EMBO/sd-character-level-ner huggingface dataset:
Figure 3A. Schematic workflow of the RNAseq experiment from FACS-purified CD34+α6+ HFSCs (CD34+α6+), cells cultured in 3C and freshly isolated epidermal cells (Epi d0). B. Heatmap and Euclidian distance dendrogram of quantified transcripts from RNAseq data generated as shown in panel A. 3C cultures cluster with purified CD34+α6+ HFSCs (n=3 biological replicates). C. Venn diagram of genes most significantly upregulated in 3C and in purified CD34+α6+ (log2FC >2, padj<0.05) together with published HFSC signature shows significant overlap between the 3 signatures (p= 0.00252451 and p=3.058928e-51, hypergeometric distribution). D. Schematic workflow of the RNAseq experiment from FACS-purified CD34+α6+ and CD34-α6+ cells from 3C cultures. E. Heatmap of selected genes differentially expressed in 3C-CD34+α6+ and 3C-CD34¯α6+ cells. Note upregulation of multiple key HFSC lineage identity transcription factors and bulge markers (triangles) in 3C-CD34+α6+ cells, and upregulation of genes implicated in lineage progression in 3C-CD34¯α6+ cells (circles). F. Venn diagram of genes upregulated in 3C-CD34+α6+ and in purified CD34+α6+ cells (log2FC >2, padj<0.05) together with a published HFSC signature shows significant overlap between the 3 groups (p= 1.381896e-09 and p= 0.01636903, hypergeometric distribution). G. RT-qPCR analyses of FACS-purified CD34+α6+ and CD34-α6+ cells from either 3C cultures or from freshly isolated epidermis show upregulation of HFSC identity in both freshly isolated and 3C-CD34+α6+ cells compared to freshly isolated CD34-α6+ progenitors. 3C- CD34-α6+ cells show intermediated expression (mean ±SEM; n=4; *p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis). H. Both 3C-CD34+α6+ and 3C-CD34-α6+ cells show upregulation of a panel of cell cycle genes and genes shown to be enriched in HFSC progeny (mean ±SEM; n=4; *p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis). See also Appendix figure S3 and Tables S1-S3.
Inhibitor effects on the local pH value at SU e in the intracristal spaceA) Mitochondrial pH at CV SU e measured in HeLa WT in glycolytic (HGlc) and respiring (Gal) conditions.Figure 7A) Mitochondrial pH at CV SU e measured in HeLa WT in glycolytic (HGlc) and respiring (Gal) conditions. Where indicated, 5 µg/mL oligomycin was added to inhibit CV activity. B) Mitochondrial pH at CV SU e in IF1-KO cells. Conditions: glycolytic (HGlc) and respiring (Gal) with and without oligomycin. C) Mitochondrial pH at CV SU e in IF1-OE cells. Conditions: glycolytic (HGlc) and respiring (Gal) with and without oligomycin. D) D) Fluorescence image showing the localization of SU e-sEcGFP in WT cells. Scale bar: 10 µm. E) pH calibration curve for testing the effect of antimycin A on local pH at SU e. F) Effect of inhibiting the respiratory chain at complex III (CIII) with antimycin A (AA) on the local pH at SU e in WT cells (glycolytic conditions). G) Local pH at SU e in IF1-KO cells after inhibiting CIII. H) Local pH at SU e in IF1-OE cells after inhibiting CIII. Da
Fig.Figure 3(A) Serine phosphorylation of cPLA2α in response to GBS strain K79 in HBMEC pretreated with gefitinib or vehicle control and in EGFR knockout HBMEC. (B) Serine phosphorylation of cPLA2α in response to GBS strain K79 in control, EGFR knockout and S1P2 knockout HBMEC. (C) Bacterial counts recovered from the blood and brain in wild type mice (n = 5) and CysLT2-/- mice (n = 5) infected with strain K79 for 1h. (D) CysLT1 protein expression in CysLT1 knockdown HBMEC using shRNA (left panel) and relative invasion frequency of 8 GBS isolates in CysLT1 knockdown and control HBMEC. t-test. From left to right, p value of K79 is 0.0039, p value of K160 is 0.0082, p value of K161 is 0.00091, p value of K181 is 0.0087, p value of K226 is 0.00020, p value of K237 is 0.0039, p value of K238 is 0.00066, p value of P539 is 1.76E-05 (right panel). (E) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in CysLT1 knockdown HBMEC compare to control. (F) Bacterial counts recovered from the blood and brain in wild type mice (n = 7) and CysLT1-/- mice (n = 7) infected with strain K79 for 1h. (G) Ezrin protein expression in ezrin knockdown HBMEC using shRNA (left panel) and relative invasion frequency of GBS strain K79 in ezrin knockdown and control HBMEC (right panel). (H Ezrin phosphorylation in HBMEC pre-treated with CysLT1 antagonist (montelukast) and infected with GBS strain K79 for 60 min (left panel) I) ezrin phosphorylation in the homogenates of brain capillaries of CysLT1-/- and control mice infected with GBS strain K79 for 1h (right panel). (J) Intracellular K79 co-localization with EGFR and ezrin in control and S1P2 knockout HBMEC (as shown by arrows, cyan). Percentage of co-localization was calculated by counting numbers of all GBS and co-localized intracellular GBS from at least three representative fields, and expressing as numbers of co-localized GBS/all GBS x 100.
Figure 2(A) Representative images of wild-type (WT) and mutant (KO) neurospheres obtained from E15.5 neocortices and cultured in vitro. The culture passage ("step") corresponds to 3 days of culture after dissociation and re-plating. KO aggregates are more numerous and larger in size. Data information: Scale bars: 50µm. (B) Graph showing the number of neurospheres per P100 cell plate at different steps, as indicated. Nr2f1KO neurospheres proliferate for a longer time (up to 30 passages tested), whereas WT cells exhaust around step 9. n ≥ 3 culture wells from n = 2 batches. Data information: data are represented as means ± SEM. 2-way ANOVA (B) (**P<0.01, ***P<0.001). (C) Panel of WT and KO isolated neurospheres grown from day 1 to day 10. Blue and red lines represent the diameter of WT and KO neurospheres, respectively. Data information Scale bars: 50µm. (D,E) PH3 (green; dividing cells) IF on cross-sections of day 4 neurospheres. White arrowheads point at mitotic figures, which are quantified in (E) as percentage of dividing cells over DAPI-stained nuclei (blue). n ≥ 3 neurospheres from n = 2 batches. Data information: data are represented as means ± SEM. Student t-test (E) (**P<0.01, ***P<0.001). Scale bars: 50µm.
Figure 3(A) Pml+/+ and Pml-/- BMDMs were treated with zVAD for 30 min, followed by AT-406 (zA) treatment at the indicated time-points, before preparing total cell lysates (TCL). TCL were immunoprecipitated with anti-FADD and the amounts of p-RIPK1[S166], RIPK1 and caspase-8 in the precipitates and TCL were analysed. (B) Pml+/+ and Pml-/- BMDMs were treated with zVAD + AT406 (zA) as in (A), and TCL were then harvested. TCL were immunoprecipitated with anti-caspase-8, and the contents of p-RIPK1[S166] and RIPK1 in the precipitates and TCL was analysed.
Fig.Figure 7Protoplasts of WT and pldα6 from 12-day old stage leaf sheath were collected and transfected with a pM999-SLR1 construct, incubated for 12 hours and then treated with 10 μM GA3 for 0, 3, and 9 hours. The first column, fluorescence from GFP; the second, red fluorescence from the nucleus marker Ghd7; the third, bright field, and the fourth, overlay of the three channels. All confocal images were scanned using similar laser gain and offset settings. Bars = 10 μm. Immunoblotting of SLR1-GFP proteins from protoplast cells. Equal amounts of proteins from each sample were used for SDS-PAGE, followed by immunoblotting with anti-GFP antibodies.
SH-BC-893 protects from ceramide-induced mitochondrial dysfunction.Figure 2TMRE (100 nM) and MitoTracker green (200 nM) staining in MEFs treated with BSA or palmitate (250 μM) for 12 h after a 3 h pre-treatment with vehicle or SH-BC-893 (893, 5 μM). TMRE intensity in (A) on a per cell basis. MitoSOX (5 μM) and MitoTracker Green (200 nM) staining in MEFs treated with vehicle (DMSO) or C2-ceramide (50 μM) for 12 h after a 3 h pre-treatment with vehicle or SH-BC-893 (893, 5 μM). MitoSOX intensity for (C) on a per cell basis. Chop, Xbp1s, Atf6, and Grp78 mRNA levels measured in MEFs treated for 16 h with BSA or palmitate (250 μM) after a 3 h pre-treatment with vehicle or SH-BC-893 (893, 5 μM); n=3. Mean ± SEM.
Silencing of TMX1 or TMX3 causes ROS production A-C (A) Thapsigargin (Tg) induced Fura-2 based cytosolic Ca2+ imaging in Ringer's buffer containing 0.Figure 3Thapsigargin (Tg) induced Fura-2 based cytosolic Ca2+ imaging in Ringer's buffer containing 0.25 mM Ca2+. (B) Quantification of basal cytosolic calcium levels and (C) SOCE quantification (plateau-basal) for WM3734 after stable silencing of TMX1 (two clones). : In (B-C), data are presented as mean ± SEM (n values: WM3734, control=939, TMX1 kds 1=988, TMX1 kds 2=508). Cytosolic Ca2+ imaging (Fura-2) and (E) SOCE quantification (plateau-basal) for WM3734 after transient silencing of TMX1 or TMX3. In (E), data are presented as mean ± SEM (n values: WM3734, control=30, TMX1 kd=49, TMX3 kd=52). Cellular H2O2 (HyPer) was evaluated in two melanoma cell lines upon TMX1 kd and TMX3 kd. (F) Exemplary ratiometric images (F505 nm / F420 nm) are shown for WM3734 and Mel Juso. Quantification of basal cellular H2O2 in WM3734 (G) and Mel Juso (H) cells. In (G-H), data are presented as mean ± SEM (n values: WM3734: control=168, TMX1 kd=209, TMX3 kd=192; Mel Juso: control=297, TMX1 kd=343, TMX3 kd=440). In (F) scale bars: 10 µm; color code: WM3734: color code: blue=0, red=3; Mel Juso: color code: blue=0, red=1.5.
Combination of R2-P2 with different phosphopeptide enrichment methods allows for deep phosphoproteomic profiling 250 µg of yeast protein extract was processed by R2-P2 using either Fe3+-IMAC, Ti4+-IMAC, Zr4+-IMAC or TiO2 magnetic beads and followed by peptide analysis with DDA-MS.Figure 3250 µg of yeast protein extract was processed by R2-P2 using either Fe3+-IMAC, Ti4+-IMAC, Zr4+-IMAC or TiO2 magnetic beads and followed by peptide analysis with DDA-MS. Phosphopeptide enrichment was performed in triplicate.   Average number of unique phosphopeptides identified by the different enrichments (n=3).   Phosphopeptide enrichment efficiency shown as the fraction of phosphorylated peptides over total peptides (n=3).   Fraction of phosphopeptides with 1, 2 or 3 phosphorylation sites (n=3).   250 µg of yeast protein extract was processed by R2-P2 using either Fe3+-IMAC, Ti4+-IMAC, Zr4+-IMAC or TiO2 magnetic beads and followed by peptide analysis with DDA-MS. Phosphopeptide enrichment was performed in triplicate. Boxplot depicting distributions of phosphopeptide MS1 signals (n=3).   Boxplot depicting distributions of phosphopeptide MS1 signal CVs (n=3).   250 µg of yeast protein extract was processed by R2-P2 using either Fe3+-IMAC, Ti4+-IMAC, Zr4+-IMAC or TiO2 magnetic beads and followed by peptide analysis with DDA-MS. Phosphopeptide enrichment was performed in triplicate. Venn diagram of identified phosphopeptides by the different phosphopeptide enrichment methods.
Unipolar insertion of Omp25 and low mobility of Omp25 and Omp2b.Figure 4Localization of initially labeled Omp25 (green) on TRSE-labeled (red) bacteria before (0 h) and after 2 h of growth in the absence of both labelings (2 h). As a control, Omp25 localization after pulse-chase labeling with TRSE is shown at the bottom panel. Scale bars: 2 μm. Model of TRSE (red) and Omp25 (green) labeling corresponding to (A). Incorporation of new material at the pole and/or at the constriction site is shown as black areas. Quantification of localization of newly inserted Omp25 represented by negative Omp25 areas from (A). Numbers are indicating mean percentages. n = 279 bacteria (5 biological replicates). Localization of initially labeled Omp2b (green) on TRSE-labeled (red) bacteria before (0 h) and after 2 h of growth in the absence of both labelings (2 h). Scale bars: 2 μm. Co-localization of Omp25 and Omp2b positive pixels with TRSE positive or negative pixels, respectively. Error bars correspond to s.d. from independent experiments. Differences were statistically analyzed by t-test. ***P < 0.001. nOmp25 = 279 bacteria, 85666 pixels (5 biological replicates). nOmp2b = 252 bacteria, 74630 pixels (3 biological replicates).
Factors contributing to incomplete penetrance (see also Table EV8).Figure 6Penetrance as a function of replicative age. Top: Bar graph showing the fraction of outliers in populations of increasing replicative age (# of divisions) for wild-type (WT), and 5 mutant strains (rrd2Δ, rpl20bΔ, cka2Δ, vac8Δ and vac17Δ). Data are presented as mean of 3 biological replicates +/- SD. Bottom: Box plot with the distribution of cell sizes for the same populations of cells. Whiskers extend to the 5th and 95th percentile. Micrographs of young (top row of images) and older (bottom row of images) wild-type (WT) cells expressing Vph1-EGFP (green vacuole) and stained with CF640R WGA (magenta bud scars). Scale bar: 5 µm. Combined effect of replicative age and a vacuole inheritance defect on penetrance. Micrographs of wild-type and vac17Δ cells expressing Vph1-EGFP (green vacuole) and Hta2-mCherry (red nucleus), stained with CF640R WGA (magenta bud scars). Cells with increasing bud-scar staining (replicative age) are shown from left to right. Scale bar: 5 µm. Relationship between stress response and penetrance. Single-cell UPRE-RFP levels were measured in ~60 different mutant strains that we had identified as penetrance mutants with intermediate penetrance with defects in actin or vacuole morphology. Cells were binned into equal-sized bins, from low to high stress response, assessed as outlier or inlier, and clustered based on their penetrance profile (composed from the fraction of outliers in each stress-response bin). Each line plot represents a penetrance profile. * denotes the cluster with a profile most similar to wild-type. Insert pie charts show the proportion of mutant strains in each cluster.
Figure 1A) GST pulldown used to analyse TBC1D14 interactors. Recombinant GST or GST-TBC1D14 was incubated with or without HEK 293A lysate. Bound proteins were eluted from the beads using Laemmli sample buffer and subjected to SDS-PAGE, with visible bands being excised from the gel and analysed by mass spectrometry. B) Lysates of HEK293A cells expressing either GFP or GFP-TBC1D14 were subjected to immunoprecipitation (IP) with GFP-Trap, and then immunoblotted for GFP, TRAPPC12 and TRAPPC4. C) Lysates of cells expressing GFP or GFP-TRAPPC3 were subjected to IP with GFP-Trap, and then immunoblotted for TRAPPC4, GFP and TBC1D14. D) Cells expressing GFP-TBC1D14, generating a tubulated endosomal compartment, were fed Alexa647 labelled Transferrin (white, blue in merge) for 15 minutes in full medium, fixed, stained for endogenous TRAPPC4 (red) and analysed by confocal microscopy. White arrowheads indicate transfected cells, yellow arrowheads depict regions of colocalisation in inset. Scale bars = 10 μm. E) Cells treated as in D were stained for endogenous TRAPPC12 (red) and analysed by confocal microscopy. Scale bars = 10 μm F) Cells in complete medium were fixed, stained for endogenous TBC1D14 (green), RAB1B (red) and TRAPPC4 (white, blue in merge) and analysed by confocal microscopy. Inset shows protein localisation in juxtanuclear area. Yellow arrowheads show regions of colocalisation Scale bars = 10 μm. Data shown are representative from 3 independent experiments.
Live Schistosoma and its miR-10 affect the expression of MAP3K7.Figure 7The putative binding sites of miR-10 in the MAP3K7 3'UTR (taken from http://www.targetscan.org/vert_72/). Above the wild type seed sequence, in red, the sequences that were placed in the mutated plasmid. HEK-293 cells were transfected with a plasmid expressing schistosomal-miR-10 at the indicated concentration together with either psiCHECK-II vector, psiCHECK-WT-MAP3K7-3'UTR or psiCHECK-mut-MAP3K7. After 24h, the cells lysates were subjected to luciferase assay. The results are presented as the ratio of renilla/luciferase expression that was normalized relative to cells transfected only with psiCHECK-II. Values are expressed as the mean+/-SD of at least 3 independent experiments. A representative experiment in which protein extracted from Jurkat-T cells stably over expressing stably control vector or miR-10 expressing vector were subjected to WB analysis with anti-MAP3K7 or anti-GAPDH antibodies. The graph presents densitometry analysis of 3 WBs analyses performed as in (C). The mean +/- SD was calculated from 4 independent experiments. Statistics were performed using t-test (*p<0.05). Jurkat-T cells stably expressing either control vector or a vector containing schistosomal-miR-10 were co-transfected with a control Luciferase plasmid or NF-κB luciferase reporter vector. In all transfections, a plasmid expressing Renilla vector as an internal control was added. 48h post-transfection, cells were harvested and were analyzed by luciferase reporter assay. Values were normalized to the Renilla activity. Data are represented as mean +/-SEM from 5 independent experiments. Statistics were performed using t-test, **p<0.01. Representative WB experiment demonstrating that MAP3K7 is downregulated in schistosomal-exposed Th cells F) or to Jurkat (H) in comparison to the control unexposed cells. (G, I) The graphs present densitometry analysis of 3 WB analyses performed as in (F and H), respectively. The mean +/- SD was calculated from 3 independent experiments. Statistics were performed using paired one-tailed t-test (*p<0.05).
Figure 4(A-C) Analysis of NRF2 ubiquitination in absence and presence of TRIM16 by Co-IP assays using transiently transfected plasmid constructs as indicated. Two different variants of ubiquitin protein are used, one that can only be ubiquitinated at Lysine 48 residue (HA-K48-UB) and other that can be only ubiquitinated at Lysine 63 residue (HA-K63-UB). All other lysine residues are mutated. L.E, Low exposure; H.E, High Exposure. Analysis of NRF2 ubiquitination in absence and presence of TRIM16 by Ni-NTA pull-down assays using transiently transfected plasmid constructs as indicated. His-tagged ubiquitin which is mutated at all lysines except 63 position is used in these assays. Analysis of NRF2 ubiquitination in absence and presence of TRIM16 by Ni-NTA pull-down assays using transiently transfected plasmid constructs as indicated. His-tagged ubiquitin which is mutated at all lysines except 63 position is used in these assays. (F) Analysis of NRF2 ubiquitination in absence and presence of TRIM16 deletion variants by Co-IP assays using transiently transfected plasmid constructs as indicated. (G) Western blot analysis of lysates from HeLa, TRIM16KO cells and TRIM16KO cells complemented with TRIM16 deletion constructs where cells were treated with MG132 (20 µM, 2h) and the blot is probed with indicated antibodies. (H) Western blot analysis of lysates from HeLa, TRIM16KO cells and TRIM16KO cells complemented with TRIM16 deletion constructs where cells were treated with MG132 (20 µM, 2h) and the blot is probed with indicated antibodies.
InlK is expressed in vivo.Figure 2A. InlK amino acid sequence. The signal sequence is underlined and the different regions of leucine rich repeats (LRRs) are outlined. The consensus pentapeptide LPXTG at the C-terminal end is boxed. B. Detection by immunofluorescence microscopy of InlK over-expressing in L. monocytogenes EGD-e (WT), ΔinlK, WT+pADc-inlK, ΔinlK+pPRT-inlK and the ΔsrtA mutant over-expressing inlK (ΔsrtA+pPRT-inlK) grown in BHI medium using the rabbit polyclonal anti-InlK antibody. InlK was detected at the surface of InlK over-expressing bacteria (WT+pADc-inlK and ΔinlK+pPRT-inlK), whereas it was undetectable at the surface WT bacteria or at the surface of the ΔstrA mutant over-expressing inlK. C. Detection of InlK by Western blot on total lysates of L. monocytogenes EGD-e (WT), ΔinlK and ΔinlK+pPRT-inlK grown in BHI using the rabbit polyclonal anti-InlK antibody. Decreased concentrations of recombinant purified InlK were used as a positive control. D. Detection of secreted InlK in the supernatant of ΔsrtA mutants over-expressing InlK. Western blotting was carried out on trichloroacetic acid precipitates of ΔsrtA and ΔsrtA+pPRT-inlK culture (OD600 = 1) supernatants using the rabbit polyclonal anti-InlK antibody. E. Detection of purified recombinant InlK protein with rabbit polyclonal anti-live Listeria antibody, rabbit polyclonal anti-killed Listeria antibody, rabbit polyclonal anti-InlK and a rabbit pre-immune serum. InlK was detected only with the rabbit polyclonal anti-live Listeria antibody indicating that it is expressed during the in vivo infectious process. BSA was used as control protein. Two different amounts of proteins were tested (500 ng and 200 ng) to access signal specificity.
Figure 5USP36 promotes SUMOylation of DKC1 and Nop56. H1299 cells transfected with indicated plasmids were subjected to Ni2+-NTA PD followed by IB to detect SUMOylated Nop56 and DKC1 respectively. The protein expression was shown in the bottom of each panel. USP36 interacts with both box C/D (C) and H/ACA (D) snoRNP complexes. H1299 cells transfected with empty vector or Flag-USP36 plasmid were assayed by IP with anti-Flag antibody, followed by IB to detect the indicated proteins. USP36 interacts with snoRNP proteins in the nucleolus. The nucleoli purified from H1299 cells transfected with empty vector or Flag-USP36 plasmid were assayed by co-IP with anti-Flag antibody, followed by IB to detect the indicated proteins. Half-life assays. The WCL from H1299 cells treated with 50 μg/ml cycloheximide (CHX) for indicated time were assayed by IB for the indicated snoRNP proteins. USP36 does not change the levels of endogenous snoRNP proteins. HeLa cells infected with scrambled or the indicated USP36 shRNA lentiviruses were assayed by IB.
a) Western analysis of whole-cell extracts (WCE), membrane (M), cytosolic (C) and peroxisome (P) fractions from parental and MCF-7 cells stably expressing constitutively active myristoylated AKT (myr-AKT) immunoblotted for TSC2, LDH, catalase and PMP70.figf3(a) Western analysis of whole-cell extracts (WCE), membrane (M), cytosolic (C) and peroxisome (P) fractions from parental and MCF-7 cells stably expressing constitutively active myristoylated AKT (myr-AKT) immunoblotted for TSC2, LDH, catalase and PMP70. Light and dark represent short and long autoradiographic exposures, respectively. (b) Western analysis of whole-cell extracts, membrane, cytosolic and peroxisome fractions from MCF-7 cells stimulated with insulin (200 nM) for 30 min after 1 h of serum starvation and immunoblotted for TSC2, LDH, catalase and PMP70. Light and dark represent short and long autoradiographic exposures, respectively. (c) Representative western analysis of HEK293 whole-cell extracts, cytosolic and peroxisome fractions, immunoblotted for phospho-TSC2 (S939; inactive), TSC2 and phospho-AKT (S473 and T308; activated). LDH and catalase were used as markers for the cytosolic and peroxisome fractions, respectively. (d) Proteinase K protection assays performed in the presence or absence of Triton X-100 to disrupt peroxisomes on equal masses of peroxisome fractions (HEK293 cells) collected at the indicated time points. The lysates were immunoblotted for TSC2, TSC1, Rheb, catalase and PMP70. Uncropped images of western blots are shown in Supplementary Fig. S6.
Figure 1A. Hierarchical clustering analysis of model organisms and their average CDS codon frequencies. B. Principal component analysis of the CDS codon frequencies of 9666 protein-coding genes. PC1 and PC2 indicate the first and second principal components. C. Heatmap of half-lives of mRNA and their CDS codon frequencies. The transcripts were ranked according to their half-lives and divided equally into quartiles. The respective codon frequencies of each group were then averaged. D. Histogram illustrating the distribution of genes and their respective GC3- and GC-content. E. Comparison of average transcript mRNA half-lives across their respective GC3- and GC-content ranges. Number of transcripts within each gene optimality range is indicated above their respective points. Data information: In (E), error bars represent the 95% confidence intervals.