent centrosomes. Zooms on the right represent a non-polarized neurite or a developing axon in stage 2 or stage 3 neurons, respectively. Scale bar = 20µm in overview, 1µm in insert, 10µm in zoom.
Figure 8Distribution of the positions of the best HOXB motif relative to the summit of the PANC1-specific FOXA2 peaks. The best PWM identified in the enrichment motif analysis and used to perform the CentriMo analysis is indicated. The graph indicates the average density in 20 bp bins in a window of 600 bp. Percentage of different sets of FOXA2 peaks overlapping HOXB8 ChIP-seq peaks. Representative snapshots showing the overlap between HOXB8 and FOXA2 ChIP-seq peaks in PANC1 cells. The FOXA2 ChIP-seq track in CFPAC1 cells is also shown for comparison. FOXA2 ChIP-seq in CFPAC1 cells transduced with a HOXB8-expressing lentivirus and in their matched mock-infected control. Gained (brown) and lost (yellow) FOXA2 peaks are shown. Pie-charts on the right show the fraction of gained, unchanged and lost peaks overlapping HOXB8 ChIP-seq peaks in transduced CFPAC1 cells. TF motif over-represented in gained or lost FOXA2-bound genomic regions. Expression of HNF1β protein in wild-type and HOXB8-expressing PANC1 cells. Representative snapshots from CFPAC1 cells transduced with the HOXB8-expressing lentivirus.
Figure 4A-C Representative images of PEX13 and PMP70 (A), PEX13 and TOMM20 (B), or PEX13-Flag and WIPI2 (C) colocalization in HeLa/Parkin cells transfected with PEX13 after 4 h DMSO or CCCP treatment. Scale bars, 20 μm (A-B) and 5 μm (C). D Western blot detection of endogenous PEX13 in HeLa/Parkin cells treated with 10 μM CCCP for the indicated time.
Figure 1(A-C) Distribution of spindle pole factors in centrosomal and acentrosomal spindles. DMSO-treated control mitotic spindles (2-centrosomes) and centrinone-treated acentrosomal spindles (0-centrosome) of HeLa cells. (A) Red, gray, green, and blue represent NuMA, α-tubulin, Cep152, and DNA, respectively. Z-projections of eight sections, every 0.3 μm. Scale bar, 5 μm. (B) Red, gray, and blue represent NuMA, Katanin p60, and DNA, respectively. Z-projections of five sections, every 0.3 μm. Scale bar, 5 μm. (C) Red, gray, and blue represent p150glued, ASPM, and DNA, respectively. Z-projections of 10 (2-centrosomes) or five (0-centrosome) sections, every 0.3 μm. Scale bar, 5 μm. (D) The structure of NuMA and microtubules in centrinone-treated acentrosomal spindles of HeLa cells. Two-way arrows indicate the bipolarity of elongated NuMA structures. Gray, red, green and blue represent α-tubulin, NuMA, Cep152 and DNA, respectively. Scale bar, 5 μm. (E) The localization of p150glued in centrinone-treated acentrosomal spindles of HeLa cells. Red, gray, green and blue represent NuMA, p150glued, Cep152 and DNA, respectively. Scale bar, 5 μm. (F) NuMA distribution in 3D. Images of NuMA (Red) and chromosomes (blue) were reconstructed in 3D and surface-rendered. (G) Representative images of NuMA (arrowheads) in centrosomal and acentrosomal cells before and after photobleaching. DMSO- or centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were observed. Time after photobleaching (sec) is indicated. Scale bar, 5 μm. (H) Normalized fluorescence intensities of NuMA-mClover at centrosomal poles (control, 2-centrosomes) or at acentrosomal spindle pole (0-centrosome) during the FRAP time course (N=20 cells for each condition). The first three time points are before photobleaching. Error bars show standard deviation (SD). (I) Mobile fractions from the normalized recovery curves of NuMA-mClover at centrosomal spindle poles (control, 2-centrosomes) or at acentrosomal spindle poles (0-centrosome). Mobile fractions (roughly corresponds to normalized fluorescence intensities at the last time point were determined by single exponential fitting. Line and error bars represent the median and interquartile range (N=20 cells for each condition). The Mann-Whitney U test (two-tailed) was used to obtain a P value.
Figure 1(A) miR-515-5p expression increases the area of the tubulin cytoskeleton. Tubulin (green) and cell nucleus (blue). Objective x20. Bar: 200 µm. (B-C) miR-515-5p inhibits random (B) and directed (C) cell migration. The indicated cell lines were transfected with miR-515-5p for 48 h before time-lapse imaging was performed for 18 h (A) or transwell migration assays were performed for 9 h (C). (B-left panel) Plots show overlays of representative trajectories travelled. (B-right panel) The distance of migration was quantified and represented as the mean ± SEM of values normalised to the respective control condition. (C-left panel) Representative field of view from the bottom of the transwell chambers. Scale bar is 20 µm. (C-right panel) Data represent percentage of the average number migratory cells per field (n=5 fields per condition). Data are mean of three experiments ± SEM. (B-C) P values were calculated by t-test between miRNA (515-5p) values and their respective non-targeting control miR (NC) values (***, P<0.001). All data shown are representative of experiments performed at least in triplicate.
Fig.Figure 7(A) Overview of the experimental procedure. (B-D) Behaviors of MPTP-PD mice assessed by pole (B), beam (C) at 3-6 weeks post-MPTP injection, and locomotor activity (total distance & average speed, D) tests at 6 weeks post-MPTP injection. n= 20 GSK-650394-treated MPTP-PD mice, 14 vehicle-treated MPTP-PD mice, and 8 MPTP-untreated non-PD mice. Data are represented as mean ± SEM. Significantly different at p=0.0012#, 0.0001*, 3.09E-08##, 8.21E-08** in graph B and p=0.0009#, 0.0035*, 1.03E-06##, 0.00003** in graph C and p=0.0001*, 0.0284**, 0.0101# (total distance), 1.49E-08*, 0.0005**, 0.0001# (average speed) in graph D. Histologic assessment of the number of TH+ mDA neurons (E, F), neurite lengths (E, G), and cell body sizes (E, H) Shown are the representative images for TH+ DA neurons in the SN TH+ DA neurons in boxed areas of (E) are enlarged in right. Histologic assessment of the number of TH+ mDA neurons in the SN and TH+ fiber intensities in the striatum Shown are the representative images for TH+ DA neurons in the TH-immunoreactivity in the striatum, Therapeutic effects of GSK-650394 in SNCA-PD model mice. Therapeutic effects of GSK-650394 in SNCA-PD model mice. Behaviors (L-N) of the SNCA-PD mice were assessed at 6-12 weeks post- SNCA injection Significantly different at p=0.008#, 0.021*, 0.00001##, 0.0009** in graph L and p=0.00002#, 0.0344*, 0.0005##, 0.0067** in graph M and p=0.0057#, 0.0345* (total distance), 0.0001#, 0.0375##, 0.0338* (average speed) in graph N. Therapeutic effects of GSK-650394 in SNCA-PD model mice. mDA neuron degeneration in the SN of the SNCA-PD mice were assessed at 12 weeks post- SNCA injection after sacrifice SNCA pathology assessed by p129-α-syn immunoreactivity in TH+ mDA neurons in the SN. n= 5 GSK-650394-treated SNCA-PD mice, 5 vehicle-treated SNCA-PD mice, and 8 SNCA-untreated non-PD mice. Therapeutic effects of GSK-650394 in SNCA-PD model mice. mDA neuron degeneration in the SN (O-U) of the SNCA-PD mice were assessed 12 weeks post- SNCA injection after sacrifice SNCA pathology assessed by p129-α-syn immunoreactivity in TH+ mDA neurons in the SN. n= 5 GSK-650394-treated SNCA-PD mice, 5 vehicle-treated SNCA-PD mice, and 8 SNCA-untreated non-PD mice.
Figure 4A Overview of the ChIRP procedure used to identify HSATIII-interacting proteins in thermal stress-exposed cells. HSATIII-interacting proteins were crosslinked and captured with a biotinylated HSATIII ASO. As a negative control, the same procedure was performed using control (37°C) cells and/or a control probe. B RT-PCR validation of the specific precipitation of HSATIII by ChIRP. The NEAT1 ncRNA and GAPDH mRNA were used as negative controls. Input: 100%. C Silver staining of the coprecipitated proteins. The predicted mobilities of five known nSB proteins are indicated. Input: 0.1%. D Western blot analyses of proteins identified by the HSATIII-ChIRP experiment. Known nSB proteins (SAFB, SRSF1, SRSF7, SRSF9, and HNRNPM) were used as positive controls, and ELAVL1, PSPC1 and α-tubulin were used as negative controls. SRSF3, SRSF5, TRA2B and THRAP3 are newly identified nSB components. Colocalization of novel nSB proteins with HSATIII. Endogenous TRA2B (E) were stained with anti-TRA2B HSATIII was visualized by RNA-FISH using a dig-labeled HSATIII ASO, and the nuclei were stained with DAPI. Scale bar: 10 μm. Colocalization of novel nSB proteins with HSATIII. THRAP3 (F), were stained with anti-THRAP3, HSATIII was visualized by RNA-FISH using a dig-labeled HSATIII ASO, and the nuclei were stained with DAPI. Scale bar: 10 μm. Colocalization of novel nSB proteins with HSATIII. FLAG-tagged PPHLN1 (G) were stained with anti-FLAG antibodies HSATIII was visualized by RNA-FISH using a dig-labeled HSATIII ASO, and the nuclei were stained with DAPI. Scale bar: 10 μm.
Figure 1(A) Semiconservative replication. Diagram of a chromosome with the telomeric C-rich and G-rich strands colored red and green, respectively. Newly synthesized strands are indicated by dotted lines. The three steps of the protocol were strand-specific, dual color, denaturing FISH (1), non-denaturing chromosome orientation (CO)-FISH (2) and denaturing FISH (3). In the second step, the nascent strands have been digested. In all steps, two sets of PNA primers specific for the G-strand and C-strand, respectively, were used to monitor the presence of both strands. The arrows indicate the color of the emitted light and its intensity (idealized).(B) Conservative replication. Diagram showing that telomeric conservative replication (shown here to involve the entire length of the telomeres at the p arms) leads to distinct staining patterns, not observed with semiconservative replication. BIR, break-induced replication.(C) Partially conservative and partially semiconservative telomeric replication. Diagram showing the staining patterns predicted for telomeres that are partially conservatively replicated (distal half) and partially semiconservatively replicated (proximal half), as might occur following fork collapse within a telomere (shown only for the telomeres at the p arms).
Perturbational Profiling Reveals Genotype-Specific Transcriptional Responses, Including Differentially Regulated Genes that Are Required for Autophagy.Figure 1(A) Gene expression analysis of PBMCs isolated from healthy individuals after infection or stimulation with Borrelia burgdorferi (Bor), MDP, or Pam3Cys (Pam). Heatmap shows log2 (fold change) relative to mock-treated control. (B) Heatmap identifying transcripts exhibiting response differences to infection or immune stimuli, stratified by ATG16L1 T300A polymorphism status. (C) HeLa GFP-LC3 cells were transfected with indicated siRNAs and infected with DsRed-labeled Salmonella. The percentage of GFP-LC3+ Salmonella at 1 hr post-infection is shown. Data are shown as means ± SD; n = 75 cells per condition from three biological replicates. Data are representative of three independent experiments. (D) Representative epifluorescent microscopy images of HeLa cells used to generate data shown in (C). The scale bars represent 10 μm. (E) Western blot of HeLa cells transfected with the indicated siRNAs and treated with Torin E64D/PepA. Data are representative of three independent experiments. (F) CLEC12A rescue constructs indicating positions of synonymous mutations and location of targeting siRNA. (G) HeLa GFP-LC3 cells were transfected with control siRNA, CLEC12A siRNA1, or CLEC12A siRNA2 as well as CLEC12A rescue constructs (R1 or R2) and infected for 1 hr with DsRed-labeled Salmonella. Data are shown as mean ± SD; n = 75 per condition. Data are representative of three independent experiments. (H) HeLa cells from (G) were lysed, and HA-CLEC12A expression levels were assessed by western blot. Data are representative of three independent experiments.
Ectopically expressed Histone H3p localizes to the nucleus during parasite asexual development and incorporates into nucleosomes.Figure 3A. Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p-HA in ring (R), trophozoite (T) and schizont (S) stages of P. falciparum asexual growth. PfH3p-HA was detected using anti-HA antibodies (green) and endogenous histone H3 with anti-histone H3 N-terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm. B. Nuclei isolated from wild-type (WT) or PfH3p-HA-expressing (WT + PfH3p-HA) schizont stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 minutes of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant blue (C.B.) or visualized by immunoblotting with anti-HA (α-HA) or anti-C-terminal histone H3 (α-H3c) antibodies. C. Co-immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild-type (WT) or transfected (WT + PfH3p-HA) schizont stage parasites were performed with either anti-HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti-HA or anti-histone H4 antibodies.
Alveolar macrophages are highly responsive to IFN-λ and good producers of IFNs.Figure 1Alveolar macrophages from different donors were purified from BAL fluid by allowing them to attach to the culture dish for 2 hours. Subsequently, they were pre-treated overnight with either type I or type III IFNs (1 ng/mL IFN-λ1 and 10 U/mL IFN-α2, respectively) before infection with Sendai virus (SeV) and influenza A virus (IAV, strain PR8) for 6 hours as indicated. Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2, IFNB1, and IFNL1. ISG response measured by ISG15 and RSAD2. Each colour corresponds to a donor; 9 different donors were analysed. The dashed line represents the detection limit. An ordinary one-way-ANOVA test was used for the statistical analysis: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Alveolar macrophages from different donors were purified from BAL fluid by allowing them to attach to the culture dish for 2 hours. Subsequently, they were pre-treated overnight with either type I or type III IFNs (1 ng/mL IFN-λ1 and 10 U/mL IFN-α2, respectively) before infection with Sendai virus (SeV) and influenza A virus (IAV, strain PR8) for 6 hours as indicated. The column labelled c shows control samples collected directly after purification. Quantification of the presence of SeV or IAV. Each colour corresponds to a donor; 9 different donors were analysed. The dashed line represents the detection limit. An ordinary one-way-ANOVA test was used for the statistical analysis: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
Figure 8(A) Micrographs of triple mutant hAPP after 15min internalization (red), BACE1 immunocytochemistry (green) and their superimposition in wild type hippocampal neurons infected with 5xFAD hAPP lentivirus. DAPI and MAP2 staining are shown in the left column. Insets show higher (3.5x) magnification of the areas indicated in the main images. Scale bar, 5μm. (B) Quantification of the proportion of BACE1 that co-localized with hAPP after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % BACE co-localized with hAPP. N=3 independent experiments, each performed in duplicate; **, P<0.01 vs. WT (one-way ANOVA followed by post hoc test). (C) Quantification of the proportion of internalized hAPP that co-localized with BACE1 after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % internalized hAPP co-localized with BACE1. N=3 independent experiments, each performed in duplicate; *, P<0.05; **, P<0.01 vs. WT (one-way ANOVA followed by post hoc test). (D) Micrographs of p75NTR after 15min internalization (red), Rab11 immunocytochemistry (green) and their superimposition in wild type hippocampal neurons. DAPI and MAP2 staining are shown in the left column. Insets show higher (3.5x) magnification of the areas indicated in the main merged images. Scale bar, 5μm. (E) Quantification of the proportion of Rab11 that co-localized with p75NTR after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % Rab11 co-localized with p75NTR. n=4; *, P<0.05; **, P<0.01 vs. WT (one-way ANOVA followed by post hoc test). (F) Quantification of the proportion of internalized p75NTR that co-localized with Rab11 after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % internalized p75NTR co-localized with Rab11. n=4; **, P<0.01 vs. WT (one-way ANOVA followed by post hoc test). (G) Micrographs of triple mutant hAPP after 15min internalization (red), Rab11 immunocytochemistry (green) and their superimposition in wild type hippocampal neurons infected with 5xFAD hAPP lentivirus. DAPI and MAP2 staining are shown in the left column. Insets show higher (3.5x) magnification of the areas indicated in the main merged images. Scale bar, 5μm. (H) Quantification of the proportion of Rab11 that co-localized with hAPP after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % Rab11 co-localized with hAPP. n=4; *, P<0.05 vs. WT (one-way ANOVA followed by post hoc test). (I) Quantification of the proportion of internalized hAPP that co-localized with Rab11 after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % internalized hAPP co-localized with Rab11. n=4; *, P<0.05 vs. WT (one-way ANOVA followed by post hoc test).
FXR is acetylated at K217 in diet‐induced obese mouse liverExperimental outline.Figure 1A Experimental outline. B An FXR peptide modified by acetylation at Lys 217 (NVKQHADQTVNEDSEGR) was identified in an MS2 spectrum and manually confirmed by a subsequent MS3 scan. Emp: control empty Ad‐Track vector. C FXR acetylated at K217 of FXR in mice fed a ND or HFD was detected by IP/IB analysis using an Ac‐K217‐specific antibody. Duplicates are shown. D Acetylated FXR or FXR acetylated at K217 in livers of dietary obese mice was detected by IP/IB. Relative levels of acetylated FXR or FXR acetylated at K217 are shown on the right. Values are presented as mean ± SEM (n = 3); two‐tailed Student's t‐test, *P 0.05. E Livers from three mice were pooled, liver extracts were prepared, and the effects of feeding a HFD on acetylation of FXR at K217 as a function of time were detected by IB. F Histology of the liver stained for Ac‐K217 by IHC to detect acetylated FXR in fasting/feeding of normal (ND) and HFD mice.