EMBO/sd-character-level-ner huggingface dataset:
Figure 5Significantly decreased MHC expression in Ddx17-depleted C2C12 cells is shown by immunocytochemistry. Nuclei were counterstained with DAPI. Bar, 100 μm. Fusion index is shown as percent of the control. n = 3, mean ± SD. **p < 0.01 (unpaired two-tailed Student's t-test). Immunocytochemistry for myogenin in Ddx17-depleted C2C12 cells. Bar, 100 μm. Myogenin-positive cells are shown as percent of the control. n = 4, mean ± SD. **p < 0.01 (unpaired two-tailed Student's t-test). Relative luciferase activities of the -1650-Luc by the combination of MyoD and Ddx17 or Ddx17 mutant (K142R). Bars indicate the average of two independent experiments, and open circles represent the values of each experiment. Ddx17 or Ddx17 mutant (K142R) was pulled down by full-length Myoparr and then detected by western blot. Reduced interaction between endogenous Ddx17 and PCAF in Myoparr-depleted C2C12 cells. After Myoparr KD, the cell lysates were subjected to immunoprecipitation (IP) with a Ddx17-specific antibody 36 h after differentiation induction (right panel). Each lysate was loaded as an input (left panel). Relative quantification of (E) is shown as IP/input ratio. n = 3, mean ± SD. *p < 0.05. n.s., not significant (unpaired two-tailed Student's t-test). Increased interaction between Ddx17 and PCAF by Myoparr. PCAF protein was pulled down by 3×Flag-Ddx17 in the presence or absence of Myoparr using a Flag antibody and then detected by western blot. ChIP-qPCR detection of Ddx17 (H) and PCAF (I) occupancies at the myogenin locus in Myoparr-depleted differentiating C2C12 cells. The data were normalized to input values. n = 3, mean ± SD. *p < 0.05 (unpaired two-tailed Student's t-test).
Figure 4A. ROS burst in pbl34/35/36 triple mutant, induced by 3-OH-C10:0. Two-week-old seedlings were treated with 1 μM 3-OH-C10:0, and the ROS burst was measured. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01). B. PTI marker gene expression was reduced in pbl34/35/36 triple mutant in response to 3-OH-C10:0. Treatment is same as in A. Samples were collected for RT-qPCR at 4 h post infiltration. Values are means ± SD (n=3 biological replicates). (Student's t-test, **P< 0.01). C. pbl34/35/36 triple mutant displayed decreased callose depositions in response to 3-OH-C10:0 treatment. Two-week-old seedlings were sprayed with 1 μM 3-OH-C10:0. The leaves were sampled for callose deposition assays 12 hrs later. The stained callose was counted for 1 mm2 of the leaves. Mock is solvent control. Scale bar=100μm.Values are means ± SD (n=6 leaves). (Student's t-test, **P< 0.01). D. Root length of pbl34/35/36 triple mutant treated with 3-OH-C10:0. The Arabidopsis seedlings were grown on ½ MS containing 2.5 μM 3-OH-C10:0 for six days, and the root length was measured. Scale bar=0.4cm. Values are means ± SD (n=3 biological replicates). (ANOVA, P <0.01). E. Disease phenotype of pbl34/35 double, pbl34/35/36 triple mutants and PBL34 overexpression plants. 35S:PBL34-FLAG was introduced to the Col-0 to generate the overexpression lines. The homozygous transgenic plants were used for the assays. The plants were sprayed with Pst DC3000 at a concentration of 1×109 cfu/ml. Plants were subjected to bacterial growth curve analysis at 3 dpi. OE-3# and OE-11# are two independent transgenic lines. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01). F. pbl34/35/36 triple mutant phenotype was rescued by PBL34. PBL34-HA was introduced to the triple mutant driven by its native promoter. Four-week-old homozygous seedlings were inoculated with 5×104 cfu/ml Pst DC3000 , and the leaves were subjected to bacterial growth curve analysis at 3 dpi. CM2-5 and CM7-3 are two independent transgenic lines. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01).
SARS-CoV-2 Spike protein primes NLRP3 inflammasome and induces pyroptosis.Figure 2Chemical inhibition of IL-1β (pg/ml) secretion after pre-incubation of macrophages from COVID-19 patients (red) for 2h with DMSO (n = 21), MMC950 (n = 21) or VX-765 (n = 8), followed by 4h incubation with S-protein and additional 2h with nigericin. For statistical analysis, one-way ANOVA with tukey post hoc test was used. Western blot analysis of total cell lysates of macrophages from a SARS-CoV-2 naïve individual (SC-naïve) (top) and COVID-19 infected individual (bottom). Macrophages were stimulated with LPS or S-protein (4h) followed by 2h of nigericin treatment. Control cells were left unstimulated. Antibodies against NLRP3 (110kDa) and β-actin (loading control) were used for detection. Representative example of three individual experiments. Representative confocal microscope pictures of macrophages from SC-naïve (top) and COVID-19 patients (bottom). Nuclei are stained with DAPI (blue) and ASC speck are labeled by immunofluorescence staining (green). Macrophages were stimulated with LPS (left) and S-Protein (right) for 4h followed by stimulation with nigericin in both cases. Pictures were taken with a 60x objective using the same microscope settings. Quantification of ASC speck from SC-naïve (n=3, blue) and COVID-19 patients (n=8, red) derived macrophages. Percentage of ASC specks was calculated per DAPI stained nuclei. For statistical analysis, two-way ANOVA with tukey post hoc test was used. IL-1β gene expression of macrophages from COVID-19 patients (n = 5; red bars) or SC-naïve individuals (n = 3 (S-protein) n = 4 (control); blue bars) stimulated with/without S-Protein were determined by qRT-PCR. Data are normalized to β-actin. Statistical significance was analyzed using the Kolmogorov-Smirnov test. Western blot analysis of total cell lysates of macrophages from SARS-CoV-2 naïve individuals (SC-naïve) (top) and COVID-19 infected individuals (bottom). Macrophages were stimulated with LPS or S-protein (1h). Control cells were left unstimulated. Antibodies against pro-IL-1β (31kDa) and β-actin (loading control) were used for detection. Representative example of three experiments performed with individual patients and SARS-CoV-2 naïve controls. Western blot analysis comparing macrophage precipitated supernatants from a SC-naïve (top) and a COVID-19 positive patient (bottom). Macrophages were treated with DMSO or MMC950 (2h) and further stimulated with LPS or S-protein (4h) followed by 2h of nigericin treatment. Antibodies binding cleaved IL-1β (17kDa) were used for detection. Representative example of three individual experiments. Western blot analysis of total cell lysates of macrophages from a SARS-CoV-2 naïve individuals (SC-naïve) (top) and COVID-19 infected individual (bottom). Macrophages were stimulated with LPS or S-protein (4h) followed by 2h of nigericin treatment. Control cells were left unstimulated. Antibodies targeting cleaved GSDMD (110kDa) and β-actin (loading control) were used for detection. Representative example of three individual experiments.
, encoded by LncBMP4, exhibits similar functions as LncBMP4.Figure 7A. Morphological observation of small peptides and LncBMP4 backbone chains, promoting PGC formation in vitro, Scale bar: 60 µm (n =3 independent experiments). B. qRT-PCR was used to detect the expression of related genes in each group, during induction (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA). C. The efficiency of PGC differentiation was detected by IF after 4 d of induction using C-KIT and CVH antibodies. Scale bar: 60 µm (n =3 independent experiments). D. The number of CVH-positive cells was determined by flow cytometry (Data are shown as mean ± SEM, n =3 independent experiments, ** p < 0.01, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA.).
Figure 7A Luciferase activity in WT and BECN1 KO 293T cells transfected with an ISRE (left) or IFN-β (right) promoter-driven luciferase reporter after infection with SeV for 12 h. B WT and BECN1 KO 293T cells were infected with SeV for indicated time points, then protein extracts were analyzed by immunoblot using the indicated antibodies. C Protein extracts of THP-1 cells infected with SeV for various time points (upper lanes) were subjected to immunoprecipitation with anti-Beclin-1 and immunoblot analysis with antibodies shown on the left. D Co-immunoprecipitation and immunoblot analysis of protein lysates of A549 cells infected with SeV. E 293T cells were transfected with vectors encoding HA-Beclin-1 and Flag-MAVS or Flag-MAVSΔCARD, followed by IP with anti-Flag beads and immunoblot analysis with anti-HA. F Co-immunoprecipitation and immunoblot analysis of WT and BECN1 KO 293T cells transfected with HA-MAVS plasmid along with vector encoding Flag-RIG-I (N) in the absence or presence of USP19. G Protein extracts of scramble or USP19-specific siRNA treated 293T cells transfected with HA-MAVS plasmid along with vector encoding for Flag-RIG-I (N) in the absence or presence of Beclin-1 were subjected to immunoprecipitation and immunoblot analysis. H Luciferase activity in 293T cells transfected with scramble or USP19-specific siRNA and ISRE luciferase reporter after SeV infection for indicated time points with or without Beclin-1. I Luciferase activity in 293T cells transfected with scramble or USP19-specific siRNA and IFN-β luciferase reporter after SeV infection for indicated time points with or without Beclin-1. J A proposed working model to illustrate how the USP19-Beclin-1 axis positively regulates autophagy and negatively regulates type I IFN signaling.
Figure 3(A-D) Compared with the control (A) flies, posterior midguts of Notch OE flies exhibit decreased ISCs and ee cells (B), while HP1c OE flies show increased ISCs and ee cells (C), and the phenotype of Notch OE can be rescued by HP1c OE (D). Arrows mark ISCs, arrowheads mark ee cells, circles mark ISC-ISC, ISC-EB or EB-EB nests. ISCs were visualized with esg-GFP alone, EBs were stained with both esg-GFP and Su(H)-lacZ, while ee cells were labeled by Pros and DNA was marked by DAPI. Scale bars, 30 μm. Images of wings from flies with the indicated genotypes. Arrows mark the ectopic veins at L5 and arrowheads mark the classical notched wing. Scale bars, 500 μm. Images of wings from flies with the indicated genotypes. Arrows mark the ectopic veins at L5 and arrowheads mark the classical notched wing. Scale bars, 500 μm. Images of wings from flies with the indicated genotypes. Arrows mark the ectopic veins at L5 and arrowheads mark the classical notched wing. Scale bars, 500 μm. Images of wings from flies with the indicated genotypes. Arrows mark the ectopic veins at L5 and arrowheads mark the classical notched wing. Scale bars, 500 μm. Images of wings from flies with the indicated genotypes. Arrows mark the ectopic veins at L5 and arrowheads mark the classical notched wing. Scale bars, 500 μm. Images of wings from flies with the indicated genotypes. Arrows mark the ectopic veins at L5 and arrowheads mark the classical notched wing. Scale bars, 500 μm.
Figure 1Immortalized brown preadipocytes were induced to mature adipocytes with supplementation of asparagine during differentiation. Gene expression was analyzed on differentiation day 6. Gene expression analyzed by quantitative PCR in brown adipocytes that were fed with or without 1mM asparagine as in (A). n=3 independent experiments. Data information: Data represent means±SEM. *P<0.05; **P<0.01; unpaired two-tailed Student's t test. Oxygen consumption rate measured by clark electrodes for mature brown adipocytes as in (B). n=4 independent experiments. Data information: Data represent means±SEM. *P<0.05; **P<0.01; unpaired two-tailed Student's t test. Left: UCP1, PGC1α and PPARγ protein levels were analyzed in brown adipocytes (day 6). Cells were supplemented with 1mM asparagine from day 4 to day 6. Right: quantification of protein levels. n=3 biological replicates. Data information: Data represent means±SEM. *P<0.05; **P<0.01; unpaired two-tailed Student's t test. Preadipocytes bearing the shRNA targeting Pgc1α were induced differentiation with or without 1mM asparagine. Protein expression was analyzed on day 6. n=3 biological replicates. Primary preadipocyes isolated from mice BAT were induced to differentiation in DMEM media with or without 1mM asparagine. Left: UCP1 and PPARγ protein levels were analyzed on day 6. Right: quantification of protein levels. n=3 biological replicates. Data information: Data represent means±SEM. *P<0.05; **P<0.01; unpaired two-tailed Student's t test. Brown preadipocytes were infected by lentiviral shRNAs against Asns. Cells were differentiated in culture media with or without 1mM asparagine. Western blot was performed on day 6.
Figure 5(A) NF-κB activation during MZT. Immunofluorescence staining of p65 shows nuclear translocation of p65 at the two-cell stage and thereafter. Scale bar = 25um. (B) The protein levels of IκBα and its phosphorylated form (p-IκBα) during mouse early embryonic development were detected by western blotting, representative western blotting of IκBα and p-IκBα was shown on the left and the quantitation analysis (right panel) indicated that the ratio of p-IκBα/total IκBα was gradually increased from MII oocytes to 4-cell stage embryos (three independent experiment replicates were performed, error bars represent s.e.m.). (C) NF-κB pathway inhibition using the specific inhibitor Bay11-7082 resulted in two cell stage arrest. Three independent experiment replicates were performed, error bars represent s.d.; ***:P<0.001 in unpaired two-tailed t-test. (D, E) Representive immunofluorescence staining of p65 after knockdown of Rnf114 by Rnf114 siRNA #1 or siRNA #2 or overexpression of Tab1 by Tab1 mRNA injection (shown in D. Scale bar = 25um.), the percentages of two-cell stage embryos with p65 nuclear translocation (shown in E.) were significantly decreased after Rnf114 knockdown or Tab1 overexpression (chi-square test was performed for statistical analysis). (F) Western blot analysis at 2-cell stage showed the levels of p-IκBα were downregulated in the Rnf114 siRNA #1, siRNA #2 injected groups. (G) Western blot analysis at 2-cell stage showed the levels of p-IκBα were downregulated in the exogenous Tab1 mRNA injected groups, ddH2O was injected as control. Dotted line indicates a cropped lane.
Figure 3A Representative image of E18.5 coronal cortical sections stained for layer V marker Ctip2 (red) from Kcc2lox/lox embryos electroporated in utero at E14.5 with plasmid constructs encoding EGFP or EGFP+Cre (Cre). Upper boundary of layer V indicted with dotted line. EGFP signal is shown as green pseudocolor. DAPI staining (blue) marks cell nuclei. Sp, subplate; IZ, intermediate zone. Scale bar = 50 µm. B Number of EGFP+Cre neurons migrating above (II-IV) and below (V-VI/IZ-SVZ) the upper border of layer V normalized to respective data from embryos electroporated with EGFP alone. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, ***P < 0.001 to EGFP. Data are presented as mean ± S.E.M., n (EGFP +Cre) = 8 embryos; n (EGFP) = 8 embryos. C Representative image of E18.5 coronal brain sections stained for layer V marker Ctip2 (red) from Kcc2lox/lox embryos electroporated in utero at E14.5 with plasmid constructs encoding mRFP or mRFP+Cre (Cre). Upper boundary of layer V indicted with dotted line. Sp, subplate; IZ, intermediate zone. Scale bar = 50 µm. mRFP signal is shown as green pseudocolor. DAPI staining (blue) marks cell nuclei. D Number of mRFP+Cre neurons migrating above (II-IV) and below (V-VI/IZ-SVZ) the upper border of layer V normalized to respective data from embryos electroporated with mRFP alone. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, ***P < 0.001 to mRFP. Data are presented as mean ± S.E.M., n (mRFP+Cre) = 5 embryos; n (mRFP) = 5 embryos. E Representative image of E18.5 coronal cortical sections stained for layer V marker Ctip2 (red) from Kcc2lox/lox embryos co-electroporated in utero at E14.5 with plasmids encoding a fluorescent marker (green) together and Cre+KCC2FL, Cre+KCC2CTD, or Cre+KCC2R952H. DAPI staining (blue) marks cell nuclei. Upper boundary of layer V indicted with dotted line. Sp, subplate; IZ, intermediate zone. Scale bar = 50 µm. F Number of transfected neurons migrating above (II-IV, left) and below (V-VI/IZ-SVZ, right) the upper border of layer V in embryos electroporated with constructs in (E) normalized to respective data from embryos electroporated with Cre. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001 to Cre. Data are presented as mean ± S.E.M., n (Cre+KCC2FL) = 13 embryos; n (Cre+KCC2CTD) = 9 embryos; n (Cre+KCC2R952H) = 9 embryos.
a) Lysates extracted from the SVZ of three different Ctrl and FIP200GFAP cKO mice treated with chloroquine, and then analyzed by western blot using antibodies to LC3 (top), p62 (middle) or vinculin (bottom).figf1(a) Lysates extracted from the SVZ of three different Ctrl and FIP200GFAP cKO mice treated with chloroquine, and then analyzed by western blot using antibodies to LC3 (top), p62 (middle) or vinculin (bottom). (b) Immunofluorescence for p62 and DAPI in the SVZ and DG of Ctrl and FIP200GFAP cKO mice at P28 (n = 3 p62 for each). Dotted lines indicate boundaries of the SVZ and granular zone (GZ). The arrows and arrowheads mark larger and smaller p62+ aggregates, respectively, in the striatum (ST) and SVZ. (c) TEM of mitochondria in SVZ tissue from Ctrl and FIP200GFAP cKO mice at P28 and P56 (n = 2 mice for each, >30 cells for each mouse). Arrows mark mitochondria. (d) TEM of mitochondria in cultured neurospheres from Ctrl and FIP200GFAP cKO mice (n = 2 mice for each, >30 cells for each mouse). Arrows mark mitochondria. (e,f) DHE and DAPI fluorescence in the dentate gyrus (e) and SVZ (f) of Ctrl and FIP200GFAP cKO mice at P28 (n = 5 mice for each). Dotted lines indicate the boundaries of the GZ (e) and SVZ (f). Arrows mark cells in the SGZ (e) and SVZ (f), and arrowheads mark cells in surround regions (the GZ in e and the ST in f). LV, lateral ventricle; ML, molecular layer. Full-length blots are presented in Supplementary Figure 11. (a) Lysates extracted from the SVZ of three different Ctrl and FIP200GFAP cKO mice treated with chloroquine, and then analyzed by western blot using antibodies to LC3 (top), p62 (middle) or vinculin (bottom).
Figure 2Flow cytometry analysis of EMPs (Ter119- Kit+ CD41+ CD16/32+) and early erythroid progenitors (Early E: Ter119- Kit+ CD41- CD71+ CD44+) in wild type and Sl/Sl E11.5 FL. EMP and early E data are the mean (±SD) of 6 wild type and 4 Sl/Sl biological replicates, with a total of 10 wild type and 6 Sl/Sl embryos analyzed in pools of 1-3 FL per genotype. Bar graphs on the right show corresponding absolute cell numbers (nr) per FL. Total FL cell counts are the mean (±SD) of 12 wild type and 13 Sl/Sl embryos. Tail somite range: 10-16 (wild type), 12-15 (Sl/Sl). Note that although EMP frequency in Sl/Sl FL is increased, their absolute number is severely decreased. CFU-C numbers in wild type and Sl/Sl E11.5 FL. Data are the mean (±SD) of 10 wild type and 10 Sl/Sl biological replicates plated in duplicate, with each replicate consisting of cell from 1-2 FLs. A total of 12 wild type and 11 Sl/Sl FL were plated over 6 independent experiments. GEMM: granulocyte, erythroid, monocyte/ macrophage, megakaryocyte; G/M/GM: granulocyte, monocyte/macrophage; Ery: erythroid. Total cell numbers (nr) of E12.5 wild type (n=7) and Sl/Sl (n=8) FL. Right: CFU-E numbers per wild type and Sl/Sl E12.5 FL. Data are the mean (±SD) of 3 biological replicates analyzed over 2 independent experiments. Each biological replicate consisted of 1-2 FL samples pooled according to genotype and was plated in duplicate into a methylcellulose based medium containing EPO; colonies were scored after 2 days. Total number of FLs analyzed: 4 wild type and 6 Sl/Sl. Quantification of erythroid lineage cells in E12.5 wild type and Sl/Sl FL. Absolute cell counts (top) and percentages (bottom) of proerythroblasts (ProE), basophilic erythroblasts (BasoE), polychromatic erythroblasts (PolyE) and orthochromatic erythroblasts (OrthoE) were determined by imaging flow cytometry analysis. The number (nr) of cells per FL was based on total cellularity and their relative proportions. Data are the mean (±SD) of 3 biological replicates for each genotype, with replicates consisting of single or 2 pooled FL samples. A total of 4 wild type, 5 Sl/+ and 6 Sl/Sl FLs were analyzed. Representative examples of maturing erythroblast series in wild type and Sl/Sl E12.5 FL, as identified by imaging flow cytometry. Scale bars: 10μm.
Figure 1A. Strategy to generate Fbxl4 knockout mice. Exon IV was targeted by loxP sites (orange arrows) and removed after Cre recombination. Black arrows indicate Frt sites. B. Genotype distribution in litters from heterozygous matings (Fbxl4+/- x Fbxl4+/-) at embryonic stage 13.5 (E13.5) and of live animals at weaning. Data is presented as a percentage. Embryos n=79, weaned mice n=339. Dashed lines indicate the expected Mendelian ratios. Chi2 test vs expected Mendelian ratios; ***, p<0.001. C. Representative images of wild-type (+/+), heterozygous knockout (+/-) and homozygous Fbxl4 knockout embryos (-/-) at E13.5. D. Relative levels of mtDNA of Fbxl4 knockout (-/-) embryos and the corresponding wild type (+/+) embryos at E13.5. Data represent mean ± SEM, n=5. Student's t-test; *, p<0.05. E. Representative images of male Fbxl4 knockout (-/-) and matched wild-type (+/+) animals at one year of age (left). A hunch-back phenotype was observed both in male and female knockout animals (right, indicated by arrow). F-I. Whole body (F), heart (G), liver (H) and kidney (I) weights of 1-year old animals. Data represent mean ± SEM, n ≥ 5 animals. Student's t-test; *, p<0.05; **, p<0.01; ***, p<0.001; n.s., not significant (p ≥ 0.05).
Acid sphingomyelinase induces clustering and activation of β1 integrins in ceramide-enriched membrane domains in human melanoma cells.Figure 6Human melanoma cells were stimulated for 10 min with 1 U/ml purified ASM or 10 μM C16 ceramide (C16-Cer) in the presence or absence of 500 ng ceramidase (CDase) or 1 μg/ml neutralizing anti-ceramide antibodies (anti-Cer), and lysed and active β1 integrin was immunoprecipitated using the HUTS-4 antibody. Octylglucopyranoside (OGP, final concentration 0.01%) served to resuspend C16 ceramide. Internalization was excluded by addition of cytochalasin B (CTB) as indicated. Samples were separated by SDS-PAGE and blotted with an activation-independent anti-β1 Integrin antibody to detect the amount of immunoprecipitated, that is active β1 integrin. Control immunoprecipitates (control Ipt) were performed with an irrelevant isotype control antibody. Activation of β1 integrin in human melanoma cells by incubation with Asm or C16 ceramide was confirmed by FACS analysis upon staining with FITC-labeled HUTS-4 antibodies. Incubation of human melanoma with human platelets or ASM for 5 min triggers co-clustering of ceramide and activated β1 integrin on the surface of the tumor cells. Cells were stained with FITC-coupled anti-active β1 integrin (HUTS-4) antibodies and Cy3-coupled anti-ceramide antibodies. Shown are representative stainings from each four independent experiments.
N-terminal region of SETDB1 contributes to its cellular localization A HEK293T cells were transfected with V5-SETDB1 and either empty or 3xF-ATF7IP expressing vector.Figure 4A HEK293T cells were transfected with V5-SETDB1 and either empty or 3xF-ATF7IP expressing vector. Over 50 transfected cells analysed per sample were analyzed. The representative data of two independent experiments is shown. Scale bar: 10 µm. B HEK293T cells were transfected with V5-SETDB1 d1 mutant and either empty or 3xF-ATF7IP expressing vector. Over 50 transfected cells per sample were analysed. The representative data of two independent experiments was shown. Scale bar: 10 µm. The yellow arrowheads indicate the cells that show nuclear (and cytoplasmic)-localization of introduced exogenous mSETDB1.