EMBO/sd-character-level-ner huggingface dataset:
Kinase activity and mitochondrial targeting of PINK1 is imperative for mitochondrial localization of Parkin.Figure 5.(A) Schematic depiction of pathogenic and deletion mutants of PINK1 used in this study. MTS, mitochondria-targeting sequence; TMD, transmembrane domain. (B) Subcellular localization of Parkin in PINK1−/− cells complemented by various pathogenic and deletion mutants of PINK1-Flag. Cells were treated with CCCP. Higher magnification views of the boxed areas are shown in the insets. (C) The number of cells with Parkin-positive mitochondria was counted as in Fig. 3 A. Error bars represent the mean ± SD values of least three experiments. (D) PINK1−/− MEFs complemented by various PINK1 mutants were treated with CCCP and subjected to immunoblotting using anti-Parkin or anti-Flag (tag of PINK1) antibodies. IB, immunoblot; Ub, ubiquitylation.
Figure 5Retinal inflammatory scores of mice immunized s.c. with PBS + DMSO (Veh), IRBP + DMSO (IRBP) or IRBP + RA + IL-2 21 and 7 days before induction of EAU by s.c. injection of IRBP emulsified in CFA. Bars are mean + SEM (n=6), *p<0.05 and ***p<0.001, IRBP + IL-2 + RA vs Veh, ###p<0.001 IRBP + IL-2 + RA vs IRBP by two-way ANOVA with Tukey post hoc test. Representative fundus images of each group of mice 35 days after induction of EAU. Arrowheads indicate area with retinal vasculitis and the asterisk indicates a region of vitreous haze, where the image appears blurred. Retinal inflammation scores in HEL-TCR transgenic mice were s.c. injected with PBS + DMSO (Veh) or HEL + RA + IL-2 on pn 18 (1x) or on pn 18 and 25 (2x). Results are mean + SEM (n=3, 4 or 5). *p<0.05 and ***p<0.001, HEL + IL-2 + RA 1x vs Veh, ##p<0.01 HEL + IL-2 + RA 2x vs Veh by two-way ANOVA with Tukey post hoc test. Representative photographs of each group of retinal fundus of HEL-TCR transgenic mice at pn 35. Arrowheads indicate representative areas with retinal vasculitis. E Representative FACS plots of IL-17+ CD4 or IL-10+ CD4 T cells in the retina on day 36 of HEL-TCR transgenic mice. Cells were gated on live single CD3+CD4+ and CD3+CD4+LAG-3+Foxp3- respectively. F Mean absolute number of IL-17+ CD4 T cells in the retina (upper panels) or mean percentages of IL-10+ LAG-3+ Foxp3- CD4 T cells in the spleens of of HEL-TCR transgenic mice with vasculitis (lower panels). Results are mean + SEM (n=3, 4 or 5). **p <0.01 and ***p <0.001 by one way ANOVA with Bonferroni post hoc test.
Figure 2 A. GAR domain deletion or mutation perturbs binding to Kif5a. Co-immunoprecipitation analysis of Kif5a and HA-Dendra2-tagged nucleolin (both overexpressed in HEK-293 cells). IP was performed with HA antibody and probed in Western blot with anti-Kif5a and anti-HA antibodies. Ncl FL - HA -Dendra2- full length nucleolin; Ncl ΔGAR - HA-Dendra2- nucleolin with a GAR domain deletion; Ncl GAR(N) - HA-Dendra2- nucleolin with all 10 arginines in the GAR domain mutated to asparagines; DEN - Dendra2. Note that HA input blots for HA-Dendra2 (Dendra) and the other three constructs are from the same membrane, but shown discontinuously owing to the different migration of these proteins in PAGE.R4, but not N4 peptide reduces Kif5a co-immunoprecipitation with nucleolin from mouse sciatic nerve axoplasm, assayed by automated capillary electrophoresis immunoassay for Kif5a. Schematic of the assay is shown in (C). Representative traces of the Kif5a immunoreactive peaks are shown R4, but not N4 peptide reduces Kif5a co-immunoprecipitation with nucleolin from mouse sciatic nerve axoplasm, assayed by automated capillary electrophoresis immunoassay for Kif5a. Schematic of the assay is shown quantifications are shown in (E). Immunoprecipitated Kif5a levels are normalized with input levels and expressed relative to N4. n = 3 independent biological repeats; means ± SEM; * p<0.05, paired Student's t-test. F. Surface plasmon resonance analysis of KLC2 binding to biotinylated R4 peptide Recombinant KLC2 was injected at different concentrations (5-160 nM) on biotinylated R4 or N4 peptides immobilized to a streptavidin sensor chip. The dissociation steady state constant KD, was determined by fitting the sensogram to a 1:1 model. G. Kinetics analyses of photoconverted Dendra fusion proteins in cultured DRG neuron axons. Adult mouse DRG neurons were transfected by electroporation with constructs expressing HA-Dendra-GAR(WT) - wild-type nucleolin GAR domain N-terminally fused with HA-Dendra, or HA-Dendra-GAR(N) with 10 arginines in the GAR domain mutated to asparagines. 48 hr after plating and transfection, Dendra fluorescence was photoconverted using a DMD module to restrict conversion within the cell body. Time-lapse images were collected from the entire field of view, and red signal intensity was analyzed within axons at distances of 60-90 µm from the cell body. Photoconverted signal sampled at 10 sec intervals is shown, normalized to signal intensity before photoconversion. N = 3 biological repeats comprising 7 cells each. Means ± SEM; **** p<0.0001 (ANOVA).
Figure 1A. KD efficiency of three different Syt11 shRNAs in DRG neurons. Cultured DRG neurons were infected with control or shRNA-expressing lentiviruses at DIV 1, and Western blotting for Syt11, Syt1, Syt4, synaptobrevin 2 (Syb2), SNAP25, complexin 1/2 (Cpx1, Cpx2), clathrin, adaptor protein 2 (AP-2), and β-actin were performed at DIV 6-7. N = 4 independent experiments. B, C. Representative Cm traces induced by 200-ms pulse depolarization (arrows) ib DRG neurons. DRG neurons were transfected with plasmids expressing shSyt11-2 (Syt11 KD, KD) or scrambled shRNA (Sc) and Cm recording was performed 5 days after transfection. Endo-5s represents the Cm decay 5 s after stimulation. Insets show Ca2+ currents recorded in the same neurons. D. Representative Cm overshoot recorded from an Syt11 KD neuron. About 45% of the KD neurons showed excessive membrane retrieval after a 200-ms depolarizing pulse. E. Averaged Cm traces recorded from Control (Ctrl), Sc, KD (from all three shRNAs, n = 39 for shSyt11-2, n = 7 for shSyt11-1, n = 14 for shSyt11-3), and rescued (with the shSyt11-2-resistant form of Syt11) DRG neurons. F-H. Statistics of Cm jumps, Endo-5s, and Ca2+ current recorded from DRGneurons as in (B-E). Data were collected from 4 (Sc, Rescue), 8 (Ctrl), and 16 (KD) independent experiments. I. Normalized Cm changes induced by two pulses of 200-ms depolarization (arrows) at a 1-s interval. Data were normalized to the capacitance jump induced by the first depolarization. J. Paired-pulse ratios of ΔCm with different interstimulus intervals as in (I). DRG neurons were stimulated with two 200-ms pulses at different intervals as indicated, and the paired-pulse ratio was calculated by normalizing the ΔCm in response to the second stimulation to that induced by the first one.
Figure 3A-B) Kinetics of S. Typhimurium replication in MEFs depleted of WIPI1 (A). Intracellular bacteria were enumerated by their ability to form colonies on agar plates. Western blot for GFP:WIPI1 upon the indicated siRNA treatments. A-B) Kinetics of S. Typhimurium replication in MEFs depleted of WIPI2 (B). Intracellular bacteria were enumerated by their ability to form colonies on agar plates. quantitative RT-PCR for WIPI2 upon the indicated siRNA treatments.
Figure 4A. FACS analysis of CD34+α6+ cells over time shows that 3C cultures reach a ~50:50 population equilibrium after 10 days of culture (mean ±SEM; n=3). B. Cells were EdU-labeled for 24 h (day 10-11) prior to analysis by flow cytometry to quantify proliferating cells. Percentage of EdU+ cells is indicated (n=4; mean ±SD; *p ≤ 0.05, Mann-Whitney U-test). C. Schematic illustration of the parameters used in the mathematical model. HFSCs (CD34+α6+) and non-HFSCs (CD34¯α6+) have a maximum proliferation rate of pSC,max and pP,max, respectively. They enter apoptosis with rates apSC and apP and are both degraded with a rate deg. Non-HFSCs can convert to HFSCs with rate tPSC and vice versa with rate tSCP. D. Simulated population proportions of CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs derived from the mathematical model describing steady state cultures. Experimental data from (A) are shown as reference (in grey). See also Appendix figure S4 and Dataset EV4.
Oligo#5 enters neurons after ICV injection, changes 5HT2C splicing and food intake.Figure 5A. Accumulation of Cy3-labeled oligo#5 in the area around the 3rd ventricle one hour post injection, shown is the ventral part of a coronal section -1.34 mm from Bregma. The injection coordinates were AP: -1.82 mm, DV: -4.5 mm, ML: 0.B. Uptake of oligo#5 in the arcuate nucleus region (arc), enlarged from panel A. C. Oligo#5 enters neurons. Enlargement of hypothalamus region taking up oligo#5 (red), stained for the neuronal marker NeuN (green). Most of the oligo#5 signal is in neurons, but some is also in NeuN negative cells (arrow), likely glia.D. Oligo#5 enters some glia cells. Enlargement of a hypothalamic region adjacent to the 3rd ventricle one hour after oligo#5 injection. The oligo (red) can be found in some GFAP-positive glia cells (arrow), but numerous GFAP-positive cells (green) show no oligo#5 uptake. E. Effect of oligo#5 on RNA1/RNA2 ratios in the injected area. The area marked by Cy3 staining in the dorsal hypothalamus, consisting mainly of the arcuate nucleus was microdissected, RNA isolated and analyzed by qPCR. NS: non-significant changes (3 hrs: p=0.01; 6 hrs: p=0.000001; 9 hrs: p=0.02; 12 hrs: p=0.06; n=3) F. Oligo#5 does not change the RNA2-editing pattern. The hypothalamic area showing oligo#5 staining was microdissected 6 hrs post injection and RNA2 was amplified and the PCR product subjected to deep sequencing. The percent of non-edited reads in total reads is shown (at least 20,000 reads per lane). G. Effect of oligo#5 on POMC expression. The RNA from panel D was analyzed using qPCR with primers against POMC, normalized to GAPDH (3 hrs: p=0.0009; 6 hrs: p=0.02; 9 hrs: p=0.1; 12 hrs: p=0.3, n=3). H. Effect of oligo#5 on food uptake in wild-type mice. C57BL/6 wild-type mice received a guide canulae and did not have access to food for 16 hours. After injection of 2 µg oligonucleotides, they could freely access food. The food consumption was measured by weighing the food used. A total of 52 animals were used, p=0.001 for the difference between oligo#5 and control oligonucleotides. Each of the six individual experiments showed statistically significant differences between oligo#5 and control (p<0.01 - 0.001). Control oligo: an oligonucleotide against human SMN2. I. Effect of oligo#5 on food uptake in ob/ob mice. Ob/obmice received oligo#5 through ICV injection, similar to panel F. However, the mice could freely access food at all times (p=0.04, n=3).
Figure 1(A) Venn diagram representing overlap of DVL3 ChIP-Seq peaks between MDA-MB-468 and MCF7 cells. (B) MEME-ChIP (Motif Analysis of Large Nucleotide Datasets) analysis of the DVL3 binding sites identified DVL3 specific motifs in MDA-MB-468 cells: TGGAATGGAATGGAATGGAAT in 622 fragments with a p value = 3.1e-4486, ATTCCATTCCATTC in 673 with a p value = 8.6e-1868, and TTCCATTCCATTCCATTCCA in 605 fragments with a p value = 2.9e-1614. The p value is the significance of the motif according to MEME, motif discovery program. (C) MEME-ChIP analysis of the DVL3 binding sites identified DVL3 specific motifs in MCF7 cells: CAGAAKVATTCTCAGAAACTYCTTTGTGA in 1394 fragments with a p value = 1.5e-2815, GTGTGYRTTCAACTCACAGAGTTGAACSTT in 1143 fragments with a p value = 3.7e-2478 and,KGAAACACTCTTTTTGTAKAWTYTGCAAG in 1359 fragments with a p value = 9.4e-2181. The p value is the significance of the motif according to MEME, motif discovery program. (D, E) Pie graph showing enriched pathways of ChIP-hits for MDA-MB-468 (D) and MCF7 (E) cell lines generated by Reactome pathway analysis. (F, G) Bar graphs showing the −log10 P-values for the Reactome terms of the most significant pathways enriched using Binomial Test in DVL3 ChIP-Seq for MDA-MB-468 (F) and MCF7 (G) cell lines.
Expanded polyglutamine repeats affect elimination of damaged mitochondria by mitophagy.Figure 3A Representative Western blots showing separation of lysosome- and mitochondria-enriched fractions by a density gradient. Total extracts from different HD models were fractionated by the density gradient. Eight fractions were collected from top (#1) to the bottom (#8). The amount of mitochondria in the lysosomal fractions was presented as a percentage of VDAC present in the entire gradient. n = 3. B, C Representative electron microscopy of PC12 cells expressing Q23 or Q74 and brain tissues from wild-type (WT) and HD transgenic R6/2 mice showing mitochondrial (as indicated by yellow arrows) association with lysosomal vacuoles (shown by red arrows). The vacuoles containing mitochondrial remnants are presented as a percentage of the total vacuoles counted. Analysis (of six independent images) was carried out by an observer blinded to the experimental conditions. *P < 0.023; **P < 0.035. Scale bar: 1 μm. D Western blot showing association of mitochondrial membrane with lysosomal membrane. The presence of membranes of each organelle was assessed using anti-MFN1, VDAC (as mitochondria markers), and LAMP1 (as a lysosomal marker). n = 3. E Representative Western blot showing mitochondrial contents (VDAC, Tom20, and aconitase) in the total lysates of PC12 cells with Q23 or Q74. Mitochondrial protein levels were normalized to beta-actin. n = 3. *P = 0.024; **P = 0.0008; ***P = 0.03. F Representative Western blot showing mitochondrial contents (VDAC, Tom20, and aconitase) in the total lysates of normal fibroblast cells and HD patient-derived fibroblasts. Mitochondrial protein levels were normalized to the cytosolic protein, enolase. n = 3. *P = 0.008; **P = 0.002; #P = 0.0008. G Representative Western blot showing mitochondrial contents (VDAC, Tom20, and aconitase) in the total lysates of mice striatal cells with Q7 and Q111. n = 3. *P = 0.0008; **P = 0.0007; #P = 0.001. H Western blot showing mitochondrial contents (Tom20 and aconitase) in the total lysates of brain tissues from wild-type (WT) and HD transgenic mice. n = 2. *P = 0.02; **P = 0.03 *§P = 0.01; **§P < 0.04.
Figure 2A Western blot gel images showing α-synuclein (α-SYN) levels in SN tissues from control (n= 9) and PD subjects (n = 18). The numbers on top of each gel panel show the ID of each sample. B The relative levels of α-synuclein between the two groups were evaluated statistically. PD subjects had moderately higher levels of α-SYN compared to controls. ID of each dot is shown in the graph. Data information: Data represent mean ± standard error of the mean. Data in (B) were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections and two-tailed p-values were calculated. Two tailed p value was calculated with Fisher's exact p. C The normalized levels of α-synuclein in the entire cohort were divided into high (n = 12) and low levels. The cut off value for determining the threshold was set at the median from all the subjects. The high levels of α-synuclein were plotted with corresponding H3K4me3 values of those subjects. The corresponding ID of each sample is shown next to each point in the graph. There was a significant correlation between high levels of α-synuclein and H3K4me3. Data information: Data represent mean ± standard error of the mean. Spearman rank correlation test was performed in (C). Two tailed p value was calculated with Fisher's exact p.
Figure 1A. Schematic representation of the experimental workflow. B. Uniform manifold approximation and projection (UMAP) embedding of single-cell RNA-Seq data from mock and SARS-CoV-2 infected colon-derived (left panels) and ileum-derived (right panels) organoids colored according to the cell type. Small insets highlight in the UMAP the cell from mock and infected organoids at 12 and 24 hpi (Red) and the rest of the cells (gray) C. Dot plot of the top marker genes for each cell type for (left) colon- and (right) ileum-derived organoids. The dot size represents the percentage of cells expressing the gene; the color represents the average relative expression across the cell type. D. Bar plot displaying the proportion of each cell type in mock and infected organoids (12 and 24 hpi).
Figure 7Schematic representation of the HOPS complex, indicating the high amino acid identity between human and zebrafish for each subunit. Comparison of control and vps16 embryos at 5 dpf. Compared to control, vps16 embryos have reduced pigmentation in the skin and retinal pigment epithelium and do not develop a swim bladder (arrows). Magnified views show altered melanocyte morphology on the head and pale, translucent eyes. Myelination, as reported using the Tg(mbp:gfp-caax) transgenic, is significantly reduced in vps16 crispants. The yellow box indicates the region assessed in D. Scale bar 100 μm. Bar graphs represent data as mean ±SEM. Statistical comparisons by Mann-Whitney U tests (n=6). ** p < 0.01.
a) Immunoblotting.f3(a) Immunoblotting. Jurkat T cells were cultured as indicated for 48 h. Only under conditions of ATG5 induction was evidence of increased autophagic activity obtained. Monomeric ATG5 (shown under the dotted line) was detected after a longer exposure time compared with the ATG5 conjugated with ATG12. Results are representative of three independent experiments. (b) Morphological analysis. Jurkat T cells were cultured as in a. Values are means±s.d. of three independent experiments. c) Morphological analysis. Jurkat T cells were treated with the indicated drugs or transduced with lentivirus ATG5, ATG5-K130R, Beclin 1 or control constructs for 48 h. Enlarged and multinucleated cells were seen as a consequence of ATG5 or ATG5-K130R lentiviral gene transfer or of etoposide treatment. Increased Beclin 1 levels or rapamycin treatment had no such effect. Representative examples of morphology are shown. Scale bar, 10 μm. Right: statistical analysis of the data. At least 100 cells were counted for each condition. Values are means±s.d. (n=3). (d) Immunoblotting. Enforced expression of ATG5, Beclin 1, or ATG5-K130R following lentiviral gene transfer. (e) Time-lapse microscopy. HeLa H2B-mCherry-α-tubulin-EGFP cells were transduced by lentiviral gene transfer as indicated. Only ATG5-overexpressing cells demonstrated evidence for mitotic catastrophe (see also Supplementary Movies). As with the GFP control, the Beclin 1 construct also included GFP, hence the intensity in green was very high. Scale bar, 10 μm.
Figure 7A) Schematic representation of the MS2-FUS (wild-type), MS2-FUS P525L, and MS2-FUS P525L fused to an SV40 NLS constructs used in the tethered splicing assay. B) Immunofluorescence microscopy on HeLa cells showing the localization of wild-type, P525L-, and P525L-SV40-NLS MS2-FUS fusion proteins. MS2 fusion proteins were visualised with anti-MS2 antibodies and the nucleus was stained with DAPI. Scale bar = 10 µm. C) Western blot documenting comparable expression levels of the MS2-FUS constructs. Cell extracts were subjected to SDS-PAGE and western blotting followed by detection of the MS2 fusion proteins by anti-MS2 antibodies (green). Tyrosine tubulin (red) served as loading control. D) RT-qPCR results depicting the ratio of spliced to unspliced p120-MS2bs reporter RNA from cells co-expressing the indicated MS2 fusion proteins. Average values and standard deviations of three biological replicates are shown, double asterisks indicate a p-value of <0.01. E) Combined FISH and immunofluorescence microscopy on HeLa cells transiently expressing MS2-FUS (upper panel) or MS2-FUS P525L (lower panel). MS2 fusion proteins were visualized with anti-MS2 antibodies (red), U11 snRNA with 6-FAM azide labelled RNA complementary to the full length snRNA (green) and nuclei were stained with DAPI. Scale bar = 10 µm. F) As in E) but with a probe detecting U12 snRNA (green).
a) Immunofluorescence of HeLa cells expressing hemagglutinin-tagged Nod1 (HA-Nod1 (red); top) or HA-Nod2 (red; bottom).figf3(a) Immunofluorescence of HeLa cells expressing hemagglutinin-tagged Nod1 (HA-Nod1 (red); top) or HA-Nod2 (red; bottom). Blue, DAPI. (b) Immunofluorescence of HeLa cells expressing Myc-tagged FL-ATG16L1 (Myc-FL-ATG16L1). (c,d) Immunofluorescence of HeLa cells coexpressing HA-Nod1 (red; c) or HA-Nod2 (red; d) and Myc-FL-ATG16L1 (green). Blue, DAPI. (e) Immunofluorescence of HeLa cells expressing a Flag-tagged truncated form of ATG16L1 (Flag-ΔN85-ATG16L1; green). Blue, DAPI. (f) Immunofluorescence of HeLa cells coexpressing HA-Nod2 (red) and Flag-ΔN85-ATG16L1 (green). Blue, DAPI. (g) Immunofluorescence microscopy of Mode-K cells stained for endogenous Nod2 (green) and ATG16L1 (red) and with DAPI (blue). For a-g, arrows indicate membrane colocalization of ATG16 and Nod2; area outlined at left is enlarged at right (a,b,e) or in other images (c,d,f,); scale bars, 8 μm. (h,i) Immunoprecipitation (IP) and immunoblot (IB) analysis of HEK293 cells transfected with Flag-ΔN85-ATG16L1 (h) or Myc-FL-ATG16L1 (i), together with HA-Nod1, HA-Nod2 or HA-tagged enhanced GFP (EGFP-HA). Ig, immunoglobulin (loading control). Results are representative of one of at least three independent experiments (a-g) or one of three independent experiments (h,i).
Figure 3(A) Tumor growth delay was measured by treating tumor with RT (12 Gy or 5x3 Gy daily; initiated at day 0) and anti-PD-L1 alone or combination of RT (n=6 mice per group). Anti-PD-L1 was given at days 0, 3, 6 and 9 (black arrows). The radiosensitizing effect of anti-PD-L1 after 12 Gy and 5x3 Gy was assessed as shown by the capped lines (means ± SD, n=1, One-way ANOVA, Bonferroni test). No weight loss was observed in the in vivo experiment. (B) Quantitative data on the percentage of gated cells are shown (n=5 per group; means ± SD, n=1, One-way ANOVA, Bonferroni test).
Figure 3(A) Schematic representation of ASY1 with the five predicted consensus Cdk phosphorylation sites. The sites found to be phosphorylated in vitro by CDKA;1-SDS complexes are highlighted in red (B) Localization patterns of different ASY1:GFP variants together with ASY3:RFP (for staging of zygotene and pachytene) in a asy1 mutant background during prophase I. Bar: 5 μ (C) Signal distribution profiles of ASY1:GFP and ASY1T142V:GFP at leptotene The many small peaks with low amplitude in ASY1T142V:GFP indicate diffused localization as opposed to the clear peaks seen in the wildtype. (D, E) Immunolocalization of ASY1 (D) and ZYP1 (E) in ASY1:GFP (asy1) and ASY1T142V:GFP (asy1) plants using anti-GFP and anti-ZYP1 antibodies, respectively. DNA was stained with DAPI (blue). Scale bars: 5 μm.