EMBO/sd-character-level-ner huggingface dataset:
Figure 7Figure 7. Suppression of Prmt1 abolishes β-catenin or Hoxa9 independent transformation in HSC-derived MLL-CSCs. (A) The relative number of colonies from Hoxa9-/- LSK-MLL-ENL leukemic cells with empty vector or shPRMT1. Data are represented as mean ± SD. (B) The % of CD45.2+ donor cells in the bone marrow of recipient mice at the indicated time points post-transplantation of Hoxa9-/- LSK-MLL-ENL leukemic cells with empty vector or shPrmt1. Data are represented as mean ± SD. (C). Kaplan-Meier survival curve of secondary recipient mice transplanted with Hoxa9-/- LSK-MLL-ENL leukemic cells with vector control or shPrmt1. (D) The relative number of colonies from Ctnnb1-/- LSK-MLL-ENL leukemic cells with empty vector or shPRMT1. Data are represented as mean ± SD. (E) The % of CD45.2+ donor cells in the bone marrow of recipient mice at the indicated time points post-transplantation of Ctnnb1-/- LSK-MLL-ENL leukemic cells with empty vector or shPrmt1. Data are represented as mean ± SD. (F). Kaplan-Meier survival curve of secondary recipient mice transplanted with Ctnnb1-/- LSK-MLL-ENL leukemic cells with vector control or shPrmt1. See also Appendix figure S6.
Analysis of MiaPaCa-2 cell organelles after ultracentrifugation.Figure 8(A) Overlay of the proton NMR spectrum fragments of the following suspensions (from top to down): untreated first (lysosomal) cell fraction after ultracentrifgation and one washing (F1*), iodixanole (Optiprep) suspension, lower (F3), middle (F2) and upper fraction (F1) of the ultracentrifugates after two washings. The chemical shifts of the spectra were calibrated to the Choline/Phosphatidylcholine (Cho/PC) signal. Apart from iodixanol, we could identify the PUFA groups (methylene groups situated between two CH = CH groups), Cho/PC and glycerophosphocholine (GPC) in F1* and F1-3. Furthermore we observed lactate and the methylene groups of fatty acids (at 1.3 ppm), citrate (at 2.55 and 2.7 ppm) and phosphoethanolamine (at 3.1 ppm) in the F1* but not in the F1-F3 groups.. (B) Western blot analysis of LAMP-1, a late endosome marker, which was nearly identically distributed over the first two fractions compared to Cathepsin-D, in the controle group, but ocurred only in the first fraction in the OMP treated group. β-COP as an indicator of the Golgi-complex, is found strongly in the lower fraction in the control group, but very weakly in the OMP treated group. Cathepsin - D, an early endosome marker, which can be found in the upper and middle fraction of the control group, but regarding the OMP group, it was only found in the first one. (E) Overlay of NMR spectra of all fractions of control and OMP. The most relevant difference is the weaning of the GPC signal after OMP treatment in the 1st fraction after only 6 hours.
Figure 3Representative images of immunostaining for p53 on small intestine sections. Arrows indicate p53-positive cells (A). Quantification of the percentage of p53 positive cells per crypt (n=5-6 mice per group) (B). Representative images of γH2AX antibody staining on small intestine paraffin sections from WT, Gadd45a-/-, G3Terc-/-, and G3-dKO mice (7 months old). Arrows indicate γH2AX positive nuclei (C). Quantification of the percentage of γH2AX positive cells per small intestine crypt (n=4-6 mice per group) (D). Representative images of small intestine crypt sections stained with antibody against pATM (Ser-1981)(E). Quantification of frequencies of pATM (Ser-1981) positive crypt cells (n=4-6 of 7-month old mice per genotype) (F). Representative image of the anaphase bridge (indicated with arrows) in the basal crypt of G3Terc-/- mice (G). The percentage of cells with anaphase bridges in pH3 positive basal crypt cell population (n=4-6 mice per genotype) (H). Heat map indicating the relative expression levels of DDR and cell cycle-related gene transcripts in MEFs from indicated genotypes (Data are extracted from RNAseq datasets).
PI(3,5)P2 attenuates spongiosis and lysosomal dysfunction.Figure 6A: Transient overexpression of Flag-tagged zDHHC9+21 (72 hours) restored PIKfyve levels in prion-infected Gt1 cells. Right: Western blot quantification. Each dot represents an individual experiment (unpaired t-test). **: p<0.01. Error bars represent s.e.m. B: Cotransfection of zDHHC9+21 reduced vacuolation in prion-infected Gt1 cells. Each dot represents a separate experiment (1000 cells/experiment, χ2: p<0.001). **: p<0.01; unpaired t-test. Error bars represent s.e.m. C: Prion-infected Gt1 cells were lentivirally transduced with GADD34 at 75 dpi. Control: empty lentiviral vector. At 79 dpi, GADD34 overexpression had normalized eIF2α phosphorylation and restored PIKfyve levels. Right: Quantification of western blots (n=3; unpaired t-test). Each dot represents an individual experiment. **: p<0.01. Error bars represent s.e.m. D: Number of vacuolated cells from the experiment shown in C. GADD34 expression rescued the total number of vacuolated cells (1000 cells/experiment, χ2: p<0.001). Each dot represents an individual experiment. **: p<0.01. Error bars represent s.e.m. E: tga20 COCS were infected with RML and optionally treated with bPIP (5µg/ml). At 45 dpi, NeuN morphometry revealed ablation of cerebellar granule layer (CGN) in prion-infected slices and its rescue by bPIP. Control: NBH treated COCS. Each dot represents an individual slice (Statistics: ANOVA). *: p<0.05. **** p<0.0001. F: tga20 COCS were treated with POM1 (optionally pre-blocked with recPrP) and treated with bPIP (5µg/ml). At 14 dpi NeuN morphometry revealed POM1-induced ablation of cerebellar granule layer (CGL) and rescue by bPIP. G: Prion-infected Gt1 cells were treated with bPIP for 3 days. The number of vacuolated cells was reduced (1000 cells/experiment, χ2: p<0.001); Statistics: Chi-square test. Each dot represents an individual experiment. **: p<0.01. Error bars represent s.e.m.
Hsp70 co‐chaperones Sis1 and Sti1 modulate protein aggregationS.Figure 5S. cerevisiae hsp42Δ and hsp42Δ tet‐off sis1 cells expressing GFP‐VHL were grown for 20 h in the absence (−Dox) or presence (+Dox) of doxycycline at 30°C and shifted for 90 min to 37°C in the presence of MG132. Changes in protein localization were monitored. DNA was stained by DAPI (blue). The total number of GFP‐VHL foci per cell and frequencies (%) of INQ formation were determined. Scale bars, 2 μm. B S. cerevisiae wt, hsp42Δ, sti1Δ, and hsp42Δ sti1Δ cells expressing GFP‐VHL were grown at 30°C and shifted to 37°C in the presence of MG132. Changes in protein localization were monitored. The total number of GFP‐VHL foci per cell and frequencies (%) of INQ formation were determined. Scale bars, 2 μm.
Figure 4(A) Weight at weaning of BrafAVKA mice from two different founders showed no significant differences. 215: n = WT = 24/ Heterozygous = 32/ AVKA = 19; 273: n = WT = 114/ Heterozygous = 117/ AVKA = 52, mean + SEM, t-test (B) Homozygous BrafAVKA mice show reduced splenic cellularity. n = 7, mean + SEM, t-test, * p < 0.05. (C-E) Relative number of CD8+ T cells. FACS analysis shows an increase in naïve cells (C), whereas differentiated TEM (D) and Tregs (E) reveal a reduction in BrafAVKA mice compared to WT. n = 8, mean + SEM, t-test, * p < 0.05, ** p < 0.01. (F) Primary B cells of BrafAVKA mice show reduced CD69 surface expression upon LPS and anti-IgM stimulation. n = 3, mean + SEM, test, ** p < 0.01. (G) Primary B cells also show reduced proliferation upon stimulation compared with WT B cells. n = 3, mean + SEM, test, ** p < 0.01
Fig.Figure 2 In yeast, the OST1- and ABF2-mediated reporter activity was inhibited by ABI1 (‑RCAR). The subsequent recovery of the response was determined by co-expressed ABA receptors RCAR1-RCAR14 belonging to three subfamilies, in the presence or absence of 30 µM ABA. The ABA response is expressed relative to the response obtained by replacing ABI1 with the catalytically inactive ABI1D177A. Regulation of an ABA-responsive LUC reporter in Arabidopsis protoplasts by ectopic expression of 1 µg ABI1-effector in combination with 5 µg ABA receptor DNA (± 10 µM ABA) per sample containing 105 protoplasts.
S100A9 expression inhibits wild-type HIV-1 infection of MoLC and eLC(A) Epidermal cells transfected with control siRNA (siCtrl) or S100A9-targetting siRNA (siS100A9) were challenged for 72h with 200 ng P24 of HIV-1-R5 (left dot-plots) or HIV-1 primary isolate (right dot-plots).Figure 5Epidermal cells transfected with control siRNA (siCtrl) or S100A9-targetting siRNA (siS100A9) were challenged for 72h with 200 ng P24 of HIV-1-R5 (left dot-plots) or HIV-1 primary isolate (right dot-plots). Cells were fixed, permeabilised and stained with langerin (Y-axis) and HIV Gag P24 (X-axis) fluorescently coupled antibodies. Samples were acquired on a Flow cytometer. The experiment shown is representative of four donors for HIV-1-R5 (n=4) and five donors for HIV-1 primary isolates (n=5). The same as above was done but on MoLC gated by size (FSC). The experiment shown is representative of four donors for HIV-1-R5 (n=4) and three donors for HIV-1 primary isolates (n=3). Data collected from all donors were compiled and represented as bar charts for cells challenged with HIV-1-R5 and HIV-1 primary isolates. Nevirapine (Nev) (10μM) was used as control when indicated and data were represented as means ± SD of HIV-Gag+ cells from at least 2 (for siS100A9-MoLC infected by HIV-1 primary isolates) to 3 donors. When applicable, student t-test was performed to determine statistical significance (*P<0.1;**P<0.01;***P<0.001).
CDC7 promotes the nucleolytic degradation of reversed forks in BRCA2 deficient cells.Figure 5A. U2OS cells were transfected with control (siLuc) or BRCA2 targeting siRNA (siBRCA2). After 48 hours cells were either mock treated or treated with 10 μM XL413 for 30 minutes and labelled consecutively with CldU (red) and IdU (green), followed by treatment with 4 mM HU for further 5 hours. B. Representative western blot of indicated proteins obtained from whole cell extracts. pS40/41 on MCM2 indicates effective CDC7 inhibition by XL413. C. Ratios of IdU/CldU tracts length is plotted. The box extends from the 25th to the 75th percentile with the line in the box representing the median. Whiskers indicate the 10-90 percentiles with data outside this range for individual outliers being plotted as dots. Mean values of IdU/CldU ratios are indicated above the plot. At least 100 replication forks were analyzed for each condition. D. The mean values of IdU/CldU tract length ratios from three independent experiments are plotted, with the standard deviation (blue lines) and mean (red line). Statistical analysis: student t-test; ns, not significant; **P<0.01, ***P < 0.001 E. Electron micrographs of representative replication forks from U2OS cells A regressed arm is labelled: P=parental strand; D=daughter strand; and R=reversed fork; the four-way junction at the reversed fork is magnified in the inset. The red arrows indicates single stranded DNA at the fork. Scale bar is 200 nm ( = 460 bp), 100 nm ( = 230 bp) in the inset. F. Frequency of reversed replication forks isolated from mock-depleted (siLuc) and BRCA2-depleted (siBRCA2) U2OS cells upon 5 hour treatment with 4 mM HU in the presence and absence of 10 μM XL413. The number of replication intermediates analyzed is indicated in parentheses. Similar results were obtained in two independent experiments (Appendix Table G. Amount of ssDNA length at the junction (Red arrows in Figure 4D) in siLuc and siBRCA2 U2OS cells treated with 4 mM of HU for 5 hours in the presence or absence of XL413. N indicates the number of forks observed, and only molecules with detectable ssDNA stretches are included in the analysis. The lines show the mean length of ssDNA regions at the fork and the value is displayed above the plot
Fig.Figure 4(a Differentially expressed transposable elements in sperm from Tesa-inj and MSUS males. Data represent genes with P < 0.05 and similar fold change. Fold change in heat map represents log2(fold change) respective to the corresponding control group. Total overlap is presented in Extended Data Fig. 11. b) mRNAs/lincRNAs in sperm from Tesa-inj and MSUS males. Data represent genes with P < 0.05 and similar fold change. Fold change in heat map represents log2(fold change) respective to the corresponding control group. Total overlap is presented in Extended Data Fig. 11. (c,d) (c) Volcano plot and (d) heat map of differentially expressed (FDR < 0.05) mRNA/lincRNA in sperm from Tesa-inj males. Dashed black lines in (c) represent y = 1.3, equivalent to FDR = 0.05, and x = ±0.03, equivalent to FC = 1.23 and 0.81. Black dots represent top candidates, which all have FC > 2 or FC < 0.5, and FDR < 0.05. Teal dots represent non-significant genes. (e) GO enrichment scores for differential RNA in Tesa-inj male sperm. Enrichments calculated with P < 0.05. For Tesa-inj n = 6 and Vehicle-inj n = 7, MSUS n = 4, Control n = 3. Dashed black line represents FDR = 0.05. FDR; false discovery rate, FC; fold change.
Figure 6A. Basolateral release of 14C-labeled fatty acids in Caco2 cells grown in a transwell system and treated with CRT0066101 at the indicated concentrations for 24 hours. n=6. B. Basolateral release of 14C-labeled fatty acids in Caco2 cells expressing shscramble or shPkd2 in a transwell system and treated with CRT0066101 for 24 hours. n=8. C. Western blot analysis of phosphorylated PKD2 in jejunum of male C57BL/6 mice in HFD and treated with CRT0066101 or control (water) for 13 weeks. n=3. D. Energy content per gram of feces of male mice in HFD and receiving daily gavage of CRT0066101 or water as control. n=6 for control and n=5 for CRT0066101. E. Lipids tolerance test after oral gavage of olive oil to male mice after 9 weeks in HFD and receiving daily CRT0066101 or control. n=6 for control and n=5 for CRT0066101. F. WB of Apolipoprotein A4 (APOA4) in jejunum of male C57BL/6 mice in HFD and treated with CRT0066101 or control (water) for 13 weeks. Quantification of bands normalized to GAPDH and relative to control (water) n=6. G. Body weight of male C57BL/6 mice in HFD and treated with CRT0066101 or control (water) for 13 weeks. n=6 for control and n=5 for CRT0066101. H. Glucose tolerance test in male C57BL/6 mice which received CRT0066101 or water (control). Measurements after 10 weeks of treatment and HFD feeding. n=6 for control and n=5 for CRT0066101. I. Insulin tolerance test in male C57BL/6 mice which received CRT0066101 or water (control). Measurements after 11 weeks of treatment and HFD. n=6 for control and n=5 for CRT0066101.
A detailed spatial map of the nucleolar human proteome.Figure 1A. Schematic overview of the nucleolus and its substructures: fibrillar center (FC), dense fibrillar component (DFC), and granular component (GC). B. UTP6 in U-2 OS cells exemplify proteins localized to whole nucleoli (HPA025936). C. Fibrillar center localization shown by a UBTF IF staining in U-2 OS cells (HPA006385). D. UMAP visualization of the IF images generated in the HPA Cell Atlas (also shown in Fig 3A). The images from singularly localizing nuclear proteins are highlighted in purple (nucleoli), blue (fibrillar center), brown (nuclear speckles) and orange (nuclear bodies). Multilocalizing nucleolar proteins are highlighted in yellow. E. Dual localization of LEO1 to both the fibrillar center and nucleoplasm in GFP-tagged HeLa cells (magenta), also supported by IF antibody staining using HPA040741 (green). F. The multilocalizing ribosomal protein RPL13 detected in the nucleoli, cytosol and ER in MCF-7 cells (HPA051702).
Figure 6A,B,C AUCell scores for the γδ TCR-induced signature (A), γδ TCR only signature (B) and αβ and γδ TCR shared signature (C) are displayed on UMAP (left) and violin (right) plots of the 7 sorted subsets corresponding to Dataset 3. As expected, AUCell scores for the 3 signatures were very low for DN3a cells, low for WT DN3b γδ- and WT DN4 γδ- cells, and high for WT DN3b γδ+ and WT DN4 γδ+ cells.
EGAD-degradation of Orm2 contributes to sphingolipid homeostasis.Figure 6(A) The levels of two major ceramide (Cer) species in lipid extracts from WT (black), tul1Δ (dark gray) or Orm2-K25,33R mutants (light grey) were measured using LC-MS and quantified using spiked non-yeast ceramides as internal standard. Data are normalized to Cer44:0;4 levels of WT cells (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. (B) Autoradiogram of sphingolipid extracts from [3H]serine radiolabelled WT cells and tul1∆ mutants were separated by thin layer chromatography. IPC, Inositolphosphorylceramide; MIPC, mannosylinositolphosphorylceramide; MIP2C, mannosyldiinositolphosphorylceramide. X indicates an unknown lipid species that is insensitive to myriocin treatment. (C) 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-WT or FLAG-Orm2-K25,33R. Control cells expressed untagged Orm2 WT. The asterisks label unspecific cross-reactions of the respective antibodies. SDS-PAGE or Phos-tag-PAGE and Western blot analysis with the indicated antibodies of total cell lysates (D) from WT cells expressing FLAG-Orm2 or FLAG-Orm2-K25,33R The asterisks label unspecific cross-reactions of the respective antibodies. SDS-PAGE or Phos-tag-PAGE and Western blot analysis with the indicated antibodies of total cell lysates (E) from WT cells and tul1∆ mutants expressing FLAG-Orm2, FLAG-Orm2-3A or FLAG-Orm2-3D with or without Myriocin treatment (1.5 µM) for one hour. (F) Schematic representation of the regulation of SPT activity via TORC2-Ypk1/2-dependent phosphorylation of Orm proteins and EGAD-dependent degradation of Orm2.
Figure 4Duolink analysis (A) and its quantification (B) of the interaction by assessing the PLA complex in mitotic cells after BCL9 or Axin1 knockdown (n=20). Scale bars represent 5 μm. Duolink analysis of the interaction of BCL9 and CLTC in mitotic cells. Scale bars represent 5 μm. Schematic diagram of 20 truncated BCL9 forms designed for subsequent immunoprecipitation analysis. Immunoprecipitation analysis of the interaction of truncated BCL9 forms in mitosis; the purple stars indicate the domain required for clathrin complex binding, and the green stars indicate the domains for β-catenin complex binding. The domain required for both clathrin and β-catenin complexes binding is indicated by a red line. Quantification of the relative binding of the truncated forms with the clathrin complex or β-catenin complex; the red frame indicates the common domains of BCL9 for binding of both complexes. Immunoprecipitation analysis of the BCL9-MF interaction in CLTC knockdown mitotic cells. Immunoprecipitation analysis of the endogenous clathrin interaction with LRP6 signalosome components in BCL9 MF-overexpressing mitotic cells.
The Tfcp2l1-FAO axis extends the lifespan of ex vivo blastocysts during paused pluripotency.Figure 7A. A schematic diagram showing the strategy of generating Tfcp2l1 knockout (KO) mice. E2, exon 2 (purple box); triangle (red arrow), loxP site; HET, heterozygotes; KO, homozygotes. B. Genotyping results of blastocysts (E3.5) from the breeding of heterozygotes. Numbers (n) of each genotype are shown. NS, not significant based on chi-square test. C. Schematics showing the procedure of testing the functions of Tfcp2l1 and FAO in the survival of paused blastocysts induced by INK-128 treatment. Two representative images for live and death blastocysts are shown. Scale bars=125 µm. D. Kaplan-Meier curve of ex vivo blastocysts untreated or treated with 200 nM of INK-128. n, number of blastocysts of each genotype. p-value was calculated using the log rank test. E. Kaplan-Meier curve of ex vivo WT blastocysts cultured in the M2 medium in the presence of 200 nM of INK-128 with or without 50 µM of etomoxir (Eto) and/or 100 µM of octanoate, as indicated. p-values were calculated using the log rank test. F. A model showing the pro-survival function of the Tfcp2l1-Cpt1a-FAO in response to mTOR inhibition. Dashed arrows, the activity is reduced; question mark, a possible connection but need further studies.
Figure 4USP1-UAF1 can efficiently deubiquitinate D2Ub in the presence or absence of FANCI (I), but not in the presence of ubiquitinated FANCI (IUb). Ubiquitinated FANCD2 (D2Ub) was mixed with a 50 base pair dsDNA and either His6-TEV-V5-FANCI (I), ubiquitinated His6-TEV-V5-FANCI (IUb) or no protein; protein-DNA mixes were subsequently incubated with USP1-UAF1 (at 25 nM or 100 nM) for indicated time periods. Deubiquitination of D2Ub and IUb was assessed, following SDS-PAGE, by Coomassie staining of the gels, as well as by western blotting of transferred blots with a specific FANCD2 antibody. USP7 and USP2 can deubiquitinate D2Ub, but their activity towards D2Ub is greatly reduced in the presence of FANCI (I) or ubiquitinated FANCI (IUb). Reactions were set-up as in A, but with 100 nM USP7 or USP2. Deubiquitination was assessed, following SDS-PAGE, by Coomassie staining. Location of the four hydrophobic residues mutated to alanine in the ubiquitin structure (PDB: 1ubq). Top: location of F4, I36, I44 and L73 (shown in red) in ubiquitin's surface (shown in yellow). Bottom: same as above but with ubiquitin's surface coloured according to charge (red: negative, blue: positive, white: no charge). Time course of FANCI ubiquitination with various ubiquitin mutants and corresponding sensitivity/resistance to USP1-UAF1 mediated deubiquitination of resulting products. Deubiquitination reactions in the presence or absence of USP1-UAF1 (100 nM) incubated for 30 minutes. Time course of USP1-UAF1 mediated deubiquitination of IUbD2Ub-DNA complexes consisting of D2Ub a 50 base pair dsDNA and IUb produced with various ubiquitin mutants or wild-type (WT) ubiquitin. Progress of deubiquitination reaction was assessed following SDS-PAGE, by both Coomassie staining of the gels, as well as by western blotting of transferred blots with a specific FANCD2 antibody. FANCI ubiquitination with indicated ubiquitin-mutants (or WT) and subsequent deubiquitination of resulting IUbD2Ub-DNA complexes were conducted twice; the residual FANCD2 ubiquitination, calculated from the FANCD2 blots for each time-point, was plotted for each type of ubiquitin in the protein complex, and corresponding deubiquitination progress curves were fitted using a one-phase decay model.