EMBO/sd-character-level-ner huggingface dataset:
Figure 4(A) Knockdown of one CCT complex member results in depletion of other CCT subunits. Following siCCT4 or siCCT7 transfection, cell lysates were evaluated by immunoblot for expression of additional CCT subunits by endogenous antibodies for CCT1, CCT4, CCT5, and CCT7. GAPDH was used as a loading control. (B) Decreased cell viability upon CCT depletion and A3A expression. CCT4 (left) and CCT7 (right) subunits were depleted by siRNA transfection in U2OS cells with dox-inducible A3A transgenes. Immunoblot analysis of cell lysates showed decreased expression of targeted CCT subunit, but no alteration in A3A expression. Scr indicates non-targeted siRNA, used as control. Ku-86 is a loading control. Following siRNA transfection to deplete CCT subunits, U2OS-A3A cells were treated with dox. Viability was determined by colorimetric change after addition of a water-soluble tetrazolium salt. Percent viability was normalized to untreated controls. Statistical analysis was performed using a paired two-tailed t-test, n=3 biological replicates; error bars, SEM. (C) Increased cell death resulting from A3A expression along with depletion of CCT. U2OS-A3A cells were depleted of CCT4 by siRNA, treated with dox, or combination. Cell death was measured by staining cells with fluorescent labeled calcein AM (live) and DNA (dead) stains. Bar chart shows quantitation of FACS results averaged over three biological replicates. Statistical analysis was performed using a paired two-tailed t test, n=3; error bars, SEM. (D) Viability of cells with catalytically inactive A3A. U2OS-A3A-C106S cells were induced with dox to express the catalytically inactive A3A mutant (C106S), depleted of CCT4 by siRNA, or combination. Cells were subsequently evaluated by live-dead staining assessed by FACS. Bar chart shows quantitation of FACS results averaged over three biological replicates. Statistical analysis was performed using a paired two-tailed t-test, n=3; error bars, SEM. Immunoblot analysis of cell lysates showed decreased expression of CCT4 after siRNA targeting. Ku-86 is a loading control. (E) Increased DNA damage signaling upon CCT depletion and A3A expression. Depletion of CCT7 in K562-A3A cells was achieved by siRNA targeting. Expression of A3A was induced by dox treatment. Intracellular staining with anti-γH2AX antibody was analyzed by flow cytometry. Bar chart shows quantification of FACS results obtained over three biological replicates. Statistical analysis was performed using a two-tailed t-test, n=3; error bars, SEM. Immunoblot analysis of cell lysates showed decreased expression of CCT7 after siRNA targeting and A3A expression upon dox treatment. Ku-86 is a loading control. **p-value <0.01, ***p-value <0.01, ****p-value <0.0001, n.s. non-significant.
Figure 2(A) Confocal fluorescent microscopy of SupT1 T cells transduced with Tet-inducible pQCXIP-FLAG-IFITM3 or pQCXIP-FLAG-IFITM3 Δ1-21 (Tet-ON) immunostained with anti-FLAG M2 antibody following overnight treatment with 500 ng/mL doxycycline. Scale bar, 10 μm. (B) Tet-ON SupT1 cell lines were treated or not with 500 ng/mL doxycycline overnight and induction of IFITM3 protein was assessed anti-FLAG M2 immunostaining and flow cytometry. (C) Tet-ON SupT1 cell lines were productively infected with NL4-3 VSV-G and then treated with 500 ng/mL doxycycline overnight to produce virus from IFITM3- and IFITM3+ cells. 25 ng p24 equivalents of purified were used to infect fresh Tet-ON SupT1 cells cell targets, which were previously treated with 500 ng/mL doxycycline or not. Infection was scored at 72h post-infection by immunostaining with KC57 and flow cytometry. Mean + SD of 3 experiments are shown. (D) 25 ng p24 equivalents of virus used in (C) was subjected to SDS-PAGE. Immunoblotting was performed using the anti-FLAG M2, anti-p24 Gag, and anti-gp120 Env (NIH #288) antibodies. (E) 293T cells were transfected with the indicated amount of pQCXIP-FLAG-IFITM3 or pQCXIP-FLAG-Δ1-21 or pCMV-MxA and 1.0 μg of HIV-1 pNL4-3. SDS-PAGE was performed on whole cell lysates followed by immunoblotting with anti-FLAG M2, anti-p24 Gag, anti-Env gp120 (NIH #288), and anti-tubulin antibodies. Western blot images are representative of 3 independent experiments. Unpaired t-test, *p<0.05, ♯p=0.12, non statistically significant. Comparisons were made between the condition indicated and the correponding condition in Tet-ON IFITM3 cells.
Identification of MOSPD2, a new FFAT motif-binding protein A: Sequence of the two peptides used for the pull-down assay.Figure 1 A: Sequence of the two peptides used for the pull-down assay. The peptides are composed of an amino-terminal biotin, a linker sequence and a random sequence (control peptide) or the FFAT sequence of ORP1 (FFAT peptide). The FFAT sequence of ORP1 is in bold and corresponds to residues 469-483 (Accession number Q9BXW6-1). Aromatic and acidic residues are in blue and red, respectively  B: Silver nitrate staining of proteins pulled-down using the control peptide (left) or the FFAT peptide (right), after SDS-PAGE. The two major differential bands are highlighted by arrows  C: Tandem mass spectrometry result table showing the three top scored proteins identified in the FFAT peptide bound fraction. Score: protein score based on the sum of the ion scores of all peptides identified; Cov.; percentage of the protein sequence covered by identified peptides; Pep.: number of unique peptide sequences identified  D: Schematic representation of MOSPD2, VAP-A, and VAP-B. Numbers correspond to the predicted positions of the beginning and the end of each domain. Calculated molecular weights (MW) of the proteins are shown  E: Western blot analysis of proteins pulled-down using the control (Ctrl) peptide (left) or the FFAT (FFAT) peptide (right). The Input, FT (flow-through), and Elution fractions correspond to HeLa cell total protein extract, unbound proteins and bound proteins, respectively. Representative illustration of at least two independent experiments
Sporozoite surface antigen induces a higher CD8+ T cell response than EEF vacuolar membrane antigen in the spleen and liver.Figure 3(A) Schematic overview of experimental design. C57BL/6 mice received 2x106 OT-I cells alone (n=2-4) or were additionally immunised with 10,000 γ-radiation attenuated WT (n=3-6), CSPSIINFEKL (n=4) or UIS4SIINFEKL (n=4) sporozoites intravenously. See Appendix Table S2 and S3 for the number of mice used for pentamer staining and peptide restimulation. Spleens and livers were harvested either at day 14 or day 42. (B Proportions and numbers of (B- Kb-SIINFEKL+ CD8+ T cells were enumerated, or (E- IFN-γ-secreting CD8+ T cells following restimulation ex vivo with SIINFEKL peptide were quantified. Flow cytometry plots show representative percentages of CD8+ T cells co-stained with CD11a and (B) Kb-SIINFEKL or (E) IFN-γ. Proportions and numbers of Kb-SIINFEKL+ CD8+ T cells were enumerated, or IFN-γ-secreting CD8+ T cells following restimulation ex vivo with SIINFEKL peptide were quantified. The upper panel of bar charts (C show the percentage of co-stained CD8+ T cells and the lower panel (D the absolute cell counts. Bar charts show mean values (±SEM) from representative experiments (*, p<0.05; **, p<0.01; ***, p<0.001; one-way ANOVA with Tukey's multiple comparison test). See Appendix Table S4 for exact p-values. Proportions and numbers of IFN-γ-secreting CD8+ T cells following restimulation ex vivo with SIINFEKL peptide were quantified. The upper panel of bar charts F) show the percentage of co-stained CD8+ T cells and the lower panel G) the absolute cell counts. Bar charts show mean values (±SEM) from representative experiments (*, p<0.05; **, p<0.01; ***, p<0.001; one-way ANOVA with Tukey's multiple comparison test). See Appendix Table S4 for exact p-values.
Figure 1A RNF123 suppresses the SeV- and EMCV-induced but not the cGAS and STING-mediated activation of the IFN-βpromoter. HEK293T cells (3×105) were transfected with the IFN-β reporter plasmid (100 ng), the pRL-TK Renilla luciferase plasmid (100 ng), the expression plasmid for cGAS (50 ng) and STING (50 ng) or empty vector (100 ng), and 0, 20, 50, or 100 ng (wedges) of RNF123 for 12 h, and then were infected with SeV or EMCV or left uninfected. Luciferase assays were performed after 18 h and results are presented relative to the luciferase activity in control cells. B RNF123 inhibits the SeV-, but not the cGAS and STING-induced activation of the NF-κB promoter. HEK293T cells (3×105) were transfected with the NF-κB reporter plasmid (100 ng), the pRL-TK Renilla luciferase plasmid (100 ng), the expression plasmids for cGAS (50 ng) and STING (50 ng), or empty vector (100 ng), and 0, 20, 50 or 100 ng (wedges) of RNF123 for 12 h, and were then infected for 24 h with SeV or left uninfected, followed by analysis as described in A. C, D RNF123 inhibits the SeV-induced, but not the cGAS and STING-mediated RANTES and IFN-β transcription. The transfection and treatment were as in A. Total RNA was isolated and RT-PCR (C) was performed using the indicated primers. C, D RNF123 inhibits the SeV-induced, but not the cGAS and STING-mediated RANTES and IFN-β transcription. The transfection and treatment were as in A. Total RNA was isolated and Q-PCR (D) was performed using the indicated primers. E RNF123 inhibits the SeV-induced, but not the cGAS and STING-mediated IFN-β secretion. The transfection and treatment were as in A. Bioassays for type I interferon in the supernatants of HEK293T cells were performed and the results are presented relative to untreated control cells. F Overexpression of RNF123 enhances NDV-eGFP replication. HEK293T cells (1×105) were transfected with the expression plasmids for Flag-RNF123 or empty vector (2 µg). At 24 h after transfection, cells were infected with NDV-eGFP. At 30 h after infection, viral replication was determined by GFP expression visualized by fluorescence microscopy and analyzed by western blot. G RNF123 enhances EMCV replication. HeLa cells (1×105) were transfected with the expression plasmids for Flag-RNF123 or empty vector (2 µg). At 24 h after transfection, cells were infected with EMCV or given DMEM with no virus. At 18 h after infection, viral replication was determined by VP1 expression, which was analyzed by western blot.
Figure 2Images and comparison of VE-cadherin LEC junctions in lacteals of jejunum between vehicle- and ABX-treated mice. Each boxed portion is highly magnified for visualization of detailed distributions of VE-cadherin to distinguish button-like (red arrowheads), zipper-like (open arrowheads) and mixed LEC junctions in lacteals (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 50 μm. Representative intravital imaging and quantification of lipid clearance from lamina propria via lacteals. Asterisks indicate the site in lamina propria where fluorescence intensity (FI) was measured. Relative FI divided by mean FI of controls at 16 min (left) and normalized FI divided by FI at 16 min in each villus (right) after initial loading of BODIPY-FA were quantified. For normalization of FI, villi that showed peak intensity earlier or later than 16 min were excluded (n = 5-7 mice/group, 5-10 villi/mouse). Scale bars, 100 μm. Representative images from transmission electron microscopic examination of lacteals and quantification of frequency of open junctions (n = 4 mice/group, 4-6 images/mouse). Jejunum of vehicle- and ABX-treated mice were harvested 2 hours after oral lipid loading. Note that the junction between LECs (red colored bidirectional arrows) is open in vehicle-treated mice, but not in ABX-treated mice. CM, chylomicron; IS, interstitium of lamina propria; LC, large caveola; Lu, lacteal lumen; Ve, vesicle containing lipoproteins. Scale bars, 1 μm. quantification of frequency of open junctions (n = 4 mice/group, 4-6 images/mouse). Note that the junction between LECs (red colored bidirectional arrows) is open in vehicle-treated mice, but not in ABX-treated mice. Diagram for ABX treatment and sampling of systemic blood at indicated time points after oral loading of corn oil into C57BL/6 adult mice. Comparisons of serum triglyceride (TG) and free fatty acids (FFA) at indicated time points in vehicle- and ABX- treated mice (n = 5-7 mice/group).
Figure 5A. Ingenuity Pathway Analysis of differential gene expression (fold-change (FC) > 1.5, p < 0.05) in wt and Sharpincpdm MSFs (passage 0). The differentially expressed genes implicated in the most significantly altered canonical pathways (leukocyte adhesion and transmigration, and tissue fibrosis) are shown. Three independent cell isolation replicates were compared pairwise. FC colour scale represents a range from 0−4. B. Analysis of collagen degradation on cross-bow-shaped micropatterns coated with the DQ collagen substrate. Mean intensity projection images of the DQ collagen fluorescence (top panel; cell area is outlined by dashed line; original integer range 0 − 255) and single confocal images of the fibrinogen-Alexa647-marked micropatterns (bottom panel) are shown (n ≥ 53 from four independent experiments). Scale bars represent 10 μm. C-D. Collagen plug contraction assay using wt and Sharpincpdm MSFs or wt MSFs with SHARPIN siRNA silencing C. Representative light microscopy images of the collagen plugs three days post cell culture. D. Quantification of the average collagen plug area 1−3 days after seeding (n = 3-6). Scale bars represent 2 mm. E. SHARPIN protein level after silencing with negative control siRNA or SHARPIN siRNA in wt MSFs. Tubulin was used as loading control. F-I. Traction force microscopy analysis of wt and Sharpincpdm MSFs on collagen-coated matrices. F. Representative images of traction forces observed in wt and Sharpincpdm cells (colour scale units: Pa; cell boundaries are indicated by dashed lines). G. Quantification of strain energy per cell (n = 8 − 11 cells from two independent experiments). F-I. Traction force microscopy analysis of wt and Sharpincpdm MSFs on collagen-coated matrices. H. Representative images of the traction forces and mCherry fluorescence of mCherry-Ctrl transfected wt cells and mCherry-Ctrl or mCherry-SHARPIN transfected Sharpincpdm cells. Scale bars represent 20 µm. I. Quantification of strain energy per cell (wt mCherry control n = 26; Sharpincpdm mCherry-Ctrl n = 21; Sharpincpdm mCherry-SHARPIN n = 24 cells from five independent experiments). J-K. Focal adhesion dynamics. J. Visualization of GFP-Paxillin dynamics in a wt and a Sharpincpdm cell over time. Colour scale represents a range from early to late occurring focal adhesions. K. Quantification of focal adhesion average size, assembly rate and disassembly rate (wt n =17527, 5250, 5311 FAs; and Sharpincpdm n= 9052, 2790, 3264 FAs, respectively from two independent experiments). Mean ± SEM.
Incorporation of cGAMP within VLPs is essential for its protective effect.Figure 6A, cGAMP-HA-VLPs and Empty-HA-VLPs + AddaVax protect against IAV challenge. One month after immunisation, animals were infected with 104 TCID50 of IAV PR8 virus. Weight loss was monitored over the following eleven days and is shown as a percentage of starting weight (A, upper graph shows mean and lower graphs show individual mice for each condition). B. cGAMP-HA-VLPs and Empty-HA-VLPs + AddaVax protect against IAV challenge. One month after immunisation, animals were infected with 104 TCID50 of IAV PR8 virus. Animals approaching the humane end-point of 20% weight loss were culled and survival to end-point curves are shown in (B). C, D. cGAMP-HA-VLPs and Empty-HA-VLPs + AddaVax induce neutralising antibody responses that accompany protection. Three weeks after immunisation with 5x104 IU of the indicated HA-VLPs, sera were collected and heat-inactivated. Titres of antibodies capable of neutralising an IAV expressing a matched HA protein were determined by micro-neutralisation (MN) assay (C). The dotted line shows the limit of detection (LOD). Correlation between the MN titers at week 3 and the weight loss at day 4.5 post-challenge is shown in (D).
transcription by sequestrating miR-200s from binding to the 5'-UTR of the ZNF596 gene A and B.Figure 6Expression of shRNA-resistant LINC00115 WT but not mutant or empty vector (EV) rescued LINC00115 shRNA-inhibited ZNF596 mRNA (A) expression in 1123 GSCs. Expression of shRNA-resistant LINC00115 WT but not mutant or empty vector (EV) rescued LINC00115 shRNA-inhibited ZNF596 protein (B) expression in 1123 GSCs. C. Schematics of the predicted binding sites of miR-200s on ZNF596 sequence. ZNF596-5'-UTR-mut contains a mutated sequence in the miR-200 binding site. D. MS2-RIP followed by qRT-PCR for endogenous microRNAs association with ZNF596. 12XMS2 empty vector, ZNF596-5'-UTR-WT-12XMS2, or ZNF596-5'-UTR-mut -12XMS2 with Flag-tagged MS2 were co-transfected into 1123 GSCs. E. Schematics of ZNF596 wild type (ZNF596-WT) and miR-200 binding site mutant (ZNF596-mut) promoter. F. Luciferase activity in 1123 GSCs co-transfected with miR-200s and luciferase reporters containing ZNF596 5'-UTR WT, mutant, or empty vector (EV).
a, Confocal micrograph of HeLa cells expressing GFP–LC3 and mCherry–galectin-8, stained for NDP52 1 h after infection with S.f4a, Confocal micrograph of HeLa cells expressing GFP-LC3 and mCherry-galectin-8, stained for NDP52 1 h after infection with S. Typhimurium. The lower right panel contains a fluorescence line scan along the yellow line in the merge inset. Arrowheads, bacteria shown in insets. b, Percentage of GFP-LC3-positive S. Typhimurium at 1 h p.i. in HeLa cells treated with the indicated siRNAs. c, Percentage of bacteria positive for NDP52. HeLa cells expressing the indicated NDP52 variants fused to YFP were infected with S. Typhimurium. d, Percentage of bacteria positive for the indicated markers. HeLa cells, either wild type or expressing YFP-galectin-8 as indicated, were infected with S. Typhimurium. Ubiquitin was detected by antibody staining. e, HeLa cells treated with the indicated siRNAs were infected with S. Typhimurium and stained for NDP52 at the indicated time points. Fluorescent bacteria were counted by microscopy at the indicated time points. Graphs, representing at least two independent repeats, show mean and s.d. of duplicate coverslips for which >200 bacteria were counted. *P  0.05, Student's t-test. Scale bar, 10 µm.
Colony stimulating factor (CSF-1) and macrophage percentages increase in response to irradiation.Figure 1A, MC38 cells in tissue culture were treated with mock (0Gy) and 10Gy irradiation. Conditioned medium (CM) was collected after 48 hours and assayed for the indicated cytokines. Fold-changes in the amounts of the cytokines are shown. B, CSF-1 mRNA expression was measured in MC38 and KPC cells exposed to 10Gy IR compared with mock irradiated cells. Cells were harvested at 24, 48 and 72 hours after irradiation and RNA expression was analysed by RT-qPCR. Data are presented as mean ± SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison (n=3). C, CSF-1 protein (pg/mg total protein) in CM collected from MC38 and KPC cells 48 hours after exposure to 10Gy IR compared to mock-irradiated cells. Data are presented as mean ± SEM and analysed by Mann-Witney (n=3). D, CSF-1 protein (pg/mg total protein) measured by ELISA in the sera of naïve mice, mice bearing mock-irradiated tumours, and mice bearing irradiated tumours analysed at time points as indicated (n=4/group). Data are presented as mean ± SEM and analysed by Kruskal-Wallis test with Dunn's multiple comparison. E-F, MC38 (E) and KPC (F) tumours were irradiated with 10Gy. Average tumour volume (red line) is shown with mean TAM infiltrate (blue line) for each time point. For TAM quantification, tumours were disaggregated and CD11b+/F480+ TAMs identified by flow cytometry. Data are presented as mean ± SEM for TAMs and SEM for tumour volume (n=6). G-H, Immunofluorescent staining of MC38 (G) and KPC (H) tumour sections; blue=DAPI, green = CD68 (TAMs). I-J, Flow cytometric analysis of immune cell populations within MC38 (I) and KPC (J) tumours 5 days following 10Gy IR compared to mock-irradiated tumours. Macrophages (CD11b+ F480+), neutrophils (CD11b+ Ly6G+), myeloid-derived suppressor cells (CD11b+ Gr1+), CD8 T cells (CD45+ CD3+ CD8+), CD4 T cells (CD45+CD3+CD4+, Natural Killer cells (CD45+ NK1.1+) were identified. Pie charts represent the proportion of CD45+ leukocytes out of the total cells.
binds active genomic loci and demarcates a subset of TAD and loop domains.Figure 2Genome browser view of HMGB1 ChIP-seq (normalized to input; mean from two replicates) from proliferating HUVEC (red) or proliferating/senescent IMR90 (grey/green) in the TMEM92 locus. Bar graphs showing the genomic distribution of HMGB1 ChIP-seq peaks in HUVEC and IMR90. The number of peaks (N) analyzed per each cell type is indicated. Venn diagram showing HMGB1 ChIP-seq peaks shared between proliferating HUVEC (red) or proliferating/senescent IMR90 (grey/green) data. *: P<0.001; more than expected by chance, hypergeometric test. Exemplary Hi-C heatmap for a subregion of IMR90 chr6 aligned to HMGB1 ChIP-seq; peaks at TAD boundaries (orange lines) are indicated (red arrowheads). Line plots showing normalised HMGB1 (red) and CTCF ChIP-seq signal (dark grey) along TADs ±20 kbp from proliferating IMR90. Line plots showing normalised HMGB1 (red) and CTCF ChIP-seq signal (dark grey) in the 160 kbp around HMGB1- (left) or CTCF-marked TAD boundaries (right). Line plots showing mean normalized insulation scores calculated from proliferating (black) and senescent IMR90 Hi-C data (blue) in the 240 kbp around HMGB1 peaks residing at TAD boundaries (top) or not (bottom). Aggregate plots showing 20 kbp-resolution Hi-C signal for proliferating (top), senescent-specific (bottom) or shared loops (middle). The number of loops in each category (N) is indicated. Line plots showing normalised HMGB1 (red) and CTCF ChIP-seq signal (grey) along proliferating (top) or shared loop domains (bottom) ±50 kbp from proliferating IMR90. The percentage of HMGB1 peaks residing at loop anchors is indicated. Box plot (left) showing significantly up/downregulated IMR90 genes harboured inside the 607 loops from panel H. The number of genes in each group (N) is indicated; center lines represent medians, box limits indicate the 25th and 75th percentile, whiskers extend 1.5 times the 25th-75th interquartile range, and outliers are represented by dots. Bar plots (right) show Go terms associated with these up/downregulated genes.
TBR expression inhibits autophagy at an early stage.Figure 5A) HEK293A cells expressing GFP, GFP-TBC1D14 224-669 or GFP-TBR were treated in duplicate with EBSS, EBSS plus 100 nM BafA1 or not for two hours, lysed and subjected to immunoblotting for LC3B, tubulin and GFP. The amount of LC3B/tubulin for each condition from three independent experiments is shown on the bar graph, ±s.e.m. * p<0.05, ** p<0.01, one way ANOVA with Sidak's multiple comparison test. B) HEK293A GFP-LC3B (2GL9) cells were transfected with mCherry or mCherry-TBR, treated with EBSS for two hours, fixed and analysed by confocal microscopy. C) HEK293A cells were treated as in (B), fixed, stained for WIPI2 and analysed by confocal microscopy. D) HEK293A cells stably expressing GFP-DFCP1 were treated as in (B), fixed and analysed by confocal microscopy. Scale bars = 20 μm. For quantification of (B), (C) and (D), 10 fields of view containing transfected cells were imaged for each of three experiments, and the number of GFP-LC3B, WIPI2 or GFP-DFCP1 puncta per cell enumerated using Imaris software (Bitplane).