EMBO/sd-character-level-ner huggingface dataset:
Figure 2A. Normal SAG-induced ciliary accumulation of Smo. MEFs serum-starved for 24 h were treated with DMSO (mock) or 100 nM SAG for an additional 24 h. Arl13b served as ciliary marker. We also examined Gpr161 but failed to observe specific immunofluorescent signals, though the antibody, prepared by using human GPR161, recognized ciliary GPR161 well in human RPE1 cells (see Fig 3E). Quantification results were pooled from four MEF strains derived from two Rabl2+/+ and two Rabl2-/- embryos. B. Reduced Gli2 accumulation at the ciliary tip. MEFs were treated as in (A). Ac-tub served as ciliary marker. Arrowheads indicate ciliary tips. Quantification results were pooled from three independent experiments, each using MEFs from different embryos. At least 100 cells were scored in each experiment and condition. C. Reduced Gli1 expression. MEFs treated as in (A) were lysed for immunoblotting. Gapdh served as the loading control. For quantification, three independent experiments were performed, each using MEFs from different embryos. D, E. Increased Gli3 FL/R ratios in E11.5 hind limb buds and E10.5 mouse embryos. Relative band intensities of the full-length (FL) and the repressor (R) forms of Gli3 were quantified (red). FL/R ratios were from four different sets of limb bud preparations (D) and three different sets of embryo preparations (E).
Figure 3Schematic representation showing the establishment of XJF1XTX ROSA26+/rtTA ESCs. The TX allele allows Xist expression from its endogenous locus by dox addition, eventually leading to XCI. Sexing of established ESCs (gDNA-qPCR, Zfy/Gapdh), performed as described in Figure 1B. Detection of the number of X-linked Atrx gene foci by DNA-FISH with or without dox addition for 96 h (N >100), performed as described in Figure 1C. Detection of the Xist cloud in #B3 by RNA-FISH, with or without dox addition for 96 h ( N >200 each). A representative image is shown [magenta: Xist (white arrowhead), cyan: DAPI. Scale bar: 10 μm]. Allele-distinguishable RT-PCR for detecting Hprt, G6pdx, and Rnf12 X-linked genes. Bands with the anticipated sizes expressed from XJF1 or XTX alleles are shown. Nanog HR assay. The percentage of GFP-positive colonies after gene targeting to the Nanog locus is shown as described in Figure 2C. #B2 or #B3 were cultured with or without dox addition for 96 h. The combined number of GFP-positive cells and total colonies from all experiments are listed and summarized at right.
Brain sAPPβ shows an age-related increase in APP23, APP24, and APP51 mice.Figure 4Brain sAPPβ shows an age-related increase in APP23, APP24, and APP51 micesAPPβ was measured in Triton X-100 brain extracts from largely the same mice as analyzed in Figs1 and2 and is expressed as percentages of levels measured in the youngest age group.Swedish sAPPβ showed an age-dependent increase in APP23 mice following a linear trend (F(1, 83) = 52.914, P < 0.001); APP23 from two independent batches were included in this analysis (see Materials and Methods and Supplementary Fig S2 for details).Swedish sAPPβ showed an age-dependent increase in APP24mice following a linear trend (F(1, 84) = 11.264, P = 0.001).Human wild-type sAPPβ showed an age-dependent increase in APP51 following a quadratic trend (F(1, 18) = 68.980, P < 0.001).Data information: Post hoc Dunnett's test group comparisons were always conducted between the youngest group and all other groups. All data are represented as group means ± SEM; *P < 0.05; **P < 0.01; and ***P < 0.001. For absolute values, see Supplementary Fig S2.
Figure 7A. Exponential cells of indicated strains (below the bars) were plated onto LB agar and either were, or were not UV-irradiated. After overnight incubation, CFU were counted. UV sensitivity of the mutant strains (KO) was then calculated as the ratio between irradiated vs non-irradiated cells and normalized to this ratio from the wt strain As a consequence, the wt ratio is 1. The experiment was conducted 4x (biological replicates) and the bars show the geometric mean. The p-values, shown above the bars, were computed using two-tailed unpaired t-test for logarithms of the ratios. The error bars show ±SEM (computed on the log scale). B. Relative expression (mRNA) of helD, mfd, rho, greA, nusA, nusB, and nusG in the ∆rnjA strain (normalized to wt [set as 1]).
a) NRK cells stably expressing CFP–LC3; LAMP1–YFP were transfected with nonspecific (NS)- or.figf2(a) NRK cells stably expressing CFP-LC3; LAMP1-YFP were transfected with nonspecific (NS)- or Cltc (clathrin heavy chain)-RNAi and starved for 12 h. Scale bar, 5 μm. (b) Cells from a were assessed for enlarged autolysosomes after starvation and quantified. n = 100 cells from three independent experiments. Error bars indicate the s.d. (c) Representative TEM micrographs of nonspecific- or Cltc-RNAi-transfected NRK cells after 12 h of serum starvation. Scale bar, 5 μm. (d) NRK cell lines stably expressing LAMP1-Cherry; clathrin-GFP were starved for 0 or 4 h and observed by confocal microscopy. As a control, clathrin-GFP-expressing cells were starved for 0 or 4 h and then stained with MitoTracker red. Scale bar, 5 μm. The insets in a, c and d represent enlarged regions of interest. (e) Cells from d were analysed for Pearson's co-localization coefficent (R(r)). n = 30 cells from three independent experiments. Error bars indicate the s.d. (f) NRK cells stably expressing LAMP1-Cherry; clathrin-GFP were starved for 4 h and time-lapse images were acquired by spinning-disc microscopy. Scale bar, 500 nm. The white arrows indicate a clathrin punctum moving along a LAMP1-positive structure. (g) NRK cells were starved for 0, 4 or 6 h and then subjected to cell fractionation. Fractions were analysed by western blotting with antibodies against LAMP2 and clathrin heavy chain. 1 is the top fraction. The outline indicates the Lamp2-positive fraction. (h) Cell lines stably expressing clathrin-GFP were starved for 4 h, stained with antibodies against GFP and LAMP1, analysed by 3D-SIM. Scale bar, 2.5 μm. The right panels show enlarged regions of interest from the left panel: T, top view; L, lateral view; B, bottom view. (i) Left panel, representative TEM micrographs of purified autolysosomes from starved clathrin-GFP-expressing cells. Scale bar, 250 nm. Right panel, SIM image of purified autolysosomes stained with GFP and LAMP1 from the same experiment. Scale bar, 5 μm. (j) NRK or stable clathrin-GFP-expressing NRK cell lines were analysed by pre-embedding-immuno-electron microscopy using antibodies against GFP. Scale bar, 100 nm. (k) Quantification of the average number of gold particles per image from control or clathrin-GFP cells. n = 25 images from three independent experiments. The error bars indicate the s.d. Uncropped images of blots are shown in Supplementary Fig. S6.
Figure 1A, B. Height (A) and body mass index (B) of the participants by age in the diet groups compared to 2 SD intervals (shaded area) and median in the Finnish reference population. C. Dietary intake of selected macro- and micronutrients of participants. The shaded areas represent Finnish dietary recommendations for 2 to 5 years olds that covers majority of participants (THL, 2019). Energy intake recommendations vary by weight and therefore unambiguous limits could not be represented.
Fig.Figure 4(A) Average z-scored metabolome profiles are clustered for all estradiol-induced overexpression strains. GO enrichment analysis is performed on all clusters with the most widely shared biological process that is significantly enriched within that cluster indicated. (B) ROC curves indicate the recovery of 7 true-positive drug-target interactions within the dataset compared to the number of false discoveries from the remaining 1280 drugs within the dataset. The recovery of expected interactions is shown using Spearman correlation, Pearon correlation, and cosine similarity. These similarity metrics allow for an area under the curve of 0.72, 0.84, and 0.66 for those similarity metrics respectively (C) The number of occurances where each drug within the library is in the top 20 ranking interactions for each target is tabulated for drugs that cause siginificant (p-values < 0.05) changes in at least 5 metabolites.
Figure 6Deletion of HO-1 in myeloid lineage did not cause expansion of LT-HSCs and did not alter their cell cycle status. Scheme of the experiment assessing long-term effect of HO-1 deficient niche on function of HSCs. Long-term PB chimerism derived from HSCs transplanted to HO-1-/- recipients is lower than from HSCs transplanted to HO-1+/+ recipients. Chimerism among BM HSPCs fractions transplanted to HO-1-/- recipients are lower than from HSCs transplanted to HO-1+/+ recipients. Data are shown as mean ± SEM, n = 5-12/group total number of cells derived from HSCs transplanted to HO-1-/- recipients are lower than from HSCs transplanted to HO-1+/+ recipients. Data are shown as mean ± SEM, n = 5-12/group HSCs that were initially transplanted to primary HO-1-/- recipients did not reconstitute secondary HO-1+/+ recipients, in contrast to HSCs that were initially transplanted to HO-1+/+ recipients, n = 5-12/group. Scheme of the experiment assessing short-term homing of HSPC to HO-1 deficient niche. Short term homing of HSPC to HO-1+/+ or HO-1-/- BM did not differ. Data are shown as mean ± SEM, n = 5/group, unpaired t-test.
SOM230 increases in vivo sensitivity to gemcitabine of tumour xenograft (MIA PaCa-2-Luc cells and CAFs or human PDAC resection).Figure 5A-C MIA PaCa-2-GLuc cells were injected with or without CAFs into the pancreas of nude mice. Mice were treated with each indicated treatment (SOM230-LAR at day 7 and gemcitabine at days 7, 10, 14 and 17), and the plasmatic luciferase activity was measured (mean ± SD) at days 7, 14 and 21 (n = 5) (P = 0.003 for gemcitabine + SOM230LAR versus untreated) (A). Tumours were excised, photographed (scale bars are 1 cm) (B), weighted (P = 0.0008) (C) and paraffin-embedded for immunohistofluorescence analyses using an anti-cleaved caspase-3 antibody (E). D Human tumours were subcutaneously xenografted in nude mice and tumour volumes (mean ± SD) measured (at day 37, P = 0.0009 for gemcitabine + SOM230LAR versus untreated and P = 0.021 for gemcitabine + SOM230LAR versus gemcitabine). Mice were treated with the indicated treatments (SOM230-LAR at days 3 and 31, and gemcitabine at day 3 and twice a week thereafter). E, F Tumours excised from mice as in (A-C) were paraffin-embedded for immunohistofluorescence analyses using an anti-cleaved caspase-3 antibody (scale bar = 100 μm) (E). Cleaved caspase-3 quantification was performed by counting the mean number of positive cells per field in five independent tumours (mean ± SD) (F) (*P = 0.036, **P = 0.009). G, H Tumours excised from mice as in (D) were paraffin-embedded for immunohistofluorescence analyses using an anti-cleaved caspase-3 antibody (scale bar = 100 μm) (G). Cleaved caspase-3 quantification was performed by counting the mean number of positive cells per field in five independent tumours (mean ± SD) (H) (*P = 0.027).
Figure 5Gene expression of GPR56, SMO (left), and TGFb targets SRC, ZEB1, and ZEB2 (right) normalized to GAPDH in AML-491 cells determined by q-RT-PCR 4 hours after treatment with THZ1 0.5µM or LDC4297 2µM. Unpaired t-test. Mean, stdev, and individual values from three individual treatments. *** p<0.0005, ** p<0.005, * p<0.05. Setup of in vivo drug treatment. NSG mice were injected with 105 AML 04H112 cells. Four weeks post injection bone marrow (BM) was analyzed for human leukemic engraftment by BM aspiration. Treatment with either THZ1 or vehicle was started in the following week as indicated. BM was analyzed again after the end of the 4-week treatment period. Overall percentage of human (huCD45+) leukemic cells in mice before and at the end of the 4-week treatment period. Individual mice and mean engraftment are shown. Unpaired t-test. *** p<0.0005. n=10 mice for each group. Left: Representative FACS plots showing the typical CD34lowGPR56high profile of sample 04H112 before injection and after the 4-week treatment with THZ1. Right: both LSC compartments, the CD34-GPR56+ and the CD34+GPR56+ fractions were significantly reduced in vivo in the THZ1 treatment group. Individual mice and mean engraftment are shown. Unpaired t-test. *** p<0.0005, ** p<0.005, * p<0.05. n=10 mice for each group. Left: Overall percentage of human (huCD45+) leukemic cells and the fraction of CD34+GPR56+ among human cells (mean, individual mice) two weeks after drug withdrawal. Right: FACS plot visualizing that the CD34+GPR56+ is re-established upon drug removal. Unpaired t-test, *** p<0.0005, ** p<0.005, * p<0.05. Correlation plot showing an anti-correlation between the percentage of CD34+GPR56+ cells in an AML sample and the corresponding IC50 for THZ1 determined in the respective sample. Pearson correlation. Each dot represents one sample. FACS histogram plot (left) and summary bar graph from three replicate wells (right) showing reduction of GPR56 surface expression on the GPR56 positive AML cell line HEL only in conditions that contain the more specific CDK7 inhibitor YKL-5-124, but not those that contain only the specific CDK12/13 inhibitor THZ531. Unpaired t-test, bars and error bars represent mean and standard deviation of three individual treatments, *** p<0.0005, ** p<0.005, * p<0.05. SRF reporter showing dose dependent suppression of the GPR56-CTF induced SRF signal by the CDK7 inhibitor YKL-5-124, but not by the CDK12/13 inhibitor THZ531. Four technical replicates. One of four individual experiments. Unpaired t-test, bars and error bars represent mean and standard deviation, *** p<0.0005, ** p<0.005, * p<0.05.
Figure 4(A Here we show the dynamics of the HPA axis during and after a prolonged pulse of stress, described in Figure 3, for KGR=2 (strong feedback), KGR=4 (moderate feedback), and KGR=8 (weak feedback). Stronger feedback from the GR attenuates the dysregulation of all HPA axis hormones. B) Here we show the dynamics of the HPA axis during and after a prolonged pulse of stress, described in Figure 3, for KGR=2 (strong feedback), KGR=4 (moderate feedback), and KGR=8 (weak feedback). Stronger feedback from the GR attenuates the dysregulation of all HPA axis hormones.
Figure 5(A) Yeast two-hybrid analysis identified that Su(H) is the only Notch signaling key component that interacts with HP1c, while lamin mated with T-antigen was used as negative control and p53 mated with T-antigen as positive control. The interactions between HP1c and Su(H) were confirmed in both orientations. (BD: binding domain, AD: activation domain, QDO/X/A: Quadruple dropout medium: SD/-Ade/-His/-Leu/-Trp supplemented with X-a-Gal and Aureobasidin A). (B) The main functional domains of HP1c and Su(H). (C) The CSD domain of HP1c and the BTD domain of Su(H) are responsible for the interaction by yeast two-hybrid analysis. (D, E) The interaction between HP1c and Su(H) is further confirmed through co-immunoprecipitation (Co-IP). Plasmids expressing HP1c-eGFP and Su(H)-flag were transfected into S2 cells. Immunoprecipitation and western blotting were performed with the indicated antibodies. (F) Plasmids expressing HP1c CSD domain and Su(H) BTD domain were transfected into S2 cells. Immunoprecipitation and western blotting were performed with the indicated antibodies.
Figure 2A. Domain structure for IDAP1 and alignment of IDAP1 homologs with the PIF/Harbinger transposase. The catalytic DDE triad in the PIF/Harbinger nucleases is indicated by black boxes. B. Domain structure for IDAP2 and alignment of the myb-like DNA-binding domains of IDAP2 homologs. The putative triple conserved W residues in the PIF/harbinger myb-like DNA-binding domain are indicated by black boxes. C. Phylogenetic analysis of IDAP1. Clade I (IDAP1) is in red, Clade II (AT3G19120) is dark blue, Clade III (AT3G55350) is pale blue, Clade IV (ALP1) is yellow, Clade V is golden, and Clade VI (Harbinger) is light green. GenBank accession numbers are prefixed gi, and the others are accession numbers for sequences retrieved from the NCBI or OneKP databases.
ATG13-mediated rescue of ATG9A accumulation at p62/SQSTM1 clusters requires the ATG13 HORMA domain but is independent of ULK1.Figure 5(A) Endogenous p62/SQSTM1 level was measured in HCT-116 ATG9A-HA KI-ATG13 WT, ATG13 KO OR ATG13 KO cells reconstituted with ATG13 WT, ATG13 ΔHORMA and ATG13 Δ2AA by immunoblotting with indicated proteins (left). Cells were grown in full DMEM media and whole cell lysates were subjected to immunoblotting (left). Quantification of normalized p62 infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests (right). (B) HCT-116 cells in A were incubated in EBSS for 4 hours then processed and analyzed as in A. (C) Confocal images of ATG9A colocalization with p62/SQSTM1 in HCT-116 ATG9A-HA KI- ATG13 WT, ATG13 KO or ATG13 KO cells reconstituted with ATG13 WT, ATG13 Δ2AA and ATG13 ΔHORMA. Cells were grown in full DMEM media, fixed, labelled with antibodies for HA and p62/SQSTM1 and imaged (Scale bar=10 µm). Images are from the same single independent experiment (D) Quantification of average surface area of p62/SQSTM1 puncta in C. Mean ± SEM, n=1 independent experiment with 30 technical replicates. A break was inserted in the Y axis to accommodate the wide range of p62/SQSTM1 puncta sizes. (E) Quantification of ATG9A colocalization with p62/SQSTM1 in C. Mean ± SEM, n=1 independent experiment with 30 technical replicates.
ER exit of Orm2 is regulated by TORC2-Ypk1 phosphorylation and essential for Orm2 degradation.Figure 7(A) Left panel: In tor1-1 avo3∆1274-1430 cells rapamycin specifically inhibits TORC2 but not TORC1 (Gaubitz et al., 2015). tor1-1 avo3∆1274-1430 cells were treated with cycloheximide (CHX) and rapamycin or vehicle (methanol) for the indicated times. Right panel: WT or Orm2 mutant cells were treated with CHX for the indicated times. The protein levels of FLAG-Orm2 or of the indicated FLAG-Orm2 mutants from total cell lysates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. (B) Graphs display the FLAG-Orm2 protein levels determined by densitometric quantification of Western blots in cell lysates of WT cells and the indicated mutants 0, 90 and 180 min after the addition of CHX. Each experiment was repeated at least three times. FLAG-Orm2 levels were normalized to Pgk1 loading controls, and each timepoint was related to t=0 min (set to 1). Data are presented as mean ±standard deviation. (C) Denaturing immunoprecipitation (IP) of FLAG-Orm2, FLAG-Orm2-3A and FLAG-Orm2-3D. The control strain expresses untagged Orm2. Input and elutions (with FLAG peptides) were analyzed by SDS-PAGE and Western blot with the indicated antibodies. (D) Epifluorescence and phase contrast microscopy of living tul1∆ mutants expressing GFP-Orm2, GFP-Orm2-3A or GFP-Orm2-3D (green) with dsRed-HDEL (red). Arrowheads point to GFP-Orm2 accumulations outside the endoplasmic reticulum (ER). Scale bars = 5 µm. (E) SDS-PAGE and Western blot analysis with the indicated antibodies of input and elution (with FLAG peptide) from native anti-FLAG co-immunoprecipitations from cells expressing FLAG-Orm2-K25,33R, FLAG-Orm2-K25,33R-3A or FLAG-Orm2-K25,33R-3D. The control strain expresses untagged Orm2. (F) The levels of the long chain bases (LCB) C18-dihydrosphingosine (C18-DHS) and C18-phytosphingosine (C18-PHS) and of two major ceramide (Cer) species in lipid extracts from WT, tul1∆, Orm2-K25,33R, Orm2-3A and Orm2-3D mutants were measured using LC-MS and quantified using spiked non-yeast LCBs/ceramides as internal standard. Data are normalized to WT levels of the most abundant LCB/ceramide species (set to 1) and presented as mean ±standard deviation from three independent experiments. Pairwise statistical significance was assessed by Student's t-test. p values are given above the respective bars. (G) Model for the dual function of the Dsc complex in EGAD-dependent proteasomal as well as ESCRT-dependent lysosomal membrane protein degradation.
Figure 3A-C Details of intramolecular F-K (A) and intermolecular F-K' (B) and K-K' (C) interactions as seen in simulations restrained by EM maps. Side chains of main interacting residues are shown in stick representation and hydrogen bonds are shown as orange dashed lines. D The kinase is oriented with the ATP binding site facing the membrane. Key interactions between the kinase and the membrane are made by K621 and K627. The side chains of K621, K627 and K216 (the latter is within the KAKTLRK motif of a neighboring FERM domain) as well as AMP-PNP bound to the ATP binding site are shown in stick representation. E Electrostatic surface potential of the symmetric FAK dimer as seen from the membrane proximal view. Both, the FERM (KAKTLRK) and the kinase (K621, K627) have extensive positive charges on this surface for interaction with negatively charged PI(4,5) P2 lipids. The kinase domains are encircled by a yellow dotted line.
Figure 2A,B Morphological changes in (A) normal colon (N1-3) and (B) surviving TA organoids upon TGFβ treatment resemble the induction of a mesenchymal phenotype in these usually well-organized epithelial structures (scale bars: 50 μm). C Genes present in an EMT signature (Gröger et al. 2012) are enriched in TGFβ-treated compared to control samples. D,E The EMT-inducing transcription factor ZEB1 is strongly induced upon TGFβ treatment both in (D) normal as well as (E) TA organoids. F The mesenchymal marker fibronectin 1 (FN1) is induced following TGFβ stimulation.
NDP52 promotes CHIKV infection in a species‐specific manner.Figure 5NDP52 promotes CHIKV infection in a species‐specific manner. MEF cells were treated with CTRL or NDP52 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (A) and viral production (B) were measured. Yeast cells expressing Gal4 DNA BD fused alone (empty vector) or to nsP2 were cotransformed with a plasmid encoding the Gal4 AD fused to hNDP52 or mouse NDP52 (C). MEF cells were infected for 3 h and transfected with empty plasmid (CTRL) or hNDP52‐3XFLAG. Immunoprecipitation experiments were performed 24 h p.i. using antibodies to FLAG or nonspecific IgG controls. Immunoprecipitated proteins were revealed with anti‐nsP2 antibody (D). MEF cells were infected for 3 h and transfected with empty plasmid (CTRL), hNDP52, hLC3‐C or hNDP52 and hLC3‐C. Viral production (E) and cell mortality (F) were assessed 24 h post transfection. Isogenic wt or Atg5−/− MEFs were infected for 24 h and cell mortality (that is, fold change relative to mock‐infected cells) (G) and viral production (H) were assessed. MEF cells were infected and immunoprecipitation experiments were performed 24 h p.i. using antibodies to p62 or nonspecific IgG controls. Immunoprecipitated proteins were revealed with anti‐capsid antibody (I). MEF cells were treated with CTRL or p62 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (J) and viral production were assessed. Whole‐cell lysates of siRNA‐treated cells were immunoblotted for p62 or actin (K).