EMBO/sd-character-level-ner huggingface dataset:
Figure 5(A) GST or GST-GSDMD, p20 (C285A)-His, p10-His and increasing amount of His-nucleocapsid were incubated for a GST-pulldown assay, followed by immunoblotting with antibodies against the indicated protein tags. Data information: Experiments were repeated for three times with similar results. (B) An in vitro cleavage assay was performed through mixing GSDMD and its mutants with caspase-1 p20/p10 complex, followed by immunoblotting with antibodies against the indicated protein tags. β-actin served as a loading control. FLAG-tagged GSDMD and its mutants were immunoprecipitated from HEK293T cells using anti-FLAG coupled magnetic beads. HA-tagged p20 and p10 were immunoprecipitated from HEK293T cells using anti-HA coupled agarose beads. Data information: Experiments were repeated for three times with similar results. (C) A GST-pulldown assay was performed through incubating GST-tagged GSDMD and mutants with His-tagged nucleocapsid, followed by immunoblotting with antibodies against the indicated protein tags. GST-GSDMD, GST-GSDMD mutants and His-nucleocapsid were purified from E. coli strain BL21. Data information: Experiments were repeated for three times with similar results. (D) GST-GSDMD and its mutant lacking amino acids 265-284 were mixed with His-tagged nucleocapsid, followed by a GST-pulldown assay. Input and precipitates were immunoblotted with antibodies against the indicated protein tags. Data information: Experiments were repeated for three times with similar results. (E) GST-tagged GSDMD linker containing amino acids 270-280, 265-284 or 260-290 were incubated with His-tagged nucleocapsid, followed by a GST-pulldown assay visualized with Coomassie blue staining. Data information: Experiments were repeated for three times with similar results. (F) Schemes for GSDMD linker substitution and GSDMD chimera. GSDMD linker spanning amino acids 265-284 were replaced by a mCherry sequence except for the amino acid D275 (GSDMDsusti., upper panel). mCherry-GSDMD-C chimera was generated by displacing GSDMD N-terminus with mCherry (lower panel). The linker was also changed as above. (G, H) Wild-type GSDMD and GSDMDsusti. (G) or mCherry-GSDMD-C (H) were purified from HEK293T cells and incubated with recombinant p20/p10 complex and His-tagged nucleocapsid in a cleavage buffer, followed by immunoblotting with antibodies against the indicated protein tags or antibody against GSDMD C-termimus. Data information: Experiments were repeated for three times with similar results. (I) Plasmids encoding wild-type GSDMD, GSDMDsusti. or mCherry-GSDMD-C were co-transfected with nucleocapsid into HEK293T cells, followed by immunoprecipitation with antibody against FLAG. Input and precipitates were immunoblotted with antibodies against the indicated protein tags. Data information: Experiments were repeated for three times with similar results.
Figure 3A. TRAPP and COPII associate more stably with SGs than with ERES. The membrane­­ association of Sec24C was evaluated in non-permeabilized or permeabilized cells with or without SA treatment, as indicated. G3BP was used as an SG marker. Left panel insets, G3BP staining in non-permeabilized cells. Dashed white lines show the outline of permeabilized SA-untreated cells. Blue, DAPI. Data information; Scale bars, 10 µm. B. Stabilizing TRAPP and COPII at the ERES prevents their relocalization to SGs. HeLa cells overexpressing GFP-Sar1H79G were treated with SA and immunostained for Sec24C, TRAPPC2, and eIF3, as indicated. Blue, DAPI. Graphs show quantification of Sec24C or TRAPPC2 at SGs in GFP-Sar1H79G-expressing cells. Data are the ratio between Sec24C or TRAPPC2 mean intensity in SG puncta and Sec24C or TRAPPC2 mean intensity at ERES, expressed as a percentage of the non-transfected (NT) cells. Mean ± s.e.m. n = 60-70, three independent experiments. ****p<0.0001, Student's unpaired two-tailed t-test. Data information; Scale bars, 10 µm. C. Effect of Sec31A depletion on TRAPPC2 recruitment to SGs. Cells were mock-treated or KD for Sec31, treated with SA, and then immunostained for TRAPPC2 and eIF3 as indicated. Graphs, quantification of TRAPPC2 at ERES and SGs after SA treatment. Mean ± s.e.m., one representative experiment; n=60-80. ** p<0.001, ****p<0.0001, Student's unpaired two-tailed t-test. Data information; Scale bars, 10 µm. D. Effect of Sec31A depletion on Sec24C recruitment to SGs. Cells were mock-treated or KD for Sec31 and then immunostained for Sec24C and cTAGE5 (to stain ERES, top panels) and for eIF3 (bottom panels). Insets show eIF3. Graphs, quantification of Sec24C at ERES and in SGs after SA treatment. n=80. ****p<0.0001, Student's unpaired two-tailed t-test. Data information; Scale bars, 10 µm.
Figure 6A. WB analysis of TALDO1 expression B. WB analysis of phosphorylation at serine residues of the immunoprecipitated TALDO1 C. Relative NADPH/NADP+ ratio (n = 3, biological replicates, mean±SEM, Dunnett-t test) D. GSH contents (n = 3, biological replicates, mean±SEM, Dunnett-t test) Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA. E. Cellular ROS was measured by flow cytometry and shown in both curve (left) and bar (right) charts (n = 3, biological replicates, mean±SEM, Dunnett-t test) Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA. F. MMP detection with down- or up-regulation of lnc-AP/pep-AP in two groups of CRC cells. Scale bar, 10 µm.
Figure 5A, B Abolishments of force-induced binding residues by 3A mutations (MICA 3A, n = 526) and TAT mutations (MICA TAT, n= 580) impairs NKG2D's catch bond with MICA WT (n = 1505) (A) and IFN-γ release (B). Data information: Bond lifetimes in (A) of MICA WT and mutants with NKG2D came from at least 21 pairs of cells and beads of at least three repeated experiments. IFN-γ release of NK cells in (B) was one representative experiment of three total independent experiments. Data points represent mean values. Error bars represent mean ± SEM for biological triplicate experiments. **P < 0.01, ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test). 50% effective concentration (EC50) of NK cells stimulated by indicated MICA mutants Data information: horizontal lines represent mean values. Error bars represent mean ± SEM for biological triplicate experiments. **P < 0.01, ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test). IFN-γ release of NK cells stimulated by indicated MICA mutants at a concentration of 11nM (D) and 22nM (E). Data information: bars in (E), and data point in (F) represent mean values. Error bars represent mean ± SEM for biological triplicate experiments. **P < 0.01, ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test).
Fig.Figure 1a, Seckel cells contain normal centrosome numbers and mitotic spindle poles. Interphase centrosomes of Seckel cells have no CPAP immunoreactivity but recruit similar levels of centrosomal proteins (ii and iii). b, Seckel cells display overly long cilia. Arl13b labels cilium. Scale bar 1 μm. Quantification of cilia length, *** p<0.001; t-test; Scatter plot showing mean with ±SD (iii). Cells displaying normal (0-5 μm), long (5-10 μm) and extra long (>10 μm) cilia. *** p<0.001; Ordinary Two-way ANOVA (iv). Average cilia length of WT and Seckel cells (v). n = 3 independent experiments, 228 cells for WT and 297 cells for Seckel. Error bars are s. e. m. *** p<0.001, t-test. c, Electron microscopy (EM) of Seckel and WT fibroblasts. In contrast to WT, Seckel basal body has disorganized microtubule of doublet and triplets (red arrows and red circles in schematics).d, Longitudinal serial-sectioning EM of WT (top) Seckel fibroblasts (bottom). In contrast to WT basal bodies that have structurally normal distal and sub-distal appendages (yellow arrows and red triangles of DA and SDA in schematics), Seckel basal body displays either lack or misplaced appendages. n> 15 basal bodies.e, Seckel cells show reduced EdU incorporation after 72 hrs. Error bars are s. e. m. ** p<0.01, ttest; n = 4 independent experiments, 727 cells for WT and 844 cells for Seckel.
Figure 5A The effect of PH compounds was analysed using clonal lines bearing single copy integrations of the mini-dual fluorescent HIV-1 LTR reporter, md-HIV, where GFP is constitutively expressed from the EIF-1α internal promoter, and DsRed from the 5' LTR. B Fluorescence-activated cell sorting (FACS) of cells bearing md-HIV provirus indicates expression of EIF-1α-GFP and LTR-dsRed. Stimulation of the md-HIV lines generally causes a shift of the fluorescence profile from GFP+/DsRed- toward GFP+/DsRed+. C-D Clone 11 were treated with the indicated concentrations of PH02 (left panels) or PH02 in combination with 10 nM PEP005 (right panels) for 24 h and cells analysed by flow cytometry. Results presented as Δmean fluorescence intensity (ΔMFI) of DsRed expression and fold increase in DsRed expression for solo and combination treatments, respectively. Data Information: Mean and SE are determined from three biological replicates (n=3) and technical duplicates. Statistical significance, as determined by one-way ANOVA is indicated: ****, P<0.00005. The exact P-values are listed in the Appendix Table S1.  clone 131 were treated with the indicated concentrations of PH02 (left panels) or PH02 in combination with 10 nM PEP005 (right panels) for 24 h and cells analysed by flow cytometry. Results presented as Δmean fluorescence intensity (ΔMFI) of DsRed expression and fold increase in DsRed expression for solo and combination treatments, respectively. Data Information: Mean and SE are determined from three biological replicates (n=3) and technical duplicates. Statistical significance, as determined by one-way ANOVA is indicated: ****, P<0.00005. The exact P-values are listed in the Appendix Table S1.
linc-Myh restricts proliferation of MuSCs in an INO80-dependent manner.Figure 3RNA in situ hybridization proximity ligation assay (rISH-PLA) detects the close proximity of a specific RNA with proteins in situ modified from Roussis et al.. RNA in situ hybridization proximity ligation assay (rISH-PLA) detects the close proximity of a specific RNA with proteins in situ modified from Roussis et al.. (B-C) rISH-PLA confirms the proximity of linc-MYH to endogenous INO80-V5 in the nucleus of proliferating WT MuSCs (B). The specific signal is lost in linc-MYH deficient MuSCs (C). (n = 3 WT/3 KO biological replicates; Mann-Whitney test, *p = 0.05). Data are mean ± S.E.M. E-I Representative images of TA muscle sections of 10 weeks old mice stained for PAX7 (green) and EdU incorporation (red). MuSCs without (green arrows) and with EdU-labeling (red arrows) are indicated. (E, F) Deletion of linc-MYH results in increased numbers of muscle stem cells and pronounced increase of the ratio of EdU-positive MuSCs. (G-I) The increase in both the number of MuSCs and EdU positive MuSCs in linc-MYH mutant muscle is abolished by constitutive (G: Pax7-Crepos / INO80-/-, Ino80 KO; H: linc-MYH-/- / Pax7-Crepos / INO80-/-, dKO) and induced (I: linc-MYH-/- / Pax7-CreERT2pos / INO80-/-, dKO CreERT) deletion of INO80 in Pax7 expressing cells. Statistical evaluation of the number of muscle stem cells observed in TA muscles (J). ; (n = 5 WT/5 linc-MYH KO/4 Ino80 KO/5 dKO/3 CreERT dKO animals in J/K, n = 6 WT/6 linc-MYH KO/5 Ino80 KO/6 dKO/3 CreERT dKO animals in L **p < 0.01, ****p <0.0001, > 31 MSCs /animal in K; ANOVA test with multiple comparisons against WT. Sidak-Holm correction was used. Data are mean ± S.E.M.). Statistical evaluation of the ratio of EdU-incorporating MuSCs relative to all Pax7-positive MuSCs (K). ; (n = 5 WT/5 linc-MYH KO/4 Ino80 KO/5 dKO/3 CreERT dKO animals in J/K, n = 6 WT/6 linc-MYH KO/5 Ino80 KO/6 dKO/3 CreERT dKO animals in L **p < 0.01, ****p <0.0001, > 31 MSCs /animal in K; ANOVA test with multiple comparisons against WT. Sidak-Holm correction was used. Data are mean ± S.E.M.). Statistical evaluation of the number of myonuclei in myofibers of TA muscles from different genotypes (L) (n = 5 WT/5 linc-MYH KO/4 Ino80 KO/5 dKO/3 CreERT dKO animals in J/K, n = 6 WT/6 linc-MYH KO/5 Ino80 KO/6 dKO/3 CreERT dKO animals in L **p < 0.01, ****p <0.0001, > 31 MSCs /animal in K; ANOVA test with multiple comparisons against WT. Sidak-Holm correction was used. Data are mean ± S.E.M.).
NMDAR mediated AMPK activation is extracellular Ca2+-dependent.Figure 5(A) Representative immunoblots describing changes in the phospho-AMPK and total-AMPK levels at various time points after NMDA treatment in cortical synaptoneurosomes in the presence or absence of extracellular Ca2+. Note in each case phospho and total AMPK levels were normalized individually to their respective tubulin levels before calculating the phospho/total AMPK ratio. (B) Line graph showing the normalized average value for the synaptic phospho/total ratio of AMPK at various time points after NMDA treatment until 5 minutes of recovery in the presence or absence of extracellular Ca2+. Data presented as mean +/- SEM. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. **p<0.01, ****p<0.0001, n= 6 animals per group. (C) Representative images depicting intracellular Ca2+ levels through Fluo8 fluorescence (Pseudo-colored) of a cortical neuron plated at low-density before stimulation, 15 sec (immediate) after NMDA treatment, 300 sec (delayed) after NMDA treatment and after ionomycin treatment in the presence of 10mM Ca2+. Scale bar 20μm. (D) Representative time trace showing the normalized change in Fluo8 fluorescence on NMDA treatment in the presence or absence of extracellular Ca2+ and D-AP5. Values in all other groups were normalized to the unsimulated condition for the corresponding experiment and all the data points are represented as a fraction of the initial time point. Ionomycin treatment in the presence of 10mM Ca2+ was used to calculate fluorescence maximum for a particular cell. (E) Box plot showing the distribution of ΔF/F0 across multiple neurons on the basal condition, on NMDA treatment in the presence Ca2+, on NMDA treatment in the absence of Ca2+, on NMDA treatment with D-AP5 and on NMDA treatment along with MK-801. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. ****p<0.0001, n≥30 neurons per group from 3 independent platings.
Dynamic 5hmC patterns during iPSC conversion a.Figure 3Representative 5hmC profiles during AA+2i conversion of WT pre-iPSCs. Illustration of the 5hmC pattern is shown in the bottom panel. Venn diagram of called peaks at day 0, 2, and 10. Inset represents 5hmC pattern and the number of peaks displaying a pattern of enrichment. Heatmap of k-means clustered 5hmC patterns with histone modifications in pre-iPSCs and mESCs from Chronis et al (2017). Cis-element annotation of 5hmC patterns in clusters obtained by k-means in Fig 3c.
Figure 4A) Manhattan plot of genome-wide association for the levels of hepatic Pex16 transcript, where the only significant locus appears directly surrounding the genomic location (red arrow). Significant cut-offs are shown for FDR (blue) and Bonferroni (red). The peak SNP (rs27364570) is highlighted with a dark red box. Y-axis shows the -log10(pvalue) vs x-axis showing each SNP measured. P-values for GWAS associations were calculated using FaST-LMM. B) Allelic distribution comparing GG vs TT (x-axis) for the peak SNP of the Pex16 association (rs27364570), where the abundance of each LPC species (y-axis) showed significantly different levels depending on the allele. P-values for GWAS associations were calculated using FaST-LMM. Boxplots show mean (middle line), 25-75% quantiles (colored box) and 5-95% quantiles (vertical lines) C) Correlations between expression of hepatic Pex16 with LPC species and phenotypic traits. Box color indicates bicor value, where all relationships are positive and number in each box shows p-value for each correlation. Pvalues were calculated based on significance of regression (students test) and adjusted for multiple comparisons (FDR = 0.05)
Figure 4A. Schematic diagram of Co-IP combined with LC-MS/MS to screen potential proteins interacted with pep-AP. B. Silver staining of gels for separating proteins by Co-IP in HEK293T cells. C. GO annotation of molecular function for pep-AP-interacted proteins. Shown in the upper table and the lower chart, most of the proteins were involved in protein binding, of which TALDO1 with transaldolase activity was quite statistically significant (marked with a red box or a red circle). D. Survival analysis correlated with TALDO1 using TCGA data. Upper: the Kaplan curve of overall survival probability for patients with high or low level of TALDO1. Lower: the expression graph of TALDO1 in patients who were dead or not. E. WB confirmation of the interaction between pep-AP-Flag and TALDO1 in HEK293T, HCT116/L-OHP and SW480/L-OHP cells. F. IF detection of the colocalization of pep-AP-Flag and TALDO1 in HCT116/L-OHP and SW480/L-OHP cells. Scale bar, 10 µm. G. WB confirmation of the interaction between HA-pep-AP and TALDO1 in HCT116/L-OHP and SW480/L-OHP cells. H. IF detection of the colocalization of HA-pep-AP and TALDO1 in HCT116/L-OHP and SW480/L-OHP cells. Scale bar, 10 µm.
Figure 6(A) Euc1 Slx5-binding mutations impair Euc1 ubiquitylation. His-ubiquitin-modified proteins were enriched by denaturing NiNTA-PDs as described in Fig. 4E. 3FLAG-tagged Euc1 constructs were expressed from plasmids under the control of the ADH promoter. NiNTA-PD eluates were probed with FLAG and ubiquitin (P4D1) antibodies. Whole cell lysates (input) were probed with FLAG and Dpm1 antibodies. (B) The Slx5-SIMs and Slx5-Md are required for Euc1 ubiquitylation. NiNTA-PDs for His-ubiquitin as in (A). All strains expressed wild-type 3FLAGEuc1 from plasmids under the ADH promoter and His-ubiquitin. Slx5 constructs were expressed from plasmids under control of the endogenous promoter. WBs were probed as described in (A), Slx5-levels were probed using an HA antibody. Asterisk denotes a non-specific band. Slx5-SIM*: SIMs 1-4 were mutated as described (Xie et al, 2010), for SIM5, aa 477-479 (IIV) were mutated to alanines. To avoid possible mislocalization by deletion of the Slx5-Md, which overlaps with a putative NLS (Westerbeck et al, 2014), an N-terminal NLS was fused to all constructs. See Appendix Fig. S7C for HU complementation. (C) Euc1 Slx5-binding mutations (SBM1/SBM2) reduce/abolish ub-HSs and recruitment of Slx5 to ub-HSs. ChIP-qPCR for ub-K48 (top) or Slx5 (using Slx5 antibody, bottom) in euc1∆ ubx5∆ cells expressing the indicated constructs from plasmids under the control of the EUC1 promoter. Data represent means ± SD (n = 3). See also Appendix Fig. S7E-G. (D) Slx5-SIMs and the Slx5-Md are required for the formation of ub-HSs and recruitment of Slx5. ChIP-qPCR for ub-K48 (left) or Slx5 (anti-HA ChIP, right) in ubx5∆ cells or slx5∆ ubx5∆ cells complemented with plasmids expressing the indicated Slx5-constructs under control of the SLX5 promoter. Data represent means ± SD (n = 2). See also Appendix Fig. S7H. (E) Schematic model for the proposed sequence of events at ub-hotspots. See main text (Discussion) for details. SIM: SUMO-interacting motif, Md: middle domain, S: SUMO, Ub: ubiquitin.
Rearrangements of endocytic coat proteins during membrane invagination.Figure 4A Scheme of suggested topology of Sla1, Ent1, Sla2 and Pan1(1-1303) proteins on the flat (left) and invaginated (right) plasma membrane. Natively unfolded parts of the proteins are shown as thick black lines. The C-termini of proteins showing FRET are depicted as yellow circles; the C-termini separated from their FRET partners during invagination are shown as gray circles. The position of the clathrin lattice and polymerizing actin cytoskeleton are shown as a pale orange and red zone, respectively. Partial loss of FRET between Ent1-mTurquoise2 and Sla1-mNeonGreen during endocytic membrane invagination. FRET values (in %) of individual endocytic patches (n=74, 75; 69, 73 for (B) and (C), respectively) sorted according to the absence/presence of Abp1 protein are shown as box plots. Center, top and bottom lines of box plots show the medians, the 25th and 75th percentiles of individual datasets, respectively. Whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Notches indicate 95% confidence intervals of the medians. presence of FRET between Ent1-mTurquoise2 and Sla2-mNeonGreen during endocytic membrane invagination. FRET values (in %) of individual endocytic patches (n=74, 75; 69, 73 for (B) and (C), respectively) sorted according to the absence/presence of Abp1 protein are shown as box plots. Center, top and bottom lines of box plots show the medians, the 25th and 75th percentiles of individual datasets, respectively. Whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Notches indicate 95% confidence intervals of the medians.
Small intestinal fetal organoids recapitulate suckling-to-weaning transition in vitro.Figure 2Real-time qPCR analysis of fetal organoids cultured for one month showing decrease in relative expression of indicated neonatal markers A Ass1, B Blimp-1, C FcRn and D Lct and increase of the adult markers E Sis, F Treh and G Arg2 (n=3 individual wells from single organoid culture (see material and methods); experiment was repeated in four to eight independent organoid cultures with similar results). Enzyme activity assay of fetal organoids for H Lactase, I sucrase, J maltase, K trehalase and L arginase. Activity is given in µM glucose/µg protein·min-1 (experiment was generated from single organoid culture (see material and methods) and repeated in 3 independent organoid cultures with similar results).
Figure 7A The Age-BPs regulated by myrf-2 in body wall muscle (BWM). Age-BPs with more than 5 regulated genes were listed. Categories 1 and 2 are differentiated by capitalisation and bold fonts. Category 2 are shown below the corresponding Category 1. B, C The BWM-specific RNAi against myrf-2 improves sarcomere in the worms at day 8 of adulthood. Arrowheads and arrows respectively denote the gaps of myofilaments and the disorganised myofilaments with GFP aggregations. Scale Bar: 10 μm. Mean ± SD. Unpaired t-test, two-tailed. n>=50. D, E Suppressing myrf-2 in BWM increases worms' motility at day 5 of adulthood. The tracks were from worms' movement in 30 sec (D). The speed of worm movement was measured by the ratio of track length versus body length (E). Mean ± SD. n>=120. Unpaired t-test, two-tailed. F BWM-specific RNAi against myrf-2 extends worms' lifespan.