EMBO/sd-character-level-ner huggingface dataset:
RGD4C/AAVP-Grp78-HSVtk plus GCV gene therapy and combination with TMZ against orthotopic murine GL261 GBM.Figure 9Immunostaining analysis for αv, β3 or β5 integrins in GL261 cells. Scale bar, 50 μm. BLI of Luc of representative tumor-bearing mice from all experimental groups at day 34 post treatment. Total tumor Luc intensities in each experimental group before treatment, on day 10 post cell implantation, and after therapy, on day 34 post cell implantation. Data shown are representative of two experiments, n=6. Data analysis was done by two-way ANOVA and Bonferroni's multiple comparison test. Survival benefit for tumor-bearing mice from all treatment groups. Data shown are representative of two experiments, n=6. Statistical significance was determined by Kaplan-Meier method (Kaplan-Meier survival fractions). Log-rank (Mantel-Cox) test was used. Weights of C57BL/6J mice bearing intracranial GL261 tumors, from all experimental groups, before and after treatment. Data shown are representative of two experiments, n=6.
Figure 1A. Rev3l+/+ and Rev3l-/- MEFs were pulse-labelled with BrdU prior to harvesting and analyzed by flow cytometry at different time points. The analysis was focused on S phase divided in 3 parts: G1/early S, middle S and late S/G2 BrdU+ cells. Red arrow represents BrdU+ cells that re-entry in S phase and green arrow shows BrdU- cells in G2 phase. Histograms represent the percentage of cells in G1/early and late S/G2 phase after BrdU pulse. Error bars represent standard error for three independent experiments. ns: not significant, * p<0.05 by Student's t-test. B. Rev3l+/+ and Rev3l-/- MEFs were pulse-labelled with EdU for 15min, permeabilized, fixed and stained for EdU incorporation (green). S phase sub-stages from I to IV were evaluated by visual inspection of the cycling population (>300 EdU+ cells, top panel). Scale bar= 5 μm. Dot plots and pie charts show the relative proportion (percentage of total S) of each sub-stage from I to IV (middle and bottom panel, respectively). Each dot represents the mean of two technical replicates. C. Seventy-two hours after transfection with non-targeting (NT) or Rev3l siRNA, HeLa cells were pulse-labelled with BrdU for 15 min, permeabilized, fixed and stained for BrdU (red). As in (B), S phase sub-stages were evaluated by visual inspection of the cycling population (>300 BrdU+ cells, top panel). Scale bar= 5 μm. Dot plots and pie charts show the relative proportion (percentage of total S) of early, middle-late and late S phase (middle and bottom panel, respectively). Each dot represents the mean of two technical replicates.
Figure 4CD1d (PBS57) tetramer+ CD3+ cells were sorted from spleens or PP of 3 different Vα14 TN mice along with CD4+CD3+CD1d tetramer- cells from PP (PP CD4). RNAseq was performed. Heatmap of FPKM values for the indicated Tfh genes across each RNAseq sample. Spleen, mLN, and PP lymphocytes from Vα14 and C57BL/6 mice were stimulated with PMA and ionomycin. Lymphocytes were stained with anti-CD3, CD1d (PBS57) tetramer, anti-IL-4, and anti-IFNγ. Percentages of iNKT cells and non-iNKT T cells within the population of CD3+IL-4+ cells and CD3+IFNγ+ cells, n = 5 mice for C57BL/6 group and n = 4 mice for Vα14 group. Error bars are SD. Vα14 TN mice were administered α-GalCer either 2μg intravenously or 5μg by oral gavage. Mice were given Brefeldin A intraperitoneally after 30 min, and tissues were harvested 3 hours later. Cells from spleen, mesenteric lymph node, and Peyer's patches of Vα14 TN mice were permeabilized, fixed, and stained with antibodies to IL-4, IFNγ, and IL-17. Plots shown are gated on CD1d-(PBS57)-tetramer+ CD3+ iNKT cells. Quantification of data from C, n=2 mice per group. Mann-Whitney test. Error bars are SEM.
TGF-β regulates S100A9 expression and its subcellular localization in Langerhans cells (LC)RNA-Seq analysis was performed using total RNA extracted from Human primary monocyte-derived LC (MoLC), DC (MoDC), and MoDC treated with TGF-β for 24h (MoDC-TGF-β).Figure 1Heat map showing differential expression of significant TGF-β regulated genes in MoDC, MoLC and TGF-β-treated-MoDC of independent experiments from three donors (n=3). Fold change normalization to autologous MoDC was done on Log2-transformed normalized FPKM data and represented by Heat map. Row clusterization was applied based on (Log2) Fold Change values. Green and red shades correspond to increased or decreased gene expression levels respectively. RT-qPCR analysis of mRNA expression of indicated genes from MoLC or MoDC treated with TGF-β for 16h normalized to MoDC. Data represent mean values ± SD of triplicates from two donors. S100A8 and S100A9 expression were determined by western blotting of lysates from MoLC, MoDC and MoDC treated with TGF-β for 24h or from cell lines as indicated. Loading controls were done with GAPDH or actin antibodies. Human primary cells results are representative of three donors and the experiment shown is representative of three (n=3). Subcellular localization analysis of S100A9 and S100A8 in MoLC, MoDC and MoDC-TGF-β. S100A9 mean fluorescence intensities (MFI) were normalized to MoLC and represented in the upper graph. The same was done for S100A9 cytoplasmic/nuclear MFI and shown in the lower graph. Scale bar: 5µm. Graphs show normalized percentages ± SD of data from four different donors for MoLC (n=4) and MoDC (n=4) and three different donors for TGF-β-treated MoDC (n=3). When applicable, student t-test was performed to determine statistical significance **P<0.01).
Scheme of metabolic pathways in BSF (a) and PCF (b) trypanosomes.f7Scheme of metabolic pathways in BSF (a) and PCF (b) trypanosomes. Arrows indicate steps of glucose, proline and threonine metabolism and glycerolipid biosynthesis; dashed arrows indicate steps for which no evidence of flux is available. A, ATPase (a) or ATP synthase (b); AcCoA, acetyl-CoA; Cit, citrate, CoASH, CoA; DHAP, dihydroxyacetone phosphate; FAS II, type II fatty-acid biosynthesis pathway; GAP, glyceraldehyde 3-phosphate; G3P, glycerol 3-phosphate; GPDH, glycerol 3-phosphate dehydrogenase; KG, 2-ketoglutarate; Mal, malate; PEP, phosphoenolpyruvate; Pyr, pyruvate; Resp. Ch., respiratory chain; TCA, tricarboxylic acid cycle; UQ, ubiquinone. Enzymes are: 1, pyruvate dehydrogenase; 2, acetyl-CoA thioesterase; 3, acetate:succinate CoA-transferase; 4, succinyl-CoA synthetase; 5, F0F1-ATP synthase; 6, acetyl-CoA synthetase; TAO, trypanosome alternative oxidase; 7, threonine dehydrogenase; 8, proline dehydrogenase. Activities potentially stimulated by Ca2+ are indicated.
Figure 2A1) Representative WB of RGS9-2, R7BP, and Gβ5 proteins on four Tor1a+/+ and four Tor1a+/- striatal lysates. The bar histogram on the right shows the quantification of the three proteins in each sample. A2) Dot plot showing that Gβ5 striatal levels are unchanged in Tor1a+/- mice (Tor1a+/+ N=11, Tor1a+/- N=10, Mann Whitney test P=0.2453). A3) Conversely, the level of the R7 binding protein R7BP is increased in Tor1a+/- striatum (Tor1a+/+ N=12, Tor1a+/- N=12, t test **P=0.0014). Values are represented as ratio of protein vs. loading control intensity level, normalized to the mean of Tor1a+/+ control values of the same experiment B1) Representative WB of lysates of Tor1a+/+ and Tor1a+/- dorsal striatum slices incubated for 5 hours in the presence (w/ leu) or absence (w/o leu; contralateral striatum) of the protease inhibitor leupeptin. Samples showing enhanced degradation of DRD2 and/or RGS9-2 proteins show additional bands at lower molecular weight. B2) Summary plot reporting mean ± SEM of RGS9-2 and DRD2 protein level values, expressed as the ratio of protein vs. loading control intensity level, normalized to the value of the Tor1a+/+ w/leu sample measured in the same experiment. DRD2: Tor1a+/+ N=4, t test P=0.4376; Tor1a+/- N=4, t test *P=0.0427; RGS9-2: Tor1a+/+ N=5, t test *P=0.0464; Tor1a+/- N=5, t test *P= 0.0190 C1, C2, C3, C4) Dot plots showing DRD2, RGS9-2, R7BP and Gβ5 protein levels measured in striatal detergent-resistant-membrane (DRM) preparations from Tor1a+/- (+/-) and WT (+/+) mice (DRD2: Tor1a+/+ N=16, Tor1a+/- N=13, t test P=0.0129; RGS9-2: Tor1a+/+ N=7, Tor1a+/- N=6, t test *P=0.0396; R7BP: Tor1a+/+ N=6, Tor1a+/- N=6, t test ***P=0.0002; Gβ5: Tor1a+/+ N=9, Tor1a+/- N=9, t test P=0.8461)
Inhibition of IRE1α reverts the fibrotic phenotype of myofibroblasts isolated from scleroderma patients.Figure 6A CTGF mRNA expression levels in skin (dSSc) and lung (LSSc) fibroblasts isolated from scleroderma patients and treated with the IRE1α inhibitor 4μ8C. * P = 0,018 for dSSc (n = 6) and P = 0,003 for LSSc (n = 3). B Collagen1α2 mRNA expression levels in the same samples as in A. * P = 0,0030 for dSSC and P = 0,0036 for LSSc. C Quantification of collagen protein levels in the same samples as in A. * P = 0,010. D Immunohistochemistry staining to detect BiP protein in fibroblasts isolated from scleroderma patients treated or not with 4μ8C. E mRNA levels of αSMA in primary lung fibroblasts isolated from patients with either cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) or asthma. * P = 0,00001 for asthma vs. healthy controls, and P = 0,00003 for ctrl vs. 4μ8C in asthma patients. F Quantification of mRNA levels for BiP in same samples as in E. * P = 0,0023 between asthma and healthy controls and P = 0,0031 between 4μ8C-treated and control treatment in fibroblasts from asthma patients.
Figure 6Representative H&E images of human lung tissues from normal background and bronchiolitis obliterans (BO) with progressive fibrosis. Representative IF images showing the expressions of nuclear YAP, DATP cell marker CLDN4, and AT1 cell marker AQP5 in the airways of normal background (top) and BO (bottom) human lungs. CLDN4 (white), AQP5 (red), YAP (green), and DAPI (blue). Of note, a flattened airway layer in BO lungs. Scale bars, 100μm. Representative IF images showing the expressions of secretory cell marker CC10 and mTORC1 activation marker p-S6 in the airways of normal background (top) and BO (bottom) human lungs. CC10 (green), p-S6 (white), and DAPI (blue). Scale bars, 100μm. Representative IF images showing the expression of nuclear YAP, ATF4, and AQP5 in the airways of normal background (top) and BO (bottom) human lungs. YAP (green), ATF4 (white), AQP5 (red), and DAPI (blue). Of note, co-expressions of nuclear YAP, nuclear ATF4, and AQP5 in the airways of BO lungs. Scale bars, 100μm. Representative IF images showing the expression of p-S6 (white) in the airway cells losing CC10 (green) expression of BO human lungs. Scale bars, 100μm.
Figure 3(A) Doses from 1.5 mg/kg (3 µmol/kg) to 51.3 mg/kg (100 µmol/kg) were given by oral gavage to rats (n = 5 per dose); animals were killed 4 hours later for determination of Aβ40 in brain tissue. Values are mean ± SEM as determined by ANOVA (GraphPad Prism). (B) Time-dependent reduction of Aβ40 in rat brain and CSF after a single 15.4 mg/kg (30 µmol/kg) oral dose of CNP520. (C) CNP520 levels in dog blood and CSF, following a 3.1 mg/kg (6 µmol/kg) oral dose (mean ± SD = 4). (D) Time-dependent reduction of Aβ40 and 42 in dog CSF, after a 3.1 mg/kg oral CNP520 dose (mean ± s.d.).
mTORC1 binds and phosphorylates TFEB on the lysosomal surface.Figure 4(A) Spinning disk confocal image of a MEF cell that co‐expresses TFEB-GFP and mRFP-Rab7 (green and red in the merge, respectively). (B) Time‐lapse of TFEB‐ and Rab7‐positive lysosomes from the boxed region in (A). Time intervals are in seconds. (C) Time‐lapse analysis of Torin 1 treatment in a MEF cell expressing TFEB-GFP. Arrow indicates the time of Torin 1 addition. Yellow arrowheads indicate Torin 1‐induced lysosomal accumulation of TFEB-GFP. Time intervals are in minutes. (D) Immunofluorescence of HEK‐293T cells expressing TFEB-3 × FLAG, treated with DMSO (top) or Torin1 (bottom) and stained with antibodies against FLAG and mTOR (green and red in the merge, respectively; DAPI is in blue). (E) FRAP analysis of TFEB-GFP‐positive lysosomes from control MEFs (blue) or MEFs treated with Torin 1 (red). Each data point represents mean±s.d. from five independent spots. (F) Time‐lapse of photobleaching and fluorescence recovery of TFEB-GFP‐positive lysosomes from control‐treated MEFs (top) or MEFs treated with Torin 1 (bottom). Red arrowheads indicate time of photobleaching. Time intervals are in seconds. (G) Torin 1 increases binding of TFEB to mTORC1. HEK‐293T cells that express TFEB-3 × FLAG along with HAGST‐Rap2A or HAGST‐RagsDN were treated with vehicle or with Torin1, lysed and subjected to FLAG immunoprecipitation followed by immunoblotting for mTOR and raptor. FLAG-Metap2 served as negative control. (H) Immunofluorescence of HEK‐293T cells that express TFEB-3 × FLAG along with Rap2A (top) or the RagsDN mutants (bottom), treated with Torin 1 and stained with antibodies against FLAG and LAMP2 (green and red in the merge, respectively; DAPI is in blue). In all images, scale bars represent 10 μm.
Proposed model for allosteric modulation.Figure 7(A, B) A free energy diagram of the transport cycle (black) and its modulation (colors) by positive or negative allosteric modulators, PAMs (A), or NAMs (B), respectively. Cartoon representations of the low-energy states are below the diagrams and are colored as in Figure 1. The barrier heights are not to scale to the measured rates. Substrate binding to the apo OFS precedes isomerization into the IFS via the highest-energy TS (‡) that structurally resembles the IFS. Substrate release and recycling into the OFS complete the cycle. PAMs, shown as squares in the cartoons of the states, bind with higher affinity to the IFS (dark cyan squares) than to the OFS (light cyan squares). Therefore, they stabilize all IFS-like states, including the TS, and smoothen the energy landscape (cyan line). PAMs that bind too tightly to the IFS may become inhibitory, as apo OFS becomes a high-energy state (dotted blue line). NAMs bind tighter to the OFS ((B), dark magenta squares) than to the IFS (light magenta squares), increase the ruggedness of the landscape (magenta line), and slow down transport. (C) Simulations of the transport rates in the absence and presence of PAMs. The left panel shows the transport cycle with the used rate constants (Materials and Methods). PAM effects were considered equal on both OFS to IFS isomerizations (blue rate constants). The acceleration of transport becomes rate-limited by the IFS to OFS isomerization of the apo transporter as PAM action increases OFS to IFS isomerizations (red rate constant). Middle and right panels show simulated uptake and fold increase of the initial rates, respectively, in the presence of PAMs that increase kOFS to IFS by the indicated number of folds (from cyan to dark blue). Simulations of cycles with kOFS to IFSbound = 0.1 s-1 illustrate higher PAM potency (grey).
In vitro analysis of the impact of SIV-infected microglia supernatant on autophagy in primary neurons.Figure 2(A) Model of experimental design, as detailed in the Materials and Methods. (B) Total AV counting results show that a significant drop in the number of AV occurs in neurons exposed to SIV-infected microglia supernatant for 3 or 24 hr. Total number of AV are significantly increased after the pretreatment with rapamycin followed by SIV-infected microglia supernatant (Sup) exposure for 24 hr. *** P<0.001, * P<0.05 for n = 6 experiments. All values are mean±SEM. AV counting using 3D model reconstruction for neurons exposed to different treatments. (C) Flattened images of multi-stack confocal optical slices of neurons transfected with GFP-LC3 (green) to label AV, and DAPI (blue) to label the nucleus. The five panels display a typical sample image of a neuron under control conditions as well as 3 hr and 24 hr exposure to SIV-infected microglia supernatant or pretreated with rapamycin prior the exposure to SIV-infected microglia supernatant. The right hand panels show a snap shot of a 3D outline of the neuron with AV marked as green spheres. Scale bar, 20 µm.
Shp2 target genes Skp2, Aurka, and Dll1/Hey1 mediate the suppression of p27‐ and p53‐dependent senescence in PyMT tumor cells SA‐β‐gal staining on control, Shp2 ko, and Shp2 ko cells with shRNA knockdown of p27 or p53.Figure 3SA‐β‐gal staining on control, Shp2 ko, and Shp2 ko cells with shRNA knockdown of p27 or p53. Scale bar, 50 μm.Quantification of the numbers of senescent cells shown in (A). Error bars represent SEM (n = 3). **P 0.01.Quantification of the numbers of spheres formed by cells shown in (A). Error bars represent SEM (n = 3). **P 0.01. Western blot analysis of p27, p53, and α‐tubulin in control, Shp2 ko, and Shp2 ko cells with overexpression of Skp2, Aurka, or N3IC cDNAs. The numbers below indicate the relative ratios of band intensities between p27 and α‐tubulin or between p53 and α‐tubulin. Western blot analysis of p27, p53, and α‐tubulin in PyMT cells treated with control (DMSO) or inhibitors against Skp2 (MLN4924), Aurora A (VX‐680), or Notch (DAPT) at the indicated concentrations. The numbers below indicate the relative ratios of band intensities between p27 and α‐tubulin or between p53 and α‐tubulin. Quantification of the numbers of senescent cells in PyMT cells treated with control, MLN4924, MLN4924 plus p27 knockdown or MLN4924 plus p53 knockdown. Error bars represent SEM (n = 3). **P 0.01; NS, not significant. Quantification of the numbers of senescent cells in PyMT cells treated with control, VX‐680, VX‐680 plus p27 knockdown, or VX‐680 plus p53 knockdown. Error bars represent SEM (n = 3). *P 0.05; NS, not significant. Quantification of the numbers of senescent cells in PyMT cells treated with control, DAPT, DAPT plus p27 knockdown, or DAPT plus p53 knockdown. Error bars represent SEM (n = 3). **P 0.01; NS, not significant. Statistical significance was assessed by Student's unpaired t‐test.
Figure 1a. Uniform Manifold Approximation and Projection (UMAP) of sequenced single cells from inflammatory pseudotumor (IPT) CSF (n=1 sample, n=4,324 single cells, left), control CSF (n=6 samples, n=15,467 single cells, middle) and multiple sclerosis (MS) patients-derived CSF (n=6, n=18,412 cells, right). Indicated immune cell subsets as identified by Seurat v4 reference (ref) mapping. b. Stacked bar chart of relative B cell abundances as identified by Seurat v4 reference mapping in inflammatory pseudotumor (IPT) CSF, control CSF, and MS patient-derived CSF. c. Circle plots representing the relative abundance of B cell subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF, control CSF, and MS patient-derived CSF. d. Pseudotime analysis of intrathecal B cell subsets in inflammatory pseudotumor CSF with the naïve B cell cluster, identified by canonical markers, as root node. Percentage of cycling naïve B cells and IgG4 B cells depicted in pie charts. e. Retrospective immunohistochemistry DAB staining of IgG4 and IgG on archival temporal lobe resection tissue. As suggested by the consensus statement on the pathology of IgG4-RD (Deshpande et al, 2012), three 40x fields with the highest number of IgG4+ and IgG+ cells were selected, counted and averaged within these fields. Cell counts as indicated. f. C-X-C motif chemokine 13 (CXCL13) concentrations measured by ELISA from inflammatory pseudotumor (IPT) CSF, control CSF, and MS patient-derived CSF. Individual values, mean ± SEM; n=4 experiment repeats with technical replicates. g. Stacked bar chart depicting mean relative expression levels of T helper cell-associated indicated cytokines in T cell subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF as in (a). TEM, T effector memory; TCM, T central memory; Treg, regulatory T cell. h. Violin plot depicting relative expression levels of IL4R (left) and IL10RA (right) in B cell subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF.
HOPS binding to p53.Figure 5A Protein binding in RKO cells. PLA showed HOPS/p53 interaction with or without etoposide treatment at the indicated time. Red spots showed a single interaction. DNA counterstaining was performed with DAPI. α-Tubulin-FITC antibody was used to identify the cellular edge. Background control was performed according to the manufacturer's instruction. Scale bars, 10µm. The graph represents the binding increase. Non treated cells binding was assumed as 1. Data information: all the experiments were performed three times, and representative examples are shown. In A data are presented as mean ±SD. B Biochemical analysis of nuclear-cytoplasmic protein distribution. RKO cells were treated with etoposide, collected after 2 and 4 hours and processed to obtain nuclear and cytoplasmic fractions. Aliquots relative to the same cell number per fraction were immunoblotted using anti-HOPS and anti-p53 antibodies. GAPDH and Lamin B1 are purity controls of cytoplasmic and nuclear fraction respectively. C HOPS and p53 binding in cytoplasm following apoptosis induction. RKO cells untreated or treated with etoposide as above were used to isolate cytoplasmic fractions. WCL and cytoplasmic fractions were immunoprecipitated with anti-HOPS antibody and subsequently immunoblotted using anti-p53 antibody.
Figure 6Profiles showing average Ca2+ influx induced by Thapsigargin (TG) in freshly sorted NKT1 (Red) and NKT2 (Black) cells. The timing of TG (1uM), ionomycin (Iono) (2uM) and CCCP (1uM) addition, and extracellular [Ca2+] in mM are shown. single-cell quantification related to the data in a and b. (c) cytoplasmic [Ca2+] after ionomycin; (d) maximal cytoplasmic [Ca2+] after TG; (e) increase rate of cytoplasmic [Ca2+] when extracellular Ca2+ is applied; (f) decrease rate of cytoplasmic [Ca2+] when extracellular Ca2+ is removed in NKT1 and NKT2 cells which have similar Ca2+ plateau level (iso-cells; 900-1200nM for control group (a), and 400-700nM for CCCP group (b)).
USP22 knockout (KO) decreases TBZ-induced necroptotic cell death in HT‑29 cells A-B HT‑29 cells were transfected with non-silencing control siRNA (sictr) or siRNAs against USP22 (siUSP22) for 48 h at 20 nM.Figure 1A-B HT‑29 cells were transfected with non-silencing control siRNA (sictr) or siRNAs against USP22 (siUSP22) for 48 h at 20 nM. After transfection, cells were treated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα either for 6 h and analyzed by Western blotting (A) or for 18 h, and the percentage of PI-positive cells was assessed by fluorescence-based PI staining (B). Vinculin served as a loading control. C HT‑29 CRISPR/Cas9 control (ctr) and USP22 KO cells were analyzed by Western blotting for USP22 expression. GAPDH served as loading control. D HT‑29 control and USP22 KO cells were treated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα after pre-incubation with 30 µM Nec1-s and 20 µM GSK'872 for 1 h and incubated for 18 h before fluorescence-based quantification of PI-positive cells. E HT‑29 control and USP22 KO cells, generated with 2 guide RNAs (2g), expressing empty vector (EV) or PAM mutated 3xFLAG-HA-USP22 WT (USP22 PAM), C185S (USP22 PAM C185S) or C185A (USP22 PAM C185A) were analyzed by Western blotting for USP22 expression levels. β-Actin was used as a loading control. F HT-29 control and USP22 KO cells, generated with 2 guide RNAs (2g), expressing empty vector (EV) or PAM mutated 3xFLAG-HA-USP22 WT (USP22 PAM), C185S (USP22 PAM C185S) or C185A (USP22 PAM C185A) were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6, 1 ng/ml TNFα for 18 h. The percentage of PI-positive cells was assessed by fluorescence-based PI staining.