EMBO/sd-character-level-ner huggingface dataset:
c-Maf is inducible in mouse ILC2s.Figure 2(A) Gating strategy used to identify ILCs and ILC subsets in mice (mILCs). (B) Representative histogram and summary from 2 pooled independent experiments of the percentage of c-Maf expression in mILC subsets in the indicated organs (each symbol represents one individual biological replicate; ANOVA lung: F(2,12)=20.86, p=0.0001; gut: F(2,12)=22.27, p<0.0001; spleen: F(2,12)=4.705 , p=0.031; Kruskal-Wallis test LN: K=9.815, p=0.0011). Bars represent mean ± SEM. Mice received the indicated compound for 3 consecutive days and lung ILCs were analyzed by flow cytometry the following day. Expression of ST2, KLRG1 and c-Maf was assessed in ILC2s. (C) Representative dot plots from 2 pooled independent experiments Mice received the indicated compound for 3 consecutive days and lung ILCs were analyzed by flow cytometry the following day. Expression of ST2, KLRG1 and c-Maf was assessed in ILC2s. (D) the percentage of ST2+ KLRG1+, ST2-KLRG1+, ST2+KLRG1-, ST2-KLRG1- ILC2s (2way ANOVA, interaction: F(12,56)=13.82, p<0.0001)) Each symbol represents one individual biological replicate. Bars represent mean ± SEM. Mice received the indicated compound for 3 consecutive days and lung ILCs were analyzed by flow cytometry the following day. Expression of ST2, KLRG1 and c-Maf was assessed in ILC2s. (E) the percentage of c-Maf+ ILC2s (Kruskal-Wallis test: K=14.56, p=0.0057). Each symbol represents one individual biological replicate. Bars represent mean ± SEM. Mice received the indicated compound for 3 consecutive days and lung ILCs were analyzed by flow cytometry the following day. Expression of ST2, KLRG1 and c-Maf was assessed in ILC2s. (F) the gMFI of c-Maf+ ILC2s (Kruskal-Wallis test: K=14.56, p=0.0057). Each symbol represents one individual biological replicate. Bars represent mean ± SEM.
Fig.Figure 2(A) Cas9 protein and the specific sgRNA targeting the human FKRP locus. The FKRP c.1364C>A (p.A455D) mutation is 43 bases upstream of the sgRNA target sequences. Red arrowheads indicate putative cleavage site. PAM, protospacer-adjacent motif. (B) A schematic diagram shows the genome editing strategy based on CRISPR/Cas9-stimulated homologous recombination, followed by positive selection with puromycin and negative selection with FIAU. Homology left and right arms on the targeting donor vector are indicated as black boxes, flanking the piggyBac (PGK-puro∆tk) selection cassette, which is under the control of PGK promoter. PCR genotyping primers are shown as blue arrows. Note that the TTAA sequences are designed to accommodation the selection cassette excision sites, yet code the same amino acids. (C) Sequence analysis shows precise biallelic correction of the FKRP mutation in three independently corrected iPSC clones (5D17, 5D23 and 3B17), compared with their parental FKRPA455D-iPSCs. Selection cassette excision sites are identified in the corrected-iPSC lines. (D) CRISPR/Cas9 corrected-iPSC lines show a normal karyotype.
POLG NSCs exhibited impaired mitochondrial function, which was partly presented in fibroblasts.Figure 5A: Representative transmission electron microscopy images of mitochondrial structures in Detroit 551 control, WS5A and CP2A NSCs (scale bar, 400 nm). Flow cytometric analysis of mitochondrial volume (MTG) (B, n=5, technical replicates per clone), total MMP (TMRE) (C, n=5, technical replicates per clone) calculated by dividing median fluorescence intensity (MFI) for total TMRE expression by MTG in NSCs generated from control lines, WS5A and CP2A iPSCs. Flow cytometric analysis of specific MMP (D, n=5, technical replicates per clone), calculated by dividing median fluorescence intensity (MFI) in NSCs generated from control lines, WS5A and CP2A iPSCs. E: Intracellular ATP production in NSCs (n=3, technical replicates per clone). Flow cytometric analysis of mitochondrial volume (MTG) (F, n=6, technical replicates for control; n=5, technical replicates for WS5A and CP2A), total MMP (TMRE) (G, n=5, technical replicates for control; n=4, technical replicates for WS5A and CP2A) in Detroit 551, WS5A and CP2A fibroblasts. Flow cytometric analysis of specific MMP (TMRE/MTG) (H, n=5, technical replicates for control; n=4, technical replicates for WS5A and CP2A) in Detroit 551, WS5A and CP2A fibroblasts. I: Intracellular ATP production in Detroit 551, WS5A and CP2A fibroblasts (n=6, technical replicates for control; n=3, technical replicates for WS5A and CP2A).
Figure 2Phospho-site clusters in the carboxyl-terminus of V2R and B2R are underlined and color-coded to reflect the PXPXXP and PXXPXXP patterns as proposed in a previous study (Zhou et al., 2017). A number of B2R mutants were generated to mimic the spatial distribution of phospho-site pattern of Ser/Thr as present in the V2R. The first three mutants were designed to target the proximal part of the phospho-site pattern (indicated in red dotted box) while the fourth mutant was designed to target the distal part (indicated in blue dotted box). Confocal microscopy reveals robust recruitment of βarr1 to B2R mutant constructs upon agonist-stimulation. HEK-293 cells expressing either B2RWT or B2R mutants along with βarr1-mCherry were stimulated with agonist (Bradykinin; 100nM) and the localization of βarr1 was visualized using confocal microscopy. Representative images from three independent experiments are shown here, and the scale bar is 10μm. Visual scoring from three independent experiments revealed agonist-induced βarr1 recruitment (i.e. membrane and endosomal localization) in approximately 75% of the cells for B2RWT (662 cells), 74% of the cells for B2RΔG368 (169 cells), 75% of the cells for B2RL370T (130 cells), 85% of the cells for B2RΔG368+L370T (132 cells) and 77% of the cells for B2RΔI374 (116 cells). HEK-293 cells expressing B2R constructs with C-terminal SmBiT and βarr1 with N-terminal LgBiT were treated with indicated concentrations of bradykinin, and ligand-induced change in luminescent signal was measured. Concentration-response curves were plotted using GraphPad Prism, and pEC50 (top) and Emax (bottom) were calculated. Data represent mean ± SEM of five independent experiments (three for B2RΔI374), each performed in duplicate.
Oxidation of HMGB1 regulates HMGB1 subcellular localization and autophagy.Figure 5.(A) The C106 mutation (C106S) of HMGB1 impairs its nuclear localization. Hmgb1−/− MEFs were transfected with wild-type and cysteine mutant HMGB1-GFP plasmids as indicated and then were treated with 1 µM rapamycin for 12 h or starved (HBSS) for 3 h. The mean nuclear (Nuc) and cytosolic (Cyt) HMGB1 intensity per cell was analyzed by imaging cytometric analysis. *, P < 0.05 versus HMGB1+/+ group; n = 3. Representative images of HMGB1 location are shown on the left (green, HMGB1; blue, nucleus). The right panel is a schematic diagram of HMGB1 structure illustrating the basic A box and B box as well as the acidic C-terminal domain, with the cysteine mutation locations identified. (B and C) Cytoplasmic HMGB1 enhances autophagy and limits apoptosis. HMGB1−/− MEFs were transfected with wild-type or cysteine mutant HMGB1-GFP plasmids as indicated and then were starved (HBSS) for the indicated time. In a parallel experiment, Hmgb1+/+ MEFs were pretreated with 5 mM ethyl pyruvate (EP) for 2 h and then starved as indicated. LC3 punctae formation was assayed by imaging cytometric analysis (B) (B and C) Cytoplasmic HMGB1 enhances autophagy and limits apoptosis. HMGB1−/−MEFs were transfected with wild-type or cysteine mutant HMGB1-GFP plasmids as indicated and then were starved (HBSS) for the indicated time. In a parallel experiment, Hmgb1+/+MEFs were pretreated with 5 mM ethyl pyruvate (EP) for 2 h and then starved as indicated. Apoptosis was assayed by FACS (C) (C) as described in Materials and methods. *, P < 0.05 and **, P < 0.005; n = 3. UT, untreated. Non, nontransfection. (D) C23/C45 is required for the binding of HMGB1 to Beclin1. Hmgb1−/− MEFs were transfected with wild-type or cysteine mutant HMGB1-GFP plasmids as indicated and stimulated with starvation (HBSS) for 3 h. These cells were then assayed for protein expression levels as indicated by IP or Western blotting. Blots are representative of three independent experiments with similar results. (E) Reducing reagents disrupt the interaction between wild-type/C106 HMGB1 and Beclin1. As a control, before IP, samples were incubated with 50 mM DTT (+DTT) and assayed for protein expression levels as indicated by IP or Western blotting. Blots are representative of two independent experiments with similar results. (F) Knockdown of Beclin1 by siRNA inhibits autophagy under conditions of HMGB1 translocation from the nucleus to the cytosol. Cells were stimulated with HBSS, rapamycin (Rap), rotenone (Rot), or thenoyltrifluoroacetone (TTFA) for 3 h or 12 h, and LC3 punctae formation was assayed as indicated. **, P < 0.005 versus Beclin1 shRNA group; n = 3. Ctrl, control. Data are means ± SEM.
Figure 3A. Table summarizing the different target genes/targeted therapies identified for each subnetworks. B. Comparison of bCSC variation after gene knockdown by ALDEFLUOR phenotyping (right) and tumor SFE (left) using the 11 siRNAs pool (3 siRNAs/pool) against the selected target genes and one scrambled siRNA pool used as control. Results for each siRNA pool (from top to bottom) are represented as opposite bars (n=3). Correlations are measured using Spearman rank correlation (R). C-D. Examples of dose-dependent ALDHbr cells variation in response to drug treatments. ALDHbr cell proportion in response to Tazemetostat in SUM159 (n=10) (C) and JQ1 in SUM149 (n=10) (D). The horizontal red dashed-line corresponds to a reduction of 50% of the ALDHbr cell proportion in the treated conditions compared to the control and the vertical red dashed-line corresponds to the target dose predicted by the K-score. On the bottom panel, corresponding flow charts for the ALDEFLUOR staining. DEAB is an ALDH inhibitor used as negative control. E. Pearson correlation analysis of the variation of the ALDHbr cells proportion upon drug treatment and gene silencing. F. Heat map for the variation of the ALDHbr cells proportion under drug treatment, in five different cell lines. Red and blue indicate the K-score in treated conditions, respectively, above and below the K-score in the untreated condition. G-H. Primary (n=4) (G) and secondary (n=4) (H) tumorsphere-forming efficiency (SFE) of SUM159 cells under treated and untreated conditions. Grey dashed-line corresponds to the level of SFE in the untreated conditions. Statistical test used is Pairwise chi-square test. Data represent mean ±SD.
Figure 8(A) Immunofluorescent staining of AEP (red) and C/EBP β (green) in CA1 region from hippocampus of brains from colonic-injected PBS, colonic-injected AD brain extracts, and pre-Vagotomy colonic injected AD brain extracts 3xTg mice. Scale bar: 20μm. (B) Quantitative analysis of AEP positive cells and C/EBP β positive cells, respectively. The density of both AEP and C/EBP β positive cells were significantly increased by colonic-injection of AD brain extracts and decreased through vagotomy before colonic-injection. Representative data of three samples, data are shown as mean ± SEM. **P =0.016 (**P < 0.01), ****P<0.0001, one-way ANOVA. (C) Immunofluorescent staining of cleaved APPC586 (red) and Aβ (green) in cerebral cortex of brains from colonic-injected PBS, colonic-injected AD brain extracts, and pre-Vagotomy colonic injected AD brain extracts 3xTg mice. (D) Quantitative analysis of cleaved APP C586 positive cells and Aβ positive cells, respectively. The density of both cleaved APP C586 positive cells and Aβ positive cells were significantly increased by colonic-injection of AD brain extracts and decreased through vagotomy before colonic-injection. Representative data of three samples, data are shown as mean ± SEM. ****P<0.0001, one-way ANOVA. (E) Immunofluorescent staining of T22 (red) and HT7 (green) in cerebral cortex of brains from colonic-injected PBS, colonic-injected AD brain extracts, and pre-Vagotomy colonic injected AD brain extracts 3xTg mice. (F) Quantitative analysis of T22 positive cells. The density of T22 positive cells was significantly increased by colonic-injection of AD brain extracts and decreased through vagotomy before colonic-injection. Representative data of three samples, data are shown as mean ± SEM. ***P=0.0007 (***P < 0.001), ****P<0.0001, one-way ANOVA.
Figure 7(A) Scatter plot of pairwise comparison of chromatin accessibility changes between TGFβ + RA + VitC vs TGFβ iTregs showing the top identified motifs. (B) CD4+ naïve T cells were sorted from Foxp3-IRES-eGFP reporter mice, labeled with cell proliferation dye cell trace violet and differentiated into iTreg cells with TGFβ alone or TGFβ + RA + VitC with different amounts of IL-2. Cells in the cultures were analyzed daily by flow cytometry. The two upper panels show representative dot plots of Foxp3 expression vs cell proliferation for TGFβ alone and TGFβ + RA + VitC iTregs with 100 U/ml IL-2 from day 2 to day 9. The two lower panels show representative dot plots of Foxp3 expression vs cell proliferation for TGFβ alone and TGFβ + RA + VitC iTregs with 0 U/ml IL-2 from day 2 to day 9. (C) Graph showing the percentage of Foxp3+ cells at each day for TGFβ iTregs and TGFβ + RA + VitC iTregs with either 0 U/ml IL-2 or 100 U/ml IL-2. Error bars show mean ± S.D. from three biological replicates. (D) Graphs depicting the percentage of viable cells for TGFβ iTregs (left) and TGFβ+RA+VitC iTregs (right) differentiated with 0 U/ml, 1 U/ml, 5 U/ml and 10 U/ml IL-2. Error bars show mean ± S.D. from three biological replicates.
Figure 4A The simulated period of ASPD models is shorter than that of the WT model in DD. Thus, they have a ~4 h more advanced phase than the WT model in LD 12:12 (inset). PER2 protein concentrations in each mutant are normalized to the maximum PER2 concentration of the WT model (dashed gray line; inset). B The phase delays induced by a single daily dosing of PF-670 were simulated. The dosing regimens inducing ~4 h phase delays in the WT model led to much larger phase delays in all ASPD models with large inter- and intra-variations (blue circles). When the dosing times are advanced by 4 h reflecting the advanced phase of the ASPD models, the phase delays become closer to but still larger than the ~4 h phase delays (red squares). C As the average PER2 level is higher in the ASPD models PF-670 induces a larger phase delay. Here, the phase shift is the average of the phase delays represented as the red squares in (B). The line represents the least-square fitting line. r and P denote the Pearson's correlation coefficient and P-value of Pearson's correlation test, respectively. α denotes the slope of the least-square fitting line. D The ASPD models simulate the larger PF-670-induced phase delay in LD 8:16 than in LD 16:8 due to the higher PER2 level in LD 8:16 than in LD 16:8 when dosing occurs E Consistently, higher experimentally measured PER2 levels in the SCN at the dosing time leads to a larger PF-670-induced phase delay which was also captured by the NHP model when 20 mpk dosing was used.
Figure 6Gene expression profiles of PBMCs from P. falciparum symptomatic and asymptomatic infected participants were compared. A. IPA canonical pathway analysis scaled by -Log10(P-value) using the 418 differentially expressed genes (DEGs) between symptomatic and asymptomatic P. falciparum malaria. Pathways with a positive z-score in red are activated, and and pathways with a negative z-zscore in blue are inhibited in symptomatic compared with asymptomatic P. falciparum malaria. The ratios of DEGs found in each pathway over the total number of genes in the pathway are listed on the right. The red line corresponds to a P value of 0.05. Gene expression profiles of PBMCs from P. falciparum symptomatic and asymptomatic infected participants were compared. B. Upstream regulator analysis of the 418 DEGs between P. falciparum symptomatic and asymptomatic infected participants. The red lines represent a significant activation z-score of ±2. Gene expression profiles of PBMCs from P. falciparum symptomatic and asymptomatic infected participants were compared. C. Heat map of the 418 DEGs in P. falciparum symptomatic and asymptomatic infected participants as well as in healthy immune controls. E. Volcano plot displaying selected genes clusters 1 and 2 from heatmap in C scaled by Log2-fold-change and -Log10(P-value) differentially expressed between P. falciparum symptomatic and asymptomatic infected individuals. Genes in red are overrepresented in symptomatic malaria and genes in blue are overrepresented in asymptomatic malaria. Gene expression profiles of PBMCs from P. falciparum symptomatic and asymptomatic infected participants were compared. D. Bar plots showing significantly enriched gene ontology (GO) terms scaled by Log10(P-value) in clusters 1 and 2 from the heatmap in C. Red GO terms are upregulated and blue GO terms are downregulated in symptomatic compared with asymptomatic P. falciparum malaria. Gene expression profiles of PBMCs from P. falciparum symptomatic and asymptomatic infected participants were compared. F. Mean RPKMs + SEM of selected genes in P. falciparum malaria symptomatic (n=6) and asymptomatic (n=5) individuals. Dotted red lines depict transcriptional levels of healthy immune controls, Mann-Whitney test of biological replicates,*p<0.05. Gene expression profiles of PBMCs from P. falciparum symptomatic and asymptomatic infected participants were compared. G. Hierarchical clustering heatmap of genes associated with CCNA2 identified by linear regression analysis (FDR<5%). Gene expression profiles of PBMCs from P. falciparum symptomatic and asymptomatic infected participants were compared. H. Spearman correlation matrix (Benjamini-Hochberg adjusted FDR<5%) between parasitemia levels and immunoregulatory genes differentially expressed between symptomatic and asymptomatic P. falciparum-infected individuals. Significant positive correlations are shown in blue and significant negative correlations are shown in red.
Figure 7(A) Western blot of whole adult human retina extracts incubated with an anti-human COCO antibody and revealing a unique band at ~36 kDa (arrow). Recombinant human COCO was used as positive control. (B) Western blot of photoreceptors produced from human embryonic stem cells incubated with an anti-mouse COCO antibody and revealing a unique band at ~36-38 kDa (arrow). Western blots are representative of 3 independent experiments. (C) Immunofluoresence analysis of adult human retina sections with an anti-mouse COCO antibody. Specific immuno-reactivity was observed in multiple areas (arrows) when compared with sections only exposed to the secondary antibody. (D) RNAscope in situ hybridization (Dand5, top; Negative probe (dapB); down) and hematoxylin staining of P5 mouse retinas. (E) Dual RNAscope in situ hybridization and visual-arrestin immunohistochemistry of P5 mouse retinas. Images are representative of 4 animals. (F) Immunofluoresence analysis of HUVECs exposed to COCO for 5 hrs prior to fixation. Exogenously added COCO was detected using an anti-human COCO antibody, showing co-localization with human mitochondria (MTCO1 antibody).
Figure 3Diagrams detailing NUP58 mutations in mutant HAP1 clones. Nucleotide and amino acid sequences of exons 4 and 13 surrounding the gRNA target site are shown for wild-type (WT) and NUP58 mutant clones. Insertions are highlighted as closed red triangles and deletions as open dotted triangles over the nucleotide sequence. Amino acid substitutions and PTC resulting from nonsense mutations are indicated in red. A large-scale deletion (largescale del) present in C2 and affecting part of intron 3-4, exon 4, intron 4-5, and part of exon 5 is represented with dashed lines. Representation of the highest growth rates (Vmax) for each technical replicate for each indicated cell line. Each dot represents a technical replicate (WT n=35, C1 n=27, C2 n=28, C3 n=34, C4 n=32, C5 n=35, C6 n=38) of one independent experimental replicate performed. Analysis of active nuclear import using an NLS-3XmCherry as reporter as described in Figure 2C-D. Dots represent analysed cells (WT n=22, C1 n=15, C2 n=15, C3 n=18, C4 n=20, C5 n=27) of one independent experimental replicate performed.
Figure 6Model of T6SS dynamics. After membrane complex has formed, TsaC assists in recruitment of baseplate components and sheath-tube copolymerization. Once extending sheath-tube has reached opposing cell membrane, TsmA replaces TsaC at the distal end and holds it in place. Shortly after TsmA disappearance, sheath contracts and releases spike-tube to the environment. Tube components are then recycled by the ClpV ATPase. TsaB localizes at the baseplate and assists in the initiation of T6SS biogenesis.
c-Jun activity up-regulation causes resistance to apoptosis in quiescent cells because of incomplete pS6 suppression.Figure 5Covariate single-cell analysis of Ki-67 (left) and pRb(Ser807/811) (right) versus p-cJun(Ser73) in WM1552C cells before and 24 h after exposure to 0.8 μM vemurafenib. Density scatter plots were generated using signal intensities for individual cells as measured by immunofluorescence microscopy. The vertical lines were used to gate p-cJunHigh versus p-cJunLow cells. The horizontal lines were used to gate Ki-67High versus Ki-67Low cells, and pRbHigh versus pRbLow cells. Analysis of drug dose-dependent changes in proportion of pRbLow/p-cJunHigh and pRbHigh/p-cJunHigh subpopulations in four melanoma cell lines (WM115, WM1552C, LOXIMVI, COLO858) after exposure to vemurafenib for 24 h. These subpopulations were gated as shown in (A). Data are represented as mean ± SD for two replicates Covariate single-cell analysis of pRb(Ser807/811) versus pS6(Ser235/236) following 24-h treatment of WM1552C cells with different doses of vemurafenib alone (top) and vemurafenib and JNK-IN-8 together (bottom). Drugs were added at a 1:1 ratio, each at indicated concentrations when used in combination. Density scatter plots were generated using signal intensities for individual cells as measured by immunofluorescence microscopy. The horizontal and vertical lines were used to gate pS6High versus pS6Low cells, and pRbHigh versus pRbLow cells, respectively. Selected immunofluorescence images of pS6(Ser235/236) and Hoechst staining in WM1552C cells in a DMSO-treated control and 24 h after exposure to 5 μM of vemurafenib, JNK-IN-8, and their combination. Analysis of the changes in proportion of pS6Highcell population in three melanoma cell lines (LOXIMVI, WM115 and WM1552C) as a function of drug concentration for single-drug vemurafenib and JNK-IN-8 treatments and their combination treatment. Drugs were added at a 1:1 ratio, each at indicated concentrations when used in combination. pS6High population of cells was gated as indicated in (C). Data are represented as mean ± SD for two replicates. Data comparison between vemurafenib treatment and vemurafenib/JNK-IN-8 combined treatment was made by using two-way analysis of variance (ANOVA). Fraction of c-JunHighcells (as measured by single-cell (immunofluorescence microscopy) after 48 h JUN knockdown followed by 24-h treatment with vemurafenib. Fold changes are shown relative to control-treated cells. Data are presented as mean ± SD. Single-cell pS6(Ser235/236) levels in the c-JunHigh and c-JunLow fractions of cells (as measured by single-cell multiplex immunofluorescence microscopy) after 48 h of JUN knockdown and 24-h treatment with 0.32 μM vemurafenib. Single-cell pS6 data are presented as box-and-whisker plots with median signal intensities and interquartile ranges; bars extending to 1.5× the interquartile range are shown for each condition as a measure of variance. P-values were calculated using a two-sided nonparametric Mann-Whitney U-test.