EMBO/sd-character-level-ner huggingface dataset:
Figure 6A. Chemical inhibition of Hsc70 activity in HEK293T cells overexpressing FLAG-DnaJC5 reduces extracellular tau levels as shown by dot blot. Intracellular levels are shown by Western blot, DnaJC5 was detected by FLAG antibody. Quantification of extracellular tau levels is shown as average ± SEM, n=3, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. B. Chemical inhibition of Hsc70 activity in HEK293T cells overexpressing FLAG-DnaJC5 reduces extracellular α-synuclein levels as shown by dot blot. Intracellular levels are shown by Western blotDnaJC5 was detected by FLAG antibody. Quantification of extracellular synuclein levels is shown as average ± SEM, n=3, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. C. Dominant-negative Hsc70 E175S blocks tau release when overexpressed in HEK293T cells as shown by dot blot. Intracellular levels are shown by Western blot. Quantification of extracellular tau levels is shown as average ± SEM, n=4, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. D. Knockdown of Hsc70 reduces extracellular tau release (dot blot) in HEK293T cells overexpressing FLAG-DnaJC5. Intracellular levels are shown by Western blot, DnaJC5 was detected by FLAG antibody. Quantification of extracellular tau levels is shown as average ± SEM, n=3, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. E. A mutant tau that does not bind Hsc70 is not released when FLAG-DnaJC5 is overexpressed in HEK293T cells. Dot blot and quantification of extracellular tau levels are shown. Intracellular protein levels are shown with representative Western blot, DnaJC5 was detected by FLAG antibody. Data are mean ± SEM, n=3 p<0.001. one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. F. Dot blot of Hsp70 isoform overexpression effect on extracellular tau release in HEK293T cells. Intracellular protein levels are shown with representative Western blot. HSPA variants were detected with V5 antibody. Data are mean ± SEM, n=3 p<0.01 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. G. Dot blot of different FLAG-DnaJ overexpression effect on extracellular tau release in HEK293T cells. Intracellular protein levels are shown with representative Western blot. DnaJ levels were detected by FLAG antibody. Data are mean ± SEM, n=3; *** p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. H. Dot blot of increasing FLAG-DnaJC7 levels on FLAG-DnaJC5 overexpression-mediated extracellular tau release in HEK293T cells. Intracellular protein levels are shown with representative Western blot, DnaJ proteins were detected by FLAG antibody. Quantification of extracellular levels are mean ± SEM, n=4. I. Schematic representation of Hsp70 control of DnaJC5-mediated tau release. When clients are unable to bind Hsc70, or ifHsc70 is inactive or knocked down, client proteins are sent for degradation instead of release. Thus, Hsc70 is important for upstream control of the DnaJC5-mediated extracellular release.
Figure 6A Representation of METTL5's role in 18S rRNA. Large ribosomal subunit (LSU) and small ribosomal subunit (SSU) of the ribosome are shown in light pink and antique pink, respectively (left). The secondary structure of 18S rRNA in the SSU (middle) was adapted from (http://apollo.chemistry.gatech.edu/RibosomeGallery/).METTL5 (shown in brown) catalyzes A1832 methylation on 18S rRNA in the SSU of the ribosome. B Schematic illustration depicting the regulation of differentiation potential for endoderm (endo), mesoderm (meso) and ectoderm (ecto) by METTL5 via the translational control of FBXW7- c-MYC axis. Dotted arrows indicate inefficient differentiation induction.
Figure 2A. Unique residue pairs mapped to the OCCM complex (PDB|5udb) and the key link Mcm2 - Orc5 (Mcm2-850-Orc5-369). B. The in vitro helicase loading assay demonstrates that an Mcm2 C-terminal deletion mutant supports complex assembly (lanes 6 and 7) and blocks formation of the final helicase loading product (lanes 8 and 9). C. Overexpression analysis of Mcm2-7ΔC2 shows that this mutant causes dominant lethality, indicating that the C-terminus of Mcm2 is essential in cell survival.
THEMIS knock‐down increases TCR‐induced phospho‐ERK in Jurkat 1G4‐CD8 cellsLentiviral knock‐down (KD) of THEMIS expression in 1G4‐CD8 cells.Figure 2A Lentiviral knock‐down (KD) of THEMIS expression in 1G4‐CD8 cells. The efficiency of the THEMIS KD was assessed using immunoblotting. B Phospho‐flow cytometry analysis of pERK responses in THEMIS KD cells. 1G4‐CD8 control and THEMIS KD cells were stimulated with NY‐ESO‐1 pMHC tetramers and analysed for ERK phosphorylation using flow cytometry. Data shown are representative of three independent experiments.C Quantification of pERK‐positive cells from (B). Data from three independent experiments were used in the analysis. n = 3, means ± SEM are shown; two‐tailed unpaired Student's t‐test, *P 0.05, **P 0.01. D Phospho‐flow cytometry analysis of pERK responses against titrated concentrations of 9V tetramer. Time point was fixed at 60 s. Data shown are representative of three independent experiments.E Quantification of pERK‐positive cells from (D). Data from three independent experiments were used in the analysis. n = 3, means ± SEM are shown.
Fig.Figure 1Ex vivo generation of CAR T cells. Activated human PBMC were left untransduced or incubated with CD8-LVCD19CAR at an MOI of 2. Five days later, expression of CD19-CAR and CD8 were determined on CD3+ cells. Numbers indicate the percentage of cells in the respective gate. Experimental outline for in vivo CAR generation. 1x107 human PBMC were engrafted into naïve NSG mice or NSG mice that had been intraperitoneally (i.p.) injected with 5x105 Raji cells (Raji +) six days before. One day later, 2x106 t.u. of CD8-LVCD19CAR (filled circles) or CD8-LVRFP (grey triangles) were i.p. injected, respectively. As further control, another group of mice received PBS (open circles). Seven days later, mice were sacrificed and organs and cells were removed for further analysis. Detection of CAR T cells by vector copy numbers (VCN). Genomic DNA was isolated from peritoneal cavity, spleen and blood cells. VCN were determined in technical duplicates by qPCR for two individual mice of each group. Presence of B cells in the transplanted PBMC is indicated below. Cells isolated from the peritoneal cavity (peritoneum), spleen, or blood were evaluated by flow cytometry for the percentages of human CD8+ in CD3+ cells (D), within the fraction of human CD45+ cells. Representative density plots are shown for the peritoneal cells. Cells isolated from the peritoneal cavity (peritoneum), spleen, or blood were evaluated by flow cytometry for the percentages of CAR+ or RFP+ cells in the CD8+ and CD8- fractions, respectively (E) within the fraction of human CD45+ cells. Representative density plots are shown for the peritoneal cells. Cells isolated from the peritoneal cavity (peritoneum), spleen, or blood were evaluated by flow cytometry for the percentages of human CD19+ cells within the fraction of human CD45+ cells. Representative density plots are shown for the peritoneal cells. Mice were transplanted with B-cell depleted human PBMC and then received CD8-LVCD19CAR (filled circle) or PBS (open circle). As control, CD8-LVCD19CAR or PBS were injected into mice transplanted with non-depleted PBMC. Data represent mean ± SD for all groups (CD8-LVCD19CAR: n=6 in D, n=4 in G; CD8-LVRFP: n=4; PBS: n=4 in G, all others n=3).
RNA interference targeting BIP or CHOP blocked sodium butyrate induced autophagy in colorectal cancer cells.Fig 9  HCT-116 cells were transfected with BIP or CHOP specific siRNAs for 48 h and then treated with or without 2mM sodium butyrate (NaB) for 24 h. Negative control (NC) scramble siRNA was used the negative control for the transfection. Representative Western blots are shown for BIP siRNA (A) and CHOP siRNA (C). The expression level of each protein was determined by densitometry and normalized to GAPDH (ratio of protein:GAPDH). (B) Normalized expression levels of BIP and LC3-II in HCT-116 cells treated with BIP specific siRNA. (C) Normalized expression levels of CHOP and LC3-II in HCT-116 cells treated with CHOP specific siRNA. Means and standard deviation (SD) of three independent experiments are shown. One-way ANOVA was used for statistical analysis. * P<0.05, ** p<0.01, compared to the respective control group.
Fig.Figure 6(a) The scheme of three batches of just-weaned mice (18 mice per batch) were fed with a chicken, corn, or mixture of corn and chicken diet for 5.5 months, representing a carnivore diet, an herbivore diet, and an omnivore diet, respectively The expression levels of GPR15, C10ORF99, FOXP3 in mice fed with corn, chicken, and mixture of corn plus chicken, commercial food chow and a positive control (propionate added to commercial food chow) at different times (weanling mice were defined as w-1) in colonic tissue were determined by q-RT-PCR. Each symbol or bar represents pooled RNA from colon tissues from three mice. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA). The expression levels of IL-10 in mice fed with corn, chicken, and mixture of corn plus chicken, commercial food chow and a positive control (propionate added to commercial food chow) at different times (weanling mice were defined as w-1) in colonic tissue were determined by q-RT-PCR. Each symbol or bar represents pooled RNA from colon tissues from three mice. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA). (f, g) SCFAs and acetic acid content in dietary experiments. Faecal samples from mice on the indicated diets were analysed for SCFAs by gas chromatography. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA). (h) TNF-α and IL-6 levels in faecal samples from mice on the indicated diet demonstrated no colitis in all mouse groups. IL-6 and TNF-α protein production levels, as determined by performing enzyme-linked immunosorbent assays. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA). (i) Luciferase activities of constructs with the mouse C10ORF99 promoter in HEK293 cells. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA). Luciferase activities of constructs with the Japanese quail C10ORF99 promoter in HEK293 cells. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).
Autophagic and lysosomal markers localize to RVs in IBMPFD tissue.Figure 6.(A) Immunofluorescence images of quadriceps muscle using p62 and Lamp1 or -2 antibodies. Quadriceps section from a 9-mo-oldVCP-WT mouse, age-matched VCP-RH transgenic, 12-mo-oldVCP-RH transgenic, and IBMPFD patientmuscle biopsy are shown. Open arrows highlight p62 and Lamp1/2 colocalization, and closed arrows show regions of only p62immunofluorescence. Single myofibers containing RVs are outlined in the merge panels. (B and C) Transmission electron microscopy of tibialis anterior muscle from one of two independent lines (B shows RH12, and C shows RH9 lines) of 12-mo-old VCP-RH-expressing transgenic mice. Note the large vacuolated structures that are perinuclear or subsarcolemmal. Bars: (A) 30 µm; (B and C) 500 nm.
Figure 5A Binary food choice assays were conducted after GRNs were ablated by expressing the cell death gene (hid), under Gr28b.c-GAL4 (n = 4). B Tip recording analysis of the indicated S-type sensilla and genotypes (n ≥ 7). The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis to compare two sets of data (**P < 0.01). C Expression of the Gr28b.c-GAL4 reporter in subsets of Gr66a-positive GRNs. First panel shows that all bitter responsive GRNs are labeled by Gr66a-I-GFP (anti-GFP, green). Second panel shows the expression of the Gr28b.c-GAL4, crossed with UAS-DsRed (anti-DsRed, red). Third panel shows merged image of first and second panels. Forth panel shows a schematic diagram of sensilla arrangement. Red-marked sensilla indicate the expression of Gr28b.c-GAL4 reporter in second panel. Scale bars represent 40 μm.
Figure 6A particle-based model of subepidermal blister healing at the basal layer. Epidermal basal cells (colored in blue), which do not divide, are placed on the basement membrane (gray). Stem cells (SCs, green) give rise to progeny (simulating HF-derived cells; red) within epidermal defects or in the surrounding epidermis (IFE-derived cells; yellow). t: arbitrary time. See Movie EV1. Contribution of each progeny cell within the epidermal defect or of surrounding epidermis to subepidermal blister healing, measured as the ratio of the area occupied by each progeny to the area of the initial epidermal defect. A model of subepidermal blister healing without SCs within epidermal defects. See Movie EV2. Time course of subepidermal blister healing in control (A) and SC-depleted epidermal defects (C). Effects of the impaired flattening of keratinocytes upon epidermal regeneration. The diameter of basal keratinocytes (long axis of the spheroid) in the regenerated vs. surrounding epidermis was calculated as 1.5:1 (in contrast to 2:1 in Figure 6A). See Movie EV3. Time course of wound healing for control (A) and less flattened keratinocytes in the regenerated epidermis (E).
The Munc18-1 domain 3a constitutes a minimal template for Syx1 and Syb2.Figure 8A Schematic depiction of a putatively tertiary Munc18-1/Habc/H3 complex. B Munc18-1, Habc and H3 construct a tertiary complex. C The tertiary complex globally resembles the Munc18-1/Syx1 complex. D Formation of the tertiary complex with Munc18-1 QKAA, KKEE, and L348R mutants. KKEE strongly impaired formation of the tertiary complex. E Munc18-1 displays interaction with Syx1-Habc. F QKAA, KKEE, and L348R all retained interaction with Syx1-Habc. G Cartoon illustration of the templating mechanism of domain 3a. The extended domain 3a (H11 and H12) binds to Syx1-H3 and Syb2 simultaneously. Color codes are as follows: Munc18-1, light gray; Munc18-1 H11 and H12, cyan; Syx1-H3, blue; Syb2, red. Key binding sites (L348, K332, and K333) on the H12 are highlighted with orange spheres.
Figure 2Cell lysates were collected from the joints of mice infected with WT CHIKV or CHIKV nsP-mutants at 15 hpi, 2 dpi or 6 dpi (n=5 per group). The concentrations of 36 immune mediators were quantified using a 36-plex microbead-based immunoassay. Immune mediators are grouped based on function. Each color represents the relative concentration of a particular analyte. Blue and red indicates low and high concentration, respectively.
Figure 3(A) Immunofluorescence of SREBP1 in MCF7 parental and PKM2 KO cells by confocal imaging. Scale bar, 5μm. (B) Quantitation of nuclear SREBP1 intensity in A. n>=25 cells as in (A) from three different fields. ( Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05 and ∗∗∗p < 0.001. (C) Western blot analyses of nuclear and cytoplasmic SREBP1 levels in parental and two PKM2 KO clones. PCNA serves as a nuclear protein loading control. (D) Western blot analyses of TMEM33, RNF5, SCAP, SREBP1, and IDI1 in PKM2 KO MCF7 and MDA-MB-231 cells stably transfected with shControl (shCtrl) or shTMEM33 constructs. Arrow indicates nuclear SREBP1, N: nucleus and P: precursor. (E) Cell proliferation assays of the PKM2 KO MCF7 cells stably transfected with shCtrl or shTMEM33 constructs (n=3). shCtrl: shControl. Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05 and ∗∗∗p < 0.001. (F) Cell migration assays of PKM2 KO MDA-MB-231 cells stably transfected with shCtrl or shTMEM33 constructs (n=3). Scale bars, 50µm. (G) Western blot analysis of TMEM33, RNF5, SCAP, SREBP1, HMGCR, LDLR, FDFT1, IDI1, and PKM2 in wild type, PKM2 KO, TMEM33 OE (pool), and two TMEM33OE clones (clones #1 and #8) MDA-MB-231 cells. Arrow indicates nuclear SREBP1. N: nucleus and P: precursor. (H, I) Gene set enrichment analysis of the top enriched gene signatures (H), including (I) Cholesterol homeostasis signature in MDA-MB-231 TMEM33 OE (#1) cells using KEGG and hallmark gene sets in GSEA databases. Adjusted p-value and normalized enrichment score (NES) were generated by GSEA. (J) Volcano plot showing the altered transcripts induced by TMEM33 OE (clone #1) (p value <0.05) and PKM2 KO (p value <0.05) in MDA-MB-231 cells. Green depicts shared down- regulated genes by both TMEM33 OE and PKM2 KO; red depicts shared up-regulated genes by both TMEM33 OE and PKM2 KO. Variance analysis (ANOVA) was used to calculate the p-value. (K) Volcano plot showing protein changes induced by TMEM33 OE (p value <0.05) and PKM2 KO (p value <0.05) in MDA-MB-231 cells. TMEM33 labeled in red dot was upregulated in both TMEM33 OE and PKM2 KO cells and PKM2 labeled in purple had no change in TMEM33 OE cells. ANOVA was used to calculate the p-value.
Figure 7(A) Western blotting of pT236- and total SOX9 following treatment of Daoy with a panel of inhibitors targeting PI3K/AKT/mTOR pathway. The whole cell lysates were collected for gel electrophoresis following 6 hours treatment with 1 μM of AZD5363, 8186, or 2014. The set of blots shown is a representative from 3 independent repeats. (B) Western blotting of endogenous SOX9 protein turnover in the presence of PI3K/mTOR dual inhibitor, AZD2014. Daoy were transfected with either siScr or siFBW7 for 48 hours prior to further treatment with 1 µM AZD2014. The cycloheximide chase of SOX9 protein turnover was examined 2 hours following the inhibitor treatment. Immunobloting of Cyclin E protein was used to indicate the efficacy of siFBW7 transfection. Changes in SOX9 protein level in the representative blots were analysed relative to GAPDH using ImageJ. The set of blots shown is representative from 3 independent experiments. (C) Quantification of resazurin-based Daoy and MB002 cell viability following treatment with Cisplatin (blue bars), AZD2014 (red bars), or their combination (green bars). Daoy were treated for 72 hours, while MB002 were subjected to 5 day course of treatment. The cellviability were calculated relative to the vehicle-treated cells. The synthetic lethality combination index (CI) for each treatment was calculated from the mean of 3 independent experiments using the Compusyn software. Error bars indicate standard deviation. (D) Time course western blotting of endogenous SOX9 and apoptotic markers including cleaved PARP and caspase 3 following MB002 treatment with cisplatin, AZD2014, or their combination. β-actin were used as a loading control. The set of blots shown is representative from 3 independent experiments. (E) Western blotting profiling of MB002 expressing EV, SOX9-WT, or SOX9-T236/240A following 48 hours treatment with cisplatin, AZD2014, and their combination. The set of blots shown is representative from 3 independent experiments. (F) Quantification of cell viability of MB002 expressing EV, SOX9-WT, or SOX9-T236/240A following 5 days treatment with either of 10 μM cisplatin (black bars), 0.5 µM AZD2014 (blue bars), or their combination (red bars). Data are mean + standard deviation from 3 independent experiments, from which statistical significance were analysed by 2-way ANOVA multiple comparisons with Bonferroni's post-test. (G) Immunoblots of ATP7A and DUSP2 protein following doxycycline treatment of Daoy-SOX9/T-FBW7α. The cells were either untreated or incubated with 5 µg/mL doxycycline for 72 hours prior to being harvested for gel electrophoresis. The set of blots shown is representative from 2 independent experiments.
Mother and infants have distinct microbiome compositions not driven by household TC exposure.Figure 2(A) PCoA of Bray-Curtis dissimilarity for all (n=221) samples shows that gut communities cluster by mothers and infants. (B) PCoA separated by time and treatment.
Figure 7(A) Venn diagram showing overlapping genes in analyzed data sets. (B-C) Schematic representations of TFE3 binding region in the ATF4 promoter in MEF cells treated with Tunicamycin (0.1 µg/ml) for 16 h (B) or starved in EBSS for 2 h (C). The distance upstream from the Transcription Start Site (TSS) of a potential E-box sequence is indicated in base pair (bp). (D-E) E-box distance from transcription start site for targets categorized as lysosomal genes in MEF treated with Tunicamycin (0.1 µg/ml) for 16 h (D) or starved in EBSS for 2 h (E). Peaks called as TFE3 binding sites were analyzed for E-box elements using UCSC genome browser and distance from transcription start site assessed. Graphs represent the number of genes with E-box distance (in kbp) from the transcription start site. (F) Relative Quantitative Real-Time PCR analysis of SUMF1, DERL1, ATF6, APEX1 and GPX1 mRNA transcript levels in ARPE-19 cells infected with either adenovirus expressing Null or TFEB-S211A (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, students t-test, **p < 0.01, ***p < 0.001). (G) Immunoblots of protein lysates from ARPE-19 cells infected with either adenovirus expressing Null or TFEB-S211A. Data are representative of three independent experiments.(H) Quantification of IRE1α and APEX1 protein levels in ARPE-19 cells infected as indicated in (G) (mean ± SD of the fold increase of the indicated protein to actin ratio from three independent experiments, students t-test, ***p < 0.001).