EMBO/sd-character-level-ner huggingface dataset:
Figure 5A. WB analysis confirmed the reduced expression of iASPP in ppp1r13l knocked down cardiomyocytes. B. qPCR showed that ppp1r13l-knocked down murine cardiomyocytes (using two alternative shRNA sequences) expressed higher level of pro-inflammatory cytokine mRNAs after 4 hrs of LPS stimulation. The expression in ppp1r13l-knocked down murine cardiomyocytes was set as 1. Genome wide expression analysis is presented in Figure 1S. Differences between knockdown and control with p values (Student's t test) are indicated with asterisks and exact values.
Figure 4Radiolabeled [35S]COA7 (A) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express MIA40FLAG. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide. Radiolabeled [ 35S]TIMM8A (B) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express MIA40FLAG. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide. (C) Mitochondria were isolated from Flp-In T-REx 293 cells induced to express MIA40FLAG and control cells. The samples were analyzed by SDS-PAGE and Western blot. Mitos, mitochondria. Radiolabeled [35S] COA7 (D) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express ALRFLAG and control cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological experiments were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). Radiolabeled [35S] TIMM8A (E) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express ALRFLAG and control cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological experiments were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). (F) Mitochondria were isolated from Flp-In T-REx 293 cells induced to express ALRFLAG and control cells. The samples were analyzed by SDS-PAGE and Western blot.
Figure 6(A) Relation of chromatin state changes and gene expression changes. For 81% of differentially expressed genes, a change in a regulatory chromatin feature was observed. (B) Scheme of chromatin feature changes and associated TFs identified in this study (C) Pathway analysis of identified core TFs and their target genes from our B cell GRN. Significantly enriched gene sets (p < 0.05) relevant to CLL pathophysiology were retrieved (BCR signaling, NF-κB signaling and MAPK signaling). (D) CLL GREN. TFs identified here, associated chromatin modifiers and differentially expressed target genes were integrated into a network. A part that includes TCF4 and EBF1 is shown. The different TSSs of the TCF4 gene were combined into a single gene target for the corresponding SE. Several enhancers of EBF1 target genes were active in NBCs (H402, H441, H464, H1000) but became silenced in CLL. As an example, H464 and SNX22 are highlighted in yellow. These enhancers represent candidate enhancers for involving EBF1 binding. Color code: active enhancers, dark, light and medium green for CLL only ("C"), only in NBCs ("H") and both cell types ("CH"), respectively; TFs, red; target genes, grey; chromatin modifier, blue. (E) Analysis of regulation of SNX22 by the intronic H464 enhancer shown in panel B. This enhancer lost its ATAC signal at the predicted EBF1 binding site. EBF1 ChIP-seq analysis validated that EBF1 is indeed lost at this site. Although H3K27ac at the locus was only slightly reduced, transcription of SNX22 was largely reduced. These findings are consistent with a mechanism where EBF1 binding drives gene expression of SNX22 by binding to H464.
Representation of reactivation status of ex vivo cultures of resting CD4+ T cells.Figure 8Figure 8: Representation of reactivation status of ex vivo cultures of resting CD4+ T cells.A-C From data of ex vivo cultures of resting CD4+ T cells isolated from HIV+ patients (Table 3), the extracellular HIV-1 genomic RNA levels for each LRA treatment are represented from all patient cell cultures (A), from patient cell cultures presenting no viral reactivation in mock condition (C), and from patient cell cultures exhibiting reactivation in mock condition (B). One night after cell purification, cells were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. Six days after 5-AzadC treatment, the concentration of viral RNA in culture supernatants was determined (in copies/ml). The results were reported as the actual HIV RNA copy numbers/ml or as an estimated value calculated as 50% of the smallest value when HIV RNA was not detected in order to assign a log value. Means are represented.
Figure 6A Representative live-cell STED super-resolution images (t=0 s), showing WT Hela cells expressing MIC13-SNAP, from a time-series of images acquired at a time interval of 2.5 s stained with silicone-rhodamine. Box marks selection shown as a zoom in panel (B). Scale bar 500 nm.C Representative live-cell STED super-resolution images (t=0 s), showing WT Hela cells expressing MIC13-SNAP, from a time-series of images acquired at a time interval of 1.3 s stained with silicone-rhodamine. Box marks selection shown as a zoom in panel (D). Scale bar 500 nm. D Time-lapse image series of a mitochondrion expressing MIC13-SNAP imaged at a time interval of 1.3 s/ frame. Green and magenta asterisks show cycles of cristae merging and splitting marked by MIC13-SNAP. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm. E First image from (D) was used for showing the intensity profiles along the length of three cristae (numbered 1-3 and marked with white arrows) in (F). F Intensity profiles of lines drawn across the length of cristae show increased intensities at the edges of the line scans representing the IBM. Magenta boxes depict intensity maxima at IBM.
Figure 1Adult mouse back skin sections immunostained for LYVE1 (red), αSMA (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. LV, lymphatic vessels; apm, arrector pili muscle. Back skin sections of P55 mouse skin immunostained for LYVE1 (red), Tenascin-C (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. TNC, Tenascin-C. Adult mouse back skin sections from different postnatal (P) days immunostained for LYVE1 (red), Lhx2 (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. epi, epidermis; der, dermis. Histogram of the quantification of the LV distance to HFSC positive for Lhx2. ). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Back skin sections of P49 mouse skin immunostained for LYVE1 (red), CD34 (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 100 μm. epi, epidermis; der, dermis. White arrowheads denote the proximity of lymphatic capillaries to HFSC. Histogram of the quantification of the LV distance to HFSC positive for CD34. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. C, Catagen; Te, early Telogen; Tm, mid Telogen; Tl, late Telogen; A, Anagen. Back skin sections of K15CrePR+/T; Wls∆/∆ and Control K15CrePR+/+; Wlsflox/flox mice treated with mifepristone from P7 during 12 weeks (P90), immunostained for LYVE1 (red), and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 100 μm. epi, epidermis; der, dermis. Histogram of the quantification of the LV angle relative to the epidermis. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.
IDH mutant gliomas display a low energy potential.Figure 2A) MALDI imaging of indicated metabolites in IDHwt PDX (P3), IDHm PDX (E478) and control brain (CB). Hex-P: hexose-phosphate; AMP, ADP, ATP: adenosine mono-, di- and triphosphate respectively. B) LC-MS quantification of indicated metabolites in 6 PDXs. Relative quantities of metabolites are expressed in log scale and normalized to tissue quantity used for extract preparation. IDHwt glioblastomas (GBM) in blue, IDHm GBM in orange, IDHm lower grade gliomas (LGG) in red, contralateral control brain (CB) in grey. Sdh7-P: sedoheptulose-7-phosphate. C) Energetic charge defined as the ratio of (ATP+1/2ADP)/(ATP+ADP+AMP) was calculated at each pixel to present its distribution throughout the tumor. (D) The energetic charge ratio was confirmed by the LC-MS data performed on separate tumor extracts (n=3/ sample/ group). Indicated statistical significant group differences are based on T test, pv: p-values.
Figure 4RNA-seq of GPR56 KD in CD34+ CB cells reveals that GPR56 enhances gene expression displayed as reads per kilobase per million mapped reads (RPKM) of MYC, TNKS2, TGFB1 and SRC (top). Previously published RNA-seq of ten sorted CD34+GPR56+ versus CD34-GPR56+ fractions (Garg et al, 2019) shows a divergent expression pattern: downregulation of Wnt targets MYC and TNKS, but upregulation of TGFB1 and SRC in the CD34+GPR56+ fraction. Unpaired t-test, bars and error bars represent mean and standard deviation of three biological replicates, *** p<0.0005, ** p<0.005, * p<0.05. Cartoon visualizing that GPR56 enhances genes and pathways differentially active in the CD34+GPR56+ fraction, which is characterized by slow cell cycle progression, high LSC frequency, and high expression of the stemness gene HLF versus the CD34-GPR56+ cells, which are more differentiated (lower LSC frequency), cycle faster, and have little HLF expression. Arrows and blocked arrows indicate activation or inhibition by the indicated small molecules, respectively, which were used in subsequent experiments. Contour FACS plots (left) and summary bar graph (right) showing CD34 and GPR56 expression after 5-day culture of purified CD34+GPR56+ cells from AML sample E218974 with CHIR99021 or vehicle DMSO, n=3. Unpaired t-test, bars and error bars represent mean and standard deviation of three technical replicates, *** p<0.0005. Left: FACS profile and sorting strategy for the CD34-GPR56+ fraction (top), and post-sort purity (bottom) of AML sample E2112376 on the day of thawing. Right: CD34 and GPR56 expression measured by flow cytometry 5 days post exposure of purified CD34-GPR56+cells to the indicated molecules or their combinations. PRI: PRI-724, arrows indicate whether the pathway is inhibited (↓) or activated (↑) by the compound. Left: Bar graph showing the distribution of CD34 and GPR56 fractions at the end of the 5-day culture of purified CD34-GPR56+ cells from AML E2112376 with indicated compounds. Right: statistical analysis shown only for the CD34+GPR56+ fraction. See Dataset EV11 for full statistical analysis. PRI: PRI-724, n = 3. Unpaired t-test, bars and error bars represent mean and standard deviation of three individual treatments, *** p<0.0005, ** p<0.005, * p<0.05. Cartoon visualizing the proposed mechanism by which both GPR56+ LSC enriched compartments are maintained: GPR56 enhances pathways, which reciprocally inhibit each other and are differentially active in the two fractions. This should result in a constant transition between the compartments and thus prevent exhaustion of the two populations.
Figure 4293T-FcR-Kb cells co-expressing empty vector or Rab mutants (Rab5ACA, Rab22ACA, Rab7ADN) individually, with either LacZ as a control or US6 were analyzed for cross-presentation of OVA using B3Z cells. The effects of US6 on cross-presentation were analyzed by plotting the mean percentage of IL-2 release by cells co-expressing US6 (A) with control vector or Rab mutants compared to cells co-expressing LacZ with control vector or Rab mutants, respectively. 293T-FcR-Kb cells co-expressing empty vector or Rab mutants (Rab5ACA, Rab22ACA, Rab7ADN) individually, with either LacZ as a control or ICP47 (B), were analyzed for cross-presentation of OVA using B3Z cells. The effects of ICP47 on cross-presentation were analyzed by plotting the mean percentage of IL-2 release by cells co-expressing ICP47 (B) with control vector or Rab mutants compared to cells co-expressing LacZ with control vector or Rab mutants, respectively. 293T-FcR-Kb cells expressing Rab5ACA were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured. 293T-FcR-Kb cells expressing Rab22ACA were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured. 293T-FcR-Kb cells expressing Rab7ADN (E) were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured.
Figure 6(A) Vero GFP-split cells were transfected with variant S proteins and imaged 18h post-transfection. Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for each of the transfected variant S proteins. Right Panel: Representative images of Vero GFP-split cells 18h post-transfection, GFP (Green) and Hoechst (Blue). Top and bottom are the same images with and without Hoechst channel. Scale bars: 200 µm. (B) Left Panel: Quantification of Delta S protein mediated fusion in Vero-GFP split cells by video microscopy. Results are mean ± SD from three fields per condition from one representative experiment. Right Panel: Fusion quantification of 3 independent video microscopy experiments, 20h post transfection, normalized to D614G. (C) 293T cells were transfected S proteins with each variant-associated mutation for 24h and stained with biotinylated ACE2 and fluorescent streptavidin before analysis by flow cytometry. Left Panel: Representative ACE2 binding dilution curves for the Delta variant in relation to Alpha and D614G. Right Panel: EC50 values (concentration of ACE2 needed for 50% binding) for Alpha for the Delta variant. (D) Caco2 GFP-split cells were transfected with variant S proteins and imaged 18h post-transfection. Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for each of the transfected variant S proteins. Right Panel: Representative images of Caco2 GFP-split cells 18h post-transfection, GFP (Green) and Hoechst (Blue). Top and bottom are the same images with and without Hoechst channel. Scale bars: 200 µm
TBK1 and mTOR S2159 phosphorylation are required for TLR3- and TLR4-stimulated mTORC1 catalytic activity and signaling.Figure 6A. TBK1 activity is required for TLR3- and TLR4-stimulated mTORC1 catalytic activity: RAW264.7 macrophages were pre-treated with amlexanox ([50 μM] or 100 μM]) (2 hr.), rapamycin [20 ng/mL] (30 min), or Ku-0063794 [1 μM] (30 min) and treated -/+ poly (I:C) [30 μg/ml] or LPS [100 ng/ml] (60 min). Raptor was immunoprecipitated (IP), and IPs and whole cell lysate (WCL) were immunoblotted (IB) as indicated. B. and C. mTOR S2159 phosphorylation is required for TLR3- and TLR4-stimulated mTORC1 signaling: RAW264.7 macrophages were co-transfected with vector control, wild type, or rapamycin-resistant (RR) AU1-mTOR alleles (RR or RR/S2159A) together with HA-S6K1. Cells were treated with rapamycin (+) to ablate endogenous mTORC1 function and stimulated with poly(I:C) (A) or LPS (B) as in A. HA-S6K1 was immunoprecipitated, and IPs and WCL was immunoblotted as indicated. B. and C. mTOR S2159 phosphorylation is required for TLR3- and TLR4-stimulated mTORC1 signaling: RAW264.7 macrophages were co-transfected with vector control, wild type, or rapamycin-resistant (RR) AU1-mTOR alleles (RR or RR/S2159A) together with HA-S6K1. Cells were treated with rapamycin (+) to ablate endogenous mTORC1 function and stimulated with poly(I:C) (A) or LPS (B) as in A. HA-S6K1 was immunoprecipitated, and IPs and WCL was immunoblotted as indicated. D. Pharmacologic inhibition of PI3Kα but not Akt reduces TLR3- and TLR4-stimulated mTORC1 signaling: RAW264.7 macrophages were pre-treated with the PI3Kα class I inhibitor BYL-719 [10 μM], the Akt inhibitor MK-2206 [10 μM], amlexanox [100 uM], or Ku-0063794 [1 μM] (30 min) and stimulated -/+poly (I:C) or LPS as in A.
PLCβ1 and PTPRN2 facilitate cofilin localization and activity.Figure 5(A) Kaplan-Meier curve representing distal metastasis-free survival of a cohort of breast cancer patients (N = 1609) as a function of their primary tumor's CFL1 expression levels (Data from KMPlot (Gyorffy et al., 2010)). Patients' primary tumors' CFL1 expression levels were classified as low (blue) or high (red) expression. (B,C) Membrane and membrane-associated proteins were purified from cells transfected with siRNA targeting PTPRN2 (B) or PLCβ1 (C) or control siRNA. Fractions were subjected to Western blot analysis for CFL1 and EGFR levels. Right, densitometry analysis of CFL1 levels normalized to EGFR levels. (B,C) Membrane and membrane-associated proteins were purified from cells transfected with siRNA targeting PTPRN2 (B) or PLCβ1 (C) or control siRNA. Fractions were subjected to Western blot analysis for CFL1 and EGFR levels. Right, densitometry analysis of CFL1 levels normalized to EGFR levels. (D,E) Membrane and membrane-associated proteins were purified from cells overexpressing PTPRN2 (D), PLCβ1 (E) or a control vector. Fractions were subjected to Western blot analysis for CFL1 and EGFR levels. Right, densitometry analysis of CFL1 levels normalized to EGFR levels. (D,E) Membrane and membrane-associated proteins were purified from cells overexpressing PTPRN2 (D), PLCβ1 (E) or a control vector. Fractions were subjected to Western blot analysis for CFL1 and EGFR levels. Right, densitometry analysis of CFL1 levels normalized to EGFR levels. (F) LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA were partially permeabilized and incubated with biotin-actin monomers. Cells were stained for incorporated biotin-actin monomers using Streptavidin-555 (red) and DAPI (blue). Right, quantification of mean fluorescence intensity of incorporated biotin-actin monomers. N = 100 cells/group. Scale bar, 20 μm.
Figure 8A. Heatmap visualization of the expressions of selected nitric oxide (NO)-related genes. B-E. Gene expression of Ca2+ channel gene (B) ryanodin receptor, RYR2, (C) sarco(endo) plasmic reticulum Ca2+-ATPase (SERCA2 or ATP2A2), (D) phospholamban (PLN), and (E) endothelial nitric oxide synthase NOS3 (n=6/group) Box plots for mRNA expressio represent quartiles, minimum and maximum value (relative fold change "FC&qu Statistics: one-way ANOVA followed by Tukey"s test for Significant differences between groups (p value) are indicated on graphs Expression of neuronal nitric oxide synthase (NOS1) mRNA (F Bar graph represent mean±SD Statistics: one-way ANOVA followed by Tukey"s test for Significant differences between groups (p value) are indicated on graphs Expression of neuronal nitric oxide synthase (NOS1 protein with a representative Western blot WT brain was used as positive control in the 1st lane (G) H. Cardiac gene expression of dimethylarginine dimethylaminohydrolase 1 (DDAH1) (n=6/group) Box plots for mRNA expressio represent quartiles, minimum and maximum value (relative fold change "FC&qu Statistics: one-way ANOVA followed by Tukey"s test for Significant differences between groups (p value) are indicated on graphs I. Cardia citrulline to arginine ratio (n=5/group) Bar graph represent mean±SD J. Cardiac protein nitrotyrosine quantified from Western blots K. Nitrite concentration in heart tissue (n=4/group) Bar graph represent mean±SD
Figure 1A Scheme of the lentiviral vector with inducible FlagOmomyc (top) and immunoblotting (bottom) of FlagOmomyc and actin loading control upon Dox treatment of BT168FOcells for 0-48 hours. B Proliferation curves of BT168FO and BT168GFPcells upon Dox treatment for 0-96 h (n = 3; mean ± SD). Viable cells were counted using a haemocytometer. C-D Self-renewal assay upon Dox treatment. C: histograms showing the percentage of cells capable of re-forming a neurosphere seven days after dissociation (n = 3; mean ± SD). D: representative micrographs of BT168FO cell neurospheres. E qRT-PCR of proliferation, stem cell and differentiation markers (CCND1, PTEN, SOX2, NOTCH1, NESTIN, MYC) in BT168FO cells after 48 h Dox treatment, compared to uninduced cells (n = 3; mean ± SD). Expression levels in non-induced cells were set as 1. F. Transwell migration assay of BT168FO cells after 3 days with or without Dox (n = 3; mean ± SD). 10 fields were counted per assay. G. Immunofluorescence analyses of GSC differentiation. To stimulate differentiation BT168FO were grown as monolayer in the presence of serum and treated with doxycycline for up to 7 days. The upper panel displays immunofluorescence images of NESTIN, GFAP, ß-tubulin III, SOX2, and FlagOmomyc expression. FlagOmomyc blunted SOX2 expression and decreased GFAP and NESTIN protein levels, while inducing the onset of ß-tubulin III. The lower panel shows the percentage of positive cells for each cell marker evaluated (n = 4; mean ± SD). 16 fields for each assay were examined; scale bar = 100 μm.
Figure 1Figure 1. Ret-eGFP is Expressed in Multiple Sensory Neuron Subsets(A−L) Ret-positive neurons largely overlap with markers for myelinated neurons (NF200), non-peptidergic nociceptors (IB4) and C-fiber low-threshold mechanoreceptors (TH). However some Ret positive neurons are negative for these markers (indicated by the arrows in D,H and L) suggesting the existence of a further subset of Ret+ neurons. (M) Quantification of the proportion of Ret-eGFP positive neurons in DRG . (N) Quantification of Ret-eGFP co-expression with other markers. (n=2278 cells from 3 mice) Scale bars, 50 μm.
Figure 1Kaplan Meier plots representing the survival difference between SKCM patients stratified according to the 40% highest or lowest BNIP3 expression. BNIP3 levels in primary melanoma and metastases within the SKCM TCGA cohort, using Mann-Whitney's non-parametric test. The box represents the median and the 25th-75th percentile with the whiskers representing the minimum and maximum values. Representative images of BNIP3 immunostaining in a benign nevus (n=5), primary melanoma and their paired metastasis (n=7). In the representative pictures, nevi or tumor tissue is circled in yellow and highlighted with a yellow asterisk (*). Histopathological scoring of BNIP3 protein levels in nevi and melanoma patients with paired primary and metastases harboring weak and in gradient (+/-), weak and homogeneous (+) or strong and homogeneous (++) BNIP3 protein levels based on histopathological staining intensity and distribution scoring. Scale bars represent 150µm. Representative images of BNIP3 immunohistochemistry in TMAs containing (n=158) melanoma metastases and related Kaplan Meier plot of survival difference comparing melanoma patients with different BNIP3 histopathological scores. Scale bars represent 50µm.
Interactome-based Pearson correlation of KZFPs Individual KZFP interactomes were compared by Pearson correlation analysis of prey protein fold-change over control (Log2FC) values.Figure 5Individual KZFP interactomes were compared by Pearson correlation analysis of prey protein fold-change over control (Log2FC) values. Accordingly, KZFP bait proteins are ordered based on the similarity of their prey protein recovery profile.