EMBO/sd-character-level-ner huggingface dataset:
Figure 2(A) STK38 interacts with XPO1. HekRasV12 cells were transiently transfected with myc-STK38(wt) together with either Flag-XPO1(wt) plasmid, Flag-control (ctrl = Sirt3) plasmid or without DNA. 24h later, cells were incubated with Okadaic Acid (OA), (final concentration = 1µM) for 1 hour or with DMSO. Flag fusions were pulled-down and co-immunoprecipited proteins were analyzed by western blotting (WB). Upper panel displays whole cell lysates (WCL) and lower panel represents immunoprecipitated proteins. STK38 accumulates in the cytoplasm upon nutrient starvation in a XPO1-dependant manner. (B) HeLa cells were transfected with myc-STK38(wt) plasmid. The next day, cells were incubated with DMEM or EBSS in the presence of XPO1 inhibitors KPT-185 or KPT-330 as indicated (final concentration = 1 µM) or DMSO for 4 hours. Cells were then fixed and stained for myc-tag. Representative images are shown and scale bars are 40 µm. STK38 accumulates in the cytoplasm upon nutrient starvation in a XPO1-dependant manner. (C) Quantification of myc-STK38(wt) nuclear/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; *p<0.05, **p<0.01, ***p<0.001, Mann-Whitney test). XPO1 activity is required for nutrient starvation-induced autophagy. (D) Immunoblotting with indicated antibodies of whole cell lysates of cells and its graphical representation (n = 3 independent experiments, mean ± SEM; ***p<0.001, ns, not significant, Student's t test). As expected, p62 degradation in starvation conditions was inhibited in presence of XPO1 inhibitors. XPO1 activity is required for nutrient starvation-induced autophagy. (E) HeLa cells stably expressing the GFP-LC3-RFP-LC3ΔG reporter autophagic probe [26] were incubated with DMEM or EBSS in the presence of XPO1 inhibitors KPT-185 or KPT-330 (final concentration = 1 µM) or DMSO for 4 hours. The GFP-LC3 (degraded upon autophagy) and RFP-LC3ΔG (non-degraded upon autophagy) signals were recorded by FACS analysis and then shown as a ratio recapitulating overall LC3 level (n=3 independent experiments, mean ± SEM; *p<0.05, **p<0.01, ns, not significant, Mann-Whitney test). Here again, incubation with XPO1 inhibitors significantly impaired the autophagy process
Figure 5A, B Quantitative real time (qRT)-PCR analysis of MdCAX3 and MdCXIP1 expression in the roots of MdCAX3 and MdCXIP1 co-overexpressing Mb. Values are means ± SD, n = 3 (biological replicates). Asterisks indicate statistically significant differences (***, P < 0.001, ANOVA, Tukey correction). C Fluorescence ratio (488/440 nm excitations) of BCECF fluorescence in the roots of MdCAX3 and MdCXIP1 co-overexpressing Mb. The central band represents the median and the box ranges showing the interquartile range and covers the central 50% of the data. The whiskers showing the minimum and maximum of the data. Three biological replicates; 3 roots were quantified for each replicate. Asterisks indicate statistically significant differences (***, P < 0.001; ANOVA, Tukey correction). D Ratio (488/440) image of BCECF fluorescence in MdCAX3 and MdCXIP1 co-overexpressing Mb. Scale Bar: 100 μm. E Rhizosphere acidification in MdCAX3 and MdCXIP1 co-overexpressing Mb. Bromocresol purple was used as a pH indicator for visualization. Scale Bar: 1 cm.
BI‐1 deficiency increases cell survival under nutrient starvation conditions.Figure 3(A) Left panel: BI‐1 WT and KO MEFs cells were incubated in EBSS, and then cell viability was monitored using the MTS assay. Right panel: a similar experiment was performed after treating cells with the indicated concentration of tunicamycin for 24 h. Mean and standard deviation are presented of triplicates representative of three independent experiments. (B) BI‐1 WT and KO cells were treated with three different starvation stimuli for 6 and 24 h. Cell death was determined after propidium iodide (PI) staining and FACS analysis. In addition, cells were treated with 100 ng/ml tunicamycin (Tm) for 24 h. Mean and standard deviation are presented of one experiment performed in triplicates. (C) Cells were exposed to EBSS for 6 h or 1 μg/ml Tm for 2 h, and then replated in normal cell culture media. After 5 days, cell viability was monitored by staining with crystal violet. Data are representative of three independent experiments. (D) BI‐1 WT MEFs were stably transduced with lentiviral expression vectors to deliver an shRNA against bi‐1 mRNA or control mRNA (luciferase shRNA). Cell survival was measured after treatment of cells as described in (B). Mean and standard deviation of an experiment made by triplicate, representative of two independent experiments. (E) BI‐1 KO cells were stably transduced with retroviruses expressing BI‐1-GFP or empty vector, and then exposed to EBSS for indicated time points. Cell viability was monitored after PI staining by FACS. Mean and standard deviation are presented of triplicates representative of two independent experiments.
Figure 2(A) Fresh bone marrow mononuclear cells were collected from acute myeloid leukemia (AML) patient. Proliferation (section i) were analyzed after cells were treated for 4 days. (A) Fresh bone marrow mononuclear cells were collected from acute myeloid leukemia (AML) patient. ROS production (section ii) were analyzed after cells were treated for 4 days. (B) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days. Transcription of RA-target genes was analyzed with qRT-PCR at day 2, 3, and 6 (sections i). (B) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days. Proliferation was assessed at day 3 in parallel (section ii). (B) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days. ROS production were assessed at day 3 in parallel (section iii).
Expression of Rab25 regulates cellular ATP and glycogen levels.Figure 2Expression of Rab25 increases endogenous ATP level in ovarian cancer cells. *p < 0.001 Rab25 versus pcDNA. The mean cellular concentration of ATP per mg of protein (based on the standard curve generated using a known amount of ATP) is 2.36−10 moles/mg for IOSE29htpcDNA cells, 6.21−10 moles/mg for IOSE29htRab25 cells, 8.14−11 moles/mg for IOSE80htpcDNA cells, 1.05−10 moles/mg for IOSE80htRab25 cells, 2.85−10 moles/mg for A2780pcDNA cells, 3.81−10 moles/mg for A2780Rab25 cells, 7.23−10 moles/mg for HEYpcDNA cells and 9.82−10 moles/mg for HEYRab25 cells. Rab25 expression increases ATP synthesis and delays fall in total cellular ATP levels after glucose and serum withdrawal, p < 0.001 Rab25 versus control pcDNA. Down-regulation of Rab25 expression decreases ability to maintain ATP after glucose and serum withdrawal (a) p < 0.05 SF-Glu versus FBS and (b) p < 0.05 versus control. Expression of Rab25 increases total cellular glycogen content while down-regulation of Rab25 expression decreases total cellular glycogen content (µg of glycogen/mg protein). Cells were cultured in complete media for 24 h before glycogen measurement. (a) p < 0.01 Rab25 versus pcDNA, (b) p < 0.05 Rab25 RNAi versus NT RNAi and (c) p < 0.05 shRAb25 versus shRNA control. Decrease in cellular glycogen content after glucose and serum withdrawal *p < 0.001 Rab25 versus pcDNA. Effect of GPi (Bay U6571) and oligomycin on ATP production. A2780pcDNA transfected and Rab25 expressing cells were pretreated with vehicle (control), 30 µM of Bay U6571 (GPi) or 25 µg/ml of oligomycin for 2 h. Cells were then cultured in serum- and glucose free RPMI 1640 in the presence or absence of Bay U6571 or oligomycin for the indicated times. (a) p < 0.01 oligomycin time 0 versus control time 0 and (b) p < 0.01 versus time 0 at each treatment group. Effect of Bay U6571 on cellular glycogen (left panel) and ATP (right panel) levels in HEY cells. (a) p < 0.01 versus pcDNA control time 0 at 5% FBS and (b) p < 0.01 GPi versus control at each corresponding treatment. Down regulation of Rab25 expression and inhibition of glycogen breakdown by GPi administration increases 2DG induced cell death *p < 0.05 versus shRNA control.
Figure 3In the ts-che-3 mutant grown at the permissive temperature (15ºC), both RPI-2/RP2 and TRAM-1/TRAM1 are localised to the PCMC, just proximal to the ciliary axoneme. At the restrictive temperature, TRAM-1 shows leakage into the axoneme with its strongest localisation at the PCMC. Intriguingly, RPI-2 now displays strong localisation to the axoneme, indicating that in the absence of IFT, RPI-2 may be targeted into the cilium. When ts-che-3 mutant animals grown at the restrictive temperature (25ºC) are shifted at young adult to the permissive (15ºC) for 24 hours, both TRAM-1 and RPI-2 exhibit restoration of their PCMC localisation. Consistent with other TZ markers, both MKS-2 and MKSR-1 show ectopic localisation in the axoneme when grown at the restrictive temperature compared to the permissive temperature. Also consistent with what was observed for other markers, after 24 hours at the permissive temperature, animals grown at the restrictive temperature show loss of the ectopic localisation. Fluorescence quantification is shown for each marker at the indicated temperature in the heat maps on the right. Each point in the plot represents one pixel along the centre of the basal body (BB), transition zone (TZ) and middle segment (MS) regions. Dotted areas (three pixels) in the MS were used to quantify ectopic localisation (MKS-2 and MKSR-1) or accumulation (RPI-2 and TRAM-1) for statistical analyses. n = 10 cilia (4-6 animals), Kruskal-Wallis test, *P<0.05, **P<0.01; scale bars are 4 µm Loss of CHE-3 function results in short, bulbous cilia containing electron dense accumulations and abnormal membrane-microtubule connections. Shown are transmission electron micrograph (TEM) cross sections of an amphid channel cilium in wild-type and che-3(e1124) null mutant animals. Representative images of wild-type cilia show intact middle segment (containing doublet microtubules) and transition zone (with Y-shaped links) (left top and bottom panels) regions. Representative images of che-3(e1124) cilia reveal apparently intact transition zones, with visible Y-link structures, but enlarged ciliary ends filled with electron-dense accumulations, which often appear vesicular. The bulbous structures reveal doublet microtubules associated with the membrane, and occasionally ectopic microtubule-to-membrane connections, which sometimes appear similar to transition zone Y-links in the region just distal to the seemingly "normal" TZ structure. Schematics show longitudinal (left images) and cross section (right images) representations of wild-type and che-3(e1124) cilia. tz, transition zone; pcmc, periciliary membrane compartment; scale bars are as indicated in (nm) for
Figure.Figure 1(A) Distribution of antimycin A resistance scores for wild isolates, compared to standard laboratory strain (red vertical line). Resistance scores are the ratio of fitness on rich glucose media with vs without 500 µg/L antimycin A. Fitness was estimated based on colony size on solid media, corrected for spatial and plate effects After quality control, we obtained quantitative fitness scores for 154 strains, with a signal-to-noise ratio of 29.8 and an unexplained variance of 0.12. (B) Volcano plot of a genome-wide association using mixed-model linear regression of antimycin A resistance for 118,527 genetic variants. Variants with moderate or high impact are shown in blue. Red dot: the variant at locus I:3845516, which causes a T343A change in the Pyk1 amino-acid sequence. This variant was among the top scoring (effect size=-0.645, rank 34; p=0.0008, rank 70). (C) Boxplot showing antimycin A resistance for 154 strains grouped by the two alleles at the pyk1 locus. Strains carrying a C at this genomic locus (orange box) generally have higher antimycin resistance than strains carrying the reference allele T (blue box). As is standard, this boxplot and all other boxplots in this manuscript show the median of the data as central line, the quartiles as box and the extend of the rest of the distribution as whiskers. Points which are 1.5 times the inter-quartile range beyond the high and low quartiles are considered outliers and shown individually. (D) Sequence alignment of the region of interest in eukaryotic PYK proteins. The threonine residue at the highlighted position 343 is unique for the reference (laboratory) S. pombe strains. For this analysis, we collected 25 Pyk1 homologues from a wide variety of eukaryotes, including animals, plants and fungi, using the HomoloGene resource (Accession 37650). We manually expanded the homologous group to include three other species of the Schizosaccharomyces genus Sequences were aligned with MAFFT and visualised with Jalview (E) Phylogenetic tree based on 31 biallelic SNPs in pyk1 and in 500bp up- and down-stream regions. Strains carrying the common A-allele are in orange, while strains carrying the unusual T-allele are in blue Strains JB22 and JB50 (underlined and bold) refer to heterothallic and homothallic versions of the laboratory strain, respectively. The pie chart shows the relative allele frequencies at the genomic locus across all wild strains. Genotype calls at the locus were checked manually and were conclusive, i.e. no strain produced reads with both alleles.
Figure 4(A) Analysis for genome-wide SRCAP binding regions in ISCs from ChIP-seq was shown. Coordinate information for sequence alignment was from UCSC (intron and exon), Ensemble GRCM38 (mm10, release-90) (upstream 2000bp, downstream 2000bp, distal intergenic region, TSS (transcription start site), TES and promoter), GSE57919 (Dnase-seq peaks, liftover to mm10), and GSE34568 (H3K4me2, liftover to mm10). Distribution of SRCAP peaks in ChIP-seq and its overlap with each kind of region was analyzed by bedtools. (B) Transcriptome microarray analysis between Srcap-/- ISCs and WT ISCs. Top ten downregulated TFs in Srcap-/- ISCs were listed. (C) Prdm16 expression was tested in Srcap+/+ and Srcap-/- ISCs by Western blotting. (D) Prdm16 expression was visualized in indicated intestinal tissues by immunofluorescence staining. Green: GFP; Red: SRCAP; Blue: Prdm16. Scale bar, 10 μm. (E) Prdm16 promoter (-875~-854) region was used as probe to perform DNA-pulldown assay with murine intestinal crypt lysates followed by immunoblotting. (F, G) Indicated promoter activation was analyzed with luciferase assay. DKK1 was used as a control. Results of relative fold changes are shown as means ± S.D. Data represent five independent replicates. . ** p<0.01 by two-tailed Student's t-test. . (H) 1×104 ISCs from indicated mice were collected for organoid formation. For gene depletion or restoration, ISCs were infected with lentivirus. Scale bar, 200 μm. Data information: All data represent five independent experiments.
Figure 3SH-SY5Y cells overexpressing Flag-TDP-43 were treated with GlcNAc, and the O-GlcNAcylated and phosphorylated (pTDP-43) levels of TDP-43 were examined by immunoblotting. Relative O-GlcNAcylation levels (top) and phosphorylation levels (bottom) of TDP-43 shown in (A) were quantified. SH-SY5Y cells overexpressing Flag-TDP-43 were treated with either 20 μM EA or 2 mM GlcNAc or a combination. The O-GlcNAcylated or phosphorylated levels of TDP-43 were examined by immunoblotting. The asterisks indicate heavy-chain bands. SH-SY5Y cells treated with the indicated compounds were immunostained with TDP-43 or pTDP-43 and observed by immunofluorescence. Quantification of signal intensities of pTDP-43 staining (left) or foci numbers per cell (right). Cell number counted: left panel, "Mock" n = 58, "GlcNAc" n = 35, "EA" n = 45, "GlcNAc+EA" n = 52; right panel, "Mock" n = 1167, "GlcNAc" n = 1051, "EA" n = 542, "GlcNAc+EA" n = 815. SDS-soluble and insoluble fractions were isolated from SH-SY5Y cells expressing WT or catalytic-inactive mutant OGT treated with 20 μM EA, and the fractions were analyzed by immunoblotting. Quantification of (F). SDS-soluble and insoluble fractions were isolated from SH-SY5Y cells expressing WT OGT and/or Flag-TDP-43 in the presence or absence of EA, and the truncated TDP-43 fragments were examined by immunoblotting. L.E.: long exposure; S.E.: short exposure. Quantification of (H).
Figure 5A, Schematic representation of sleep deprivation (SD) experiments. Zeitgeber time 0 (ZT0) corresponding to lights ON. Western blot analysis of (B) p845GluA1 and GluA1 expression (S n=9, SD n=10) in the prefrontal cortex of sleeping and sleep deprived mice. Student's t-test *p < 0.05, **p < 0.01, ***p < 0.001. Western blot analysis of , (C) p880GluA2 and GluA2 expression (S n=9, SD n=8) in the prefrontal cortex of sleeping and sleep deprived mice. Student's t-test *p < 0.05, **p < 0.01, ***p < 0.001. Western blot analysis of (D) Fxr1 expression (S n=11, SD n=10) in the prefrontal cortex of sleeping and sleep deprived mice. Student's t-test *p < 0.05, **p < 0.01, ***p < 0.001. Western blot analysis of (E) Fmrp expression (S n=11, SD n=10) in the prefrontal cortex of sleeping and sleep deprived mice. Student's t-test *p < 0.05, **p < 0.01, ***p < 0.001. F-H, RNAseq measurement of mRNA for (F) Fxr1, (G) Fxr2, (H) Fmr1 during SD. n=3 in each condition, Student's T-test *p<0.05.
Endocytosis synergizes with autophagy to mediate necrotic cell death.Figure 4(A) Synaptotagmin (SNT‐1) deficiency further suppresses MEC‐4(d)‐induced necrosis in animals with impaired autophagy. (B) Endophilin dysfunction enhances suppression of necrosis in mec‐4(d);lgg‐1(RNAi) animals, where autophagy is impaired. Error bars denote s.e.m. values (n>250 for all populations examined; P0.01, compared with single mutant control animals, unpaired t‐test).
a) The proportions of EGFP-positive COS-7 cells with aggregates or cell death, after transfection with EGFP-HDQ74 and rheb or pcDNA3.figf7(a) The proportions of EGFP-positive COS-7 cells with aggregates or cell death, after transfection with EGFP-HDQ74 and rheb or pcDNA3.1 (empty vector) (1:3 ratio) for 4 h and treatment with or without 10 μM calpastatin or 50 μM calpeptin for 48 h post-transfection. The control condition for assessing the effect of calpain inhibitors in rheb-transfected cells was taken as 1. Error bars show 95% confidence interval. (b) The proportions of EGFP-positive COS-7 cells with >5 EGFP-LC3 vesicles, transfected with EGFP-LC3 and pcDNA3.1 (empty vector) or rheb (1:3 ratio) for 4 h and treated with or without 10 μM calpastatin or 50 μM calpeptin for 24 h post-transfection. Error bars show 95% confidence interval. (c) The proportions of EGFP-positive COS-7 cells with aggregates, transfected with EGFP-HDQ74 and constitutively active (CA) m-calpain or pcDNA3.1 (empty vector) (1:3 ratio) for 4 h and treated with or without 0.2 μM rapamycin for 48 h post-transfection. Error bars show 95% confidence interval. (d) The proportions of EGFP-positive COS-7 cells with >5 EGFP-LC3 vesicles, transfected with EGFP-LC3 and CA m-calpain or pcDNA3.1 (empty vector) (1:3 ratio) for 4 h and treated with or without 0.2 μM rapamycin for 24 h post-transfection. Error bars show 95% confidence interval. (e,f) The proportions of EGFP-positive COS-7 cells with aggregates (e) and cell death (f) as in Figure 1c, treated with or without 0.2 μM rapamycin, 10 μM calpastatin or both for 48 h. Error bars show 95% confidence interval. (g) Clearance of soluble EGFP-HDQ74 in stable PC12 cells as in Figure 1b, treated with or without 0.2 μM rapamycin, 10 μM calpastatin or both for the 48 h switch-off period. Error bars show s.e.m. (h) Clearance of A53T α-synuclein in stable PC12 cells as in Figure 1a, treated with or without 0.2 μM rapamycin, 10 μM calpastatin or both for the 8 h switch-off period. Error bars show s.e.m. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.
Figure 3A, B, C Transmission electron microscopy image analysis was used to quantify mitochondrial size, circularity and roundness in DX/REL neuroblastoma pairs (mean +/- SD shown). Data information: , n=137-241 mitochondria per cell line statistical analyses were performed using a two-tailed Mann-Whitney U test, with significance p<0.05 (trend p<0.10). D Electron micrograph illustrating organelle masking of MAM interfaces; scale bar= 500nm. Data information: er, endoplasmic reticulum; m, mitochondria. E Proportion of mitochondria with 0 to 6 or more MAMs are shown for each cell line, as DX/REL pairs. Data information: n=137-241 mitochondria per cell line F Percentage of mitochondria perimeter with an apposed ER within defined gap-widths for DX/REL neuroblastoma cell line pairs; dotted-lines denote +10% and -10% change. Data information: n=137-241 mitochondria per cell line
Figure 5A: Western-blot analysis of amyloid-precursor protein (APP) in 6 mo male mice. GAPDH was used as a loading control. Densitometric analysis on a total of 5 independent samples is shown in the histogram on the right. B: morphological analysis of an AD subject and of Pitrm1+/- and Pitrm1+/+ mouse brains. TF: Thioflavin T (thalamus); CR: Congo-red (brain cortex); PL: polarised light (same sections as those stained with CR); Aβ1-42 immunostaining (pons); Ub: ubiquitin immunohistochemistry shown as brownish staining (brain cortex). The white bar indicates 20 µm.
Figure 7 (A) Adhesion assay of B220+CD11c+NK1.1+ cells to fibrinogen coated plates. The rhodamin-labeled liver B220+CD11c+NK1.1+ cells were separately cultured with TCM-stimulating liver tissues, and seeded on a fibrinogen-coated plate  (B) Relative mRNA levels of FX, Vtn and TSP1 in liver B220+CD11c+NK1.1+cells stimulated with LiCM or LuCM. Shown are averages (N = 6/group) with SEM and one-way ANOVA  (C) Relative mRNA levels of FX in liver B220+CD11c+NK1.1+cells stimulated with various factors. Shown are averages (N = 4) with SEM and one-way ANOVA  (D) In vitro adhesion assay. Liver B220+CD11c+NK1.1+cells were seeded on a fibrinogen plate. Cells attached to the plate were counted. Twentyμg/mL of anti-Vtn Ab or isotype control IgG were used to block the adhesion. Shown are averages (N = 6/group) with SEM and one-way ANOVA  (E) Lung B220+CD11c+NK1.1+cells were seeded on ECM coated (fibrinogen, fibronectin, or collagen I) plates. Shown are averages (N = 5/group) with SEM and one-way ANOVA
Cloning and characterization of APG9.Figure 2Cloning and characterization of APG9. A, Wild-type (WT; SEY6210), apg9 (THY154), apg9Δ (JKY007), and the apg9Δ strain transformed with single copy (CEN; pAPG9(414)) or multicopy (2μ; pAPG9(424)) plasmids encoding APG9 were grown to log phase in SMD. Protein extracts were prepared and analyzed by immunoblot using antiserum to API as described in Materials and Methods. The positions of precursor and mature API are indicated. The APG9 gene complements the prAPI accumulation phenotype of the apg9Δ mutant. B, WT (SEY6210), apg9Δ (JKY007), and the apg9Δ strain transformed with pAPG9(414) centromeric plasmid were grown in SMD and transferred to SD(−N) as described in Materials and Methods. Aliquots were removed at the indicated times and spread onto YPD plates in triplicate. Numbers of viable colonies were determined after two to three days. The APG9 gene complements the starvation-sensitive phenotype of the apg9Δ mutant.
Figure 3Localization of CNTN1 in semi-thin (0.5μm-thin) sections of retinas from either MOG/CFA-injected mice (EAE mice) or CFA-injected control mice (9 days after injection). (A,B) Sections were double-immunolabelled with antibodies against CNTN1 and RIBEYE proteins in the OPL and IPL. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, nuclear layer; IPL, inner plexiform layer Quantification of the immunosignals of the indicated proteins in the OPL and IPL. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, nuclear layer; IPL, inner plexiform layer; n.s., non-significant; ***, p<0.001 (Mann-Whitney U test, actual p-values are given in C) (N=6 independent set of experiments; n=295 images (CFA); n=274 images (MOG/CFA) for quantification of RIBEYE immunosignals; n=95 (CFA); n=92 (MOG/CFA) for quantification of CASPR1 immunosignals; n=91 (CFA), n=82 (MOG/CFA) for CNTN1 quantification; n=109 (CFA); n=100 (MOG/CFA) for quantification of v-arrestin. Scale bars: 20μm