EMBO/sd-character-level-ner huggingface dataset:
Figure 6Using CRISPR/Cas9, TLR2KO and AAVS1KO (control) pDCs were generated, cellular DNA was sequenced for ICE analysis Using CRISPR/Cas9, TLR2KO and AAVS1KO (control) pDCs were generated and cells were evaluated functionally by exposure to two different TLR2 agonists; Pam2CSK4 (5 ng/mL, yellow) and Pam3CSK4 (50 ng/mL, orange) Data information: Bars represent mean values and equal symbols represent equal donors (n=4). Statistical significance was determined using the ratio paired student T test for agonist or virus treated cells and compared to the mock treated condition, or by unpaired T test when comparing matched conditions between different KOs. * C-D: Subsequently, AAVS1KO and TLR2KO pDCs were either mock treated (mock, grey), exposed to SARS-CoV-2 (SARS-2, 0.5 MOI, purple) or TLR7/8 agonist (2.5 μg/mL R848, red) and supernatants were collected after 24 hrs to quantify type I IFNα (C) and IL-6 (D) protein concentrations. Data information: Bars represent mean values and equal symbols represent equal donors (n=4). Statistical significance was determined using the ratio paired student T test for agonist or virus treated cells and compared to the mock treated condition, or by unpaired T test when comparing matched conditions between different KOs. * E-F: To investigate if pDCs sensed the spike or envelope SARS-CoV-2 proteins, AAVS1KO and TLR2KO pDCs were exposed to SARS-CoV-2 (SARS-2, 0.5 MOI, purple) recombinant SARS-CoV-2 spike (S, 1 μg/mL, dark green) or envelope (E, 1 μg/mL, light green) proteins and IL-6 (E) and type I IFNα (F) protein concentrations were quantified. Data information: Bars represent mean values and equal symbols represent equal donors (n=4). Statistical significance was determined using the ratio paired student T test for agonist or virus treated cells and compared to the mock treated condition, or by unpaired T test when comparing matched conditions between different KOs. * G-H: Peripheral blood pDCs were isolated from PBMCs by negative selection and exposed to SARS-CoV-2 (1 MOI, purple), TLR7 agonist (2.5 μg/mL R837, blue) agonist, or E protein (1 μg/mL, light green) for 24 hrs and the concentration of IL-6 (G) and type I IFNα (H) was quantified in cell culture supernatants by ELISA. Data information: Bars represent mean values and equal symbols represent equal donors (n=4). Statistical significance was determined using the ratio paired student T test for agonist or virus treated cells and compared to the mock treated condition, or by unpaired T test when comparing matched conditions between different KOs. *
Figure 3In vitro co-culture of activated T cells with increasing ratios of neutrophils, monocytes or macrophages. T cell proliferation index is numbers of proliferated T cells after three days of indicated co-culture condition relative to the number of proliferated T cells when cultured alone. CD4+ and CD8+ T cells were derived from lymph nodes of wild-type mice. Neutrophils, monocytes and macrophages were derived from colon tumors of Apcfl/fl-Cdx2CreERT2 mice. Each dot represents an individual neutrophil (n=4), monocyte (n=3) or macrophage (n=4) sample. Bars represent mean +/- SD. Experimental setup. Eight weeks after Tamoxifen treatment of Apcfl/fl-Cdx2CreERT2 mice, animals were treated with anti-Gr1 antibody (αGr1, 3 times/week) plus CXCR2 inhibitor (CXCR2i, 5 times/week) or with IgG (3 times/week) plus DMSO control (5 times/week) for one to three weeks. Tumor neutrophil (left panel) and monocyte (right panel) content after αGr1 + CXCR2i (n=7) or IgG + DMSO (n=10) treatments. Average tumor volume per mouse at the end of αGr1 + CXCR2i (n=9) or IgG + DMSO (n=12) treatments as indicated in B. Anti-CD3 immunostaining on representative tumor sections derived from mice after indicated treatments. FACS analysis of CD45+ TCRβ+ cells, CD45+ TCRβ+ CD8+ cells and CD45+ TCRβ+ CD4+ Foxp3+ cells in extracted tumors after αGr1 + CXCR2i (n=8) or IgG + DMSO (n=7) treatments. FACS analysis of IFNγ (left panel) and IL17A (right panel) expression in tumor extracted T cells after αGr1 + CXCR2i (n=7) or IgG + DMSO (n=7) treatments.
a) siRNA constructs directed at IRGM reduced endogenous IRGM transcripts by six- to eightfold in HeLa cells.figf3(a) siRNA constructs directed at IRGM reduced endogenous IRGM transcripts by six- to eightfold in HeLa cells. Cells were transfected with control (siControl) or IRGM-targeted (siIRGM1, siIRGM2) siRNA duplexes and assayed 48 h later by quantitative RT-PCR. Error bars, s.d. (b) IRGM knockdown reduces the efficiency of anti-bacterial autophagy. HeLa cells stably expressing LC3-GFP (a marker for autophagic vesicles) were transfected with control or IRGM-directed siRNA oligos, then infected with S. typhimurium after 48 h. The percentage of bacteria encapsulated in LC3-positive vesicles was determined by microscopy. Reductions in IRGM expression resulted in a significant loss of anti-bacterial autophagy compared to control cells. (c) Microscopic examination reveals a decreased number of autophagically encapsulated bacteria in IRGM knockdown cells. Control siRNA-treated (siControl) and IRGM siRNA-treated (siIRGM1, siIRGM2) cells were infected with S. typhimurium SL1344 and imaged by confocal microscopy. Control cells show numerous bacteria (red in merged image) surrounded by LC3-GFP membranes (green in merged image). Such bacteria-containing autophagosomes (indicated by dashed boxes) were almost completely absent in IRGM-deficient cells. The actin cytoskeleton was visualized using phalloidin (blue in merge). Images are flat projections of confocal z-stacks. Scale bars, 10 μm. ( (d) HeLa cells stably expressing LC3-GFP were transfected with increasing amounts of an IRGM expression construct and infected 24 h later with S. typhimurium. The percentage of bacteria found within LC3-positive vesicles increased with increasing amounts of exogenous IRGM. Protein expression levels were confirmed by blotting lysates for Flag-IRGM expression, using an antibody to Flag to detect exogenous IRGM and actin as a loading control (lower panel). Error bars in b and d, s.e.m.
Figure 7(A) Representative single channel recordings from hippocampal neurons at 7-14 DIV with 500 nM BayK-8644, 1 µM ω-conotoxin GVIA, and 1 µM MVIIC in patch pipette upon depolarization from -80 to -30, -20, -10, and 0 mV. The right panel depicts the single-channel current-voltage relationship. Mean amplitude of unitary currents for different membrane potential studied are -2.07 pA (-30 mV), -1.75 (-20 mV), -1.53 (-10 mV) and -1.25 (0 mV; n=5 patches per test potential). Solid line represents best-fit of data using a linear equation (R2=0.92) revealing a slope conductance for these channels of 27 ± 2 pS. (B-D) Representative single channel traces and summary plot upon depolarization from -80 to 0 mV under control conditions and in the presence of ISO in the patch pipette. Cultures were pre-incubated for 15 min with 10 M 11R-PKI if indicated. The patch pipette contained either vehicle for control, nifedipine (nif; 1M), ISO (1M), or ISO plus nifedipine, which blocked all currents. The ISO-induced increase in NPo was prevented by 11R-PKI. (E-H) Representative single channel currents upon depolarization from -80 to 0 mV and summary plot after pretreatment of whole cultures with ISO. Cultures were pre-incubated with vehicle (H2O, mock wash, E) or 1 M ISO for 5 min (F,G) and washed for 3 (E,F) or 10 min (G) before forming the cell-attached patch with ISO present in the patch pipette. The upregulation of NPo to ~0.4 (cf. C,D) occurred only if neurons were pretreated with vehicle instead of ISO (E,H; mock wash) or if ISO wash out duration was 10 min (G,H) but not if wash out was only 3 min (F,H).
mice.Figure 8A-C. Iba1 was used as a marker for microglia and PIP2 levels were detected by measuring the intensity of florescence per cell. The average fluorescence was calculated above background in the cortex and hippocampus at 2 (A), 6 (B) and 9 (C) months. All data shows the mean±SD of 3 independent experiments analysed using 2 way ANOVA with Sidak's multiple comparison tests performed **=p<0.01, ****=p<0.0001.
is required for the clearance of damaged mitochondria upon viral infection (A) Immunoblot showing MAVS, IRF3-p, LC3, TOM20 and HSP60 levels in siCtrl- or siRNF34-1 cells infected with VSV and cultured in the presence of Z-VAD (50 μM) for the indicated times.Figure 7(A) Immunoblot showing MAVS, IRF3-p, LC3, TOM20 and HSP60 levels in siCtrl- or siRNF34-1 cells infected with VSV and cultured in the presence of Z-VAD (50 μM) for the indicated times. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results. (B) Immunoblot showing MAVS, LC3, TOM20 and HSP60 levels in cells transfected with Flag-RNF34 or its H342A mutant and subsequently infected with VSV for the indicated times. Cell-based studies were performed independently at least three times with comparable results. Representative confocal images (C) of immunofluorescence staining for NDP52 colocalization with MAVS in shCtrl- or shRNF34-1 cells infected with VSV for the indicated times. Red represents MAVS and green represents NDP52. Scale bar, 10 μm. Cell-based studies were performed independently at least three times with comparable results. quantification (D) of immunofluorescence staining for NDP52 colocalization with MAVS in shCtrl- or shRNF34-1 cells infected with VSV for the indicated times. Scale bar, 10 μm. Cell-based studies were performed independently at least three times with comparable results. (E) Transmission electron microscopy images of shCtrl- and shRNF34-1 HEK293T cells cultured in the presence or absence of VSV. Arrows indicate mitochondria engulfed in autophagosomes. Double arrows indicate swollen mitochondria with disrupted cristae. M, mitochondria and N, nucleus. 40000× magnification. Cell-based studies were performed independently at least three times with comparable results. (F) Representative confocal images of the shift in the fluorescence emission of Keima from 458 nm to 543 nm. Scale bar, 10 μm. Cell-based studies were performed independently at least three times with comparable results.
Figure 4(A) Representative immunofluorescence staining of α-Actinin in NRVMs transfected with siRNA for 48h and then treated with vehicle (H2O) or PE for another 48h. Scale bar: 50μm. siMEF2a indicates MEF2a siRNA. (B) Quantification of the surface area of α-Actinin-positive NRVMs with or without knockdown of NCoR1 and/or MEF2a. A total of 15-20 NRVMs was randomly chosen from 3 replicate coverslips for each group and used for statistical analysis. (C) Representative immunofluorescence staining of α-Actinin in NRVMs transfected with siRNA for 48h and then treated with vehicle or PE for another 48h. Scale bar: 50μm. siMEF2d indicates MEF2d siRNA. (D) Quantification of the surface area of α-Actinin-positive NRVMs with or without knockdown of NCoR1 and/or MEF2d. A total of 15-19 NRVMs was randomly chosen from 3 replicate coverslips for each group and used for statistical analysis. (E) qRT-PCR analysis of Acta1 and Nppa in NRVMs. n=4. Data represent three independent experiments. Student's t test was used for statistical analysis.
HCV induces Parkin-mediated mitophagosome formation.Figure 4Confocal microscopy showing mitophagosome formation associated with Parkin in HCV-infected cells. (A) Huh7 cells transiently expressing GFP-LC3 protein were infected with HCVcc in the absence or presence of 3-MA (10 mM) and BafA1 (100 nM), respectively, for 12 h before fixation. At 3 day post-infection, cells were immunostained with antibodies against HCV E2 (orange), TOM20 (blue), and Parkin (red). In the zoom images, the arrows (white puncta) indicate GFP-LC3 puncta (green) colocalized with TOM20 and Parkin. Nuclei are demarcated with white dot circles. ( (B) Quantification of the number of GFP-LC3 puncta colocalized with TOM20 in the panel (A) (mean ± SEM; n≥10 cells; *p<0.01). (C) Quantitative analysis of the area of GFP-LC3 puncta (white) representing merge of GFP-LC3 puncta, TOM20, and Parkin in the panel (A) (mean ± SEM; n≥10 cells; *p<0.001). (B and C) P values were calculated by using an unpaired Student's t-test. . (D) Ultrastructure of HCV-infected cells showing the formation of mitophagosome. Control naïve Huh7 cells (uninfected), HCV FLR-JFH1 replicon cells, and Huh7 cells infected with HCVcc (Jc1) were examined by electron microscopy. In the zoomed image, the formation of mitophagosome in HCV-infected cells is shown. Organelle mark: N, nucleus; MF, mitophagosome formation. Scale bar = 0.2 µM (uninfected); 0.1 µM (infected). (E) Immunoelectron microscopic analysis showing the formation of mitophagosome in HCV-infected cells. Huh7 cells infected with HCVcc were stained by triple immunogold labeling method, as described in Materials and Methods. The immunogold particles of GFP-LC3 (white arrow, 18 nm), HCV E2 (blue arrow, 12 nm), and TOM20 (red arrow, 6 nm) are shown. Fluorescence image of HCV core identifies HCV-infected cells (red). Nuclei were stained with DAPI (blue). Scale bar = 0.2 µM.
Figure 7A Salivary glands dissected from late 3rd instar larvae (Gbe Su(H)-lac-Z/w-;+;Fkh-GAL4/+) show less notch activity (left) compared to glands from 14 hours after puparium formation (APF) animals (middle). Down-regulation of ral in salivary glands from 14 hours APF animals (Gbe Su(H)-lac-Z/w-;UAS-ralIR/+;Fkh-GAL4/+) reduces the levels of notch activity (right). Scale bar represents 20µm.B Quantification of data from (A). Data are represented as means ± SEM; n≥ 4. Statistical significance was determined using a Student's t test. C Salivary glands dissected from 14 hours after puparium formation animals expressing mCherry-Atg8a in all cells, and notchIR specifically in GFP-marked clone cells (yw, hsflp/w,UAS-notchIR; pmCherry-Atg8a/+; act< FRT, cd2, FRT> GAL4, UAS-GFP/+) analyzed for mCherry-Atg8a puncta. Scale bar represents 20µm.D Quantification of data from (C). Data are represented as means ± SEM; n> 10. Statistical significance was determined using a Student's t test.
Convergent acetylproteome modification after resveratrol or spermidine treatment.Figure 5.Convergent acetylproteome modification after resveratrol or spermidine treatment. (A-C) Colon carcinoma HCT 116 cells were cultured for 2 wk in three different SILAC media containing different arginine and lysine isotopes. Cells were treated with 100-µM resveratrol (Resv) or spermidine (Spd) for 2 h, fractioned into cytoplasmic, nuclear, and mitochondrial extracts, processed for acetyl lysine peptide enrichment, and analyzed by MS. (A) Hierarchical clustering of drug-specific organellar distributions of all acetylated sites quantified in at least one fraction. Fold changes are calculated relative to untreated cells. Only sites regulated >1.5-fold were included in statistical analyses. (B) Graphical representation of peptides whose acetylation status was affected in a convergent or divergent way. n = 560.
Interaction of human or mouse melanoma cells with platelets results in Asm secretion and surface Asm activity independent of Asm expression in melanoma cells.Figure 3Incubation of human melanoma (HM) cells with human platelets results in the release of Zn2+-dependent ASM into the supernatant, Zn2+-dependent activity of ASM on cell surfaces, and the formation of ceramide. The assay buffer contained 100 μM Zn2+. Addition of human or mouse recombinant ASM to human melanoma or B16F10 cells, respectively, results in binding of ASM to the tumorcell surfaces as determined by flow cytometry. Cytochalasin B was added to control for internalization of added ASM. Suppression of Asm in B16F10 tumor cells using siRNA technology reduces Asm activity in B16F10 cells (right panel), but does not alter release or surface activity of the Asm after co-incubation with wild-type platelets (left panels).
Fig.Figure 7(A) WT (BY4741), tgl1∆, yeh1∆ ,and yeh2∆ cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for the indicated time periods. Cells were lysed and subjected to SDS−PAGE, followed by western blot analysis using anti-GFP antibodies. Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test). (B) WT (BY4741) and tgl3∆tgl4∆tgl5∆ cells expressing GFP−Atg8 were grown, treated, lysed, and western blotted as in (A). Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test). ( (C) WT (BY4741), ayr1∆ and ldh1∆ cells expressing GFP−Atg8 were grown, treated, lysed, and western blotted as in (A). Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test). (D) WT (BY4741), ldh1∆ayr1∆ cells expressing GFP−Atg8 were grown, treated, lysed, and western blotted as in (A). Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. **P < 0.01 (Student's-t test). (E) WT (BY4741), ice2∆ and ldb16∆ cells expressing GFP−Atg8 were grown, treated, lysed, and western blotted as in (A). Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05, **P < 0.01 ***P < 0.001 (Student's t-test). (F) WT (BY4741), ice2∆ and yeh1∆ cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for 4 h. GFP−Atg8 was visualized by fluorescence microscopy. Scale bar, 5 μm. DIC, Differential interference contrast; SD-N, nitrogen-starvation medium; WT, wild type.
Figure 1(A) The relative fold change of carbon metabolites with or without HU treatment for 60 min. Error bars represent standard deviations (SD) from six independent experiments. (B) The diagram of carbon metabolism rewiring focused on glycolysis and PPP (pentose phosphate pathway). (C) The relative ZWF1 mRNA levels were measured by qPCR. Cells were grown into an exponential phase and treated by 200 mM HU for 0, 30, 60 min. mRNA were prepared just as described in Methods and Materials. The relative mRNA levels of ZWF1 to ACTIN1 were determined by qPCR analyses. The value of the untreated WT sample was normalized to 1. Error bars represent standard deviations (SD) from at least three biological repeats. Statistical significance was evaluated based on Student's t-test (*0.01< P-values <0.05). (D, E) The relative levels of endogenous Zwf1 protein with a GFP tag were analyzed by immunoblots in WT, snf1Δ or snf1Δmig1Δ cells (D). Protein extracts were probed with an anti-GFP antibody. Mcm2 was applied as a loading control. Three independent experiments were carried out and the relative fold changes were measured by Image J (E). Error bars represent standard deviations (SD) from three biological repeats. Statistical significance was evaluated based on Student's t-test (*0.01< P-values <0.05; **0.001< P-values <0.01).
Figure 5A, PTPN3 wildtype, phosphatase-dead D811A and ICC-derived mutant L232R exhibit similar patterns of subcellular localization and co-localization with TβRI. HaCaT cells were transfected with HA-TβRI and Flag-PTPN3 (wildtype or its mutants). 24 h later, cells were harvested for immunofluorescence with the indicated antibodies. Scale bar = 10 μm. B, PTPN3 (L232R) does not increase the TβRI protein level. TβRI and PTPN3 were determined by Western blotting in HaCaT cells stably expressing PTPN3, PTPN3 (D811A) or PTPN3 (L232R). C, PTPN3 (L232R) fails to rescue the Smurf2-mediated degradation of TβRI. HEK293T cells were transfected with FLAG- TβRI, Myc-Smurf2 and the increasing anount of PTPN3 (wildtype and mutants). Cells were harvested 24 h post-transfection. Protein levels were examined by Western blotting with the indicated antibodies. D, PTPN3 (L232R) does not block the interaction between TβRI and Smurf2. HEK293T cells were co-transfected with plasmids as indicated. 20 h later, cells were treated with MG132 (20 μM) for 4 h and analyzed by IP-Western blotting. E, Ectopic expression of PTPN3 wildtype and PTPN3 (D811A), but not PTPN3 (L232R), completely abolishes the Smad7-TβRI interaction. HEK293T cells were co-transfected with plasmids as indicated. 20 h later, cells were treated with MG132 (20 µM) for 4 h, followed by IP and Western blotting. F, PTPN3 and PTPN3 (D811A), but not PTPN3 (L232R), attenuate ubiquitination of TβRI. HEK293T cells were transfected with the indicated plasmids. 24 h after transfection, IP-Western blotting analysis was performed with anti-Flag antibody IP and followed by Western blotting with the indicated antibodies.
YAP signaling regulates SOX9.Figure 6 A, B Immunofluorescence of activated mouse HSCs cultured on plastic (A) or hydrogels (A, B). Individual fluorescent channels showing localization and expression of SOX9 (left), YAP (middle) and composite image for SOX9 (red) and YAP (green) in right panel. (B) Individual and composite images for αSMA (green) and TAZ (red) or TEAD (red). Size bar, 25μm. C Quantified decrease in SOX9 protein levels following inhibition of YAP using verteporfin (VP) in activated mouse HSCs expressed relative to DMSO control (n=3). D Immunohistochemistry for SOX9 (brown) following CCl4 or BDL induced fibrosis in mice treated with DMSO (CCl4 n=4, BDL n=7) or verteporfin (CCl4 n=3, BDL n=5). E, F quantification of SOX9 cell numbers in the HSC/Hep population. Size bar, 50 μm. E, F quantification of SOX9 cell numbers in the HSC/Hep population. Size bar, 50 μm.
a) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells transfected for 3 h with Cy3-labeled poly(dA:dT) (1.figf2(a) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells transfected for 3 h with Cy3-labeled poly(dA:dT) (1.5 μg/ml). Yellow dots (Merge) indicate colocalization of green (LC-3) and red (Cy3) signals. Scale bars, 10 μm. (b) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells transfected to express Cy3-poly(dA:dT), imaged every 15 min starting 1 h after transfection (numbers in bottom left corners indicate time in min (m)). Arrow indicates coalescence of the Cy3 and GFP signals (also in bottom right image). (c) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells sham-transfected with no poly(dA:dT) (Control) or transfected with poly(dA:dT), then fixed and immunostained for ASC (red). Insets (bottom right), 5× enlargement of areas indicated by arrowheads at left. Scale bars, 10 μm (top) or 20 μm (bottom). (d) Confocal microscopy of differentiated THP-1 cells transfected with poly(dA:dT) and immunostained for LAMP-1 and ASC; below, z-axis projections. Scale bars, 10 μm. (e) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells treated for 2 h with LPS (500 ng/ml) and ATP (3 mM), then immunostained for ASC (red): left image in each pair, three-dimensional volume renderings reconstructed from z-stack images; right image in each pair, surface-segmentation models (2× enlargement). Scale bars, 10 μm. (f) Electron microscopy of differentiated THP-1 cells stimulated with LPS and ATP (left) or transfected with poly(dA:dT) (right) for 2 h and immunostained for ASC, visualized with 15-nm gold-conjugated protein A. Red arrows indicate ASC immunostaining. Right image in each pair, 4.5× enlargement of outlined area at left. Scale bars, 500 nm. Data are representative of three experiments (a,c) or two experiments (b,d-f).