EMBO/sd-character-level-ner huggingface dataset:
Figure 5Schematic diagram for generating PAX6A (PAX6A TetOn in PAX6KO) and PAX6D (PAX6D TetOn in PAX6 KO) inducible cell lines in PAX6 KO hESCs. With this design, PAX6A or PAX6D is induced separately with the doxycycline (DOX) treatment. qPCR quantification of PAX6A and PAX6D gene expression under the treatment of DOX (n=3 biological replicates for each sample). Western blot analysis of PAX6A and PAX6D protein expression under the treatment of DOX. Schematic diagram of DOX treatment in PAX6A and PAX6D TetOn cell lines for (E,F,G). qPCR quantification of SOX1 gene expression in the PAX6A TetOn and PAX6D TetOn cells (n=3 biological replicates for each group). qPCR quantification of retinal marker expression in PAX6D TetOn cells w/o DOX treatment (n=3 biological replicates for each group). Cell population composition analysis with SOX1 and SOX2 staining from flow cytometry. SOX1/SOX2 double positive cells represent the neuroepithelia fate while the SOX1 negative and SOX2 positive cells represent the neuroretina fate.
Figure 5(A) Gating strategy of brain-infiltrating immune cells including CD4+ T cells (CD3+ CD4+), CD8+ T cells (CD3+CD8+), B cells (CD3-CD19+), NK cells (CD3-NK1.1+), monocyte/macrophages (CD11b+CD45highF4/80+), neutrophils (CD11b+CD45highLy6G+), and microglia (CD11b+CD45int) and their expression of IL (interleukin)-6, TNF-α (tumor necrosis factor-α), TGF-β (transforming growth factor-β), and IL-4. FMO, fluorescence minus one. (B) Quantitative analysis of the counts of brain-infiltrating leukocytes in the brains of MCAO mice receiving indicated treatment. (C) Bar graph shows counts of leukocytes subtype in the spleens of MCAO mice receiving indicated treatment. n = 6 mice per group. (D) Quantitative analysis of the microglia in the brains of MCAO mice receiving indicated treatment. n = 6 mice per group. * (E) Bar graph shows percentages of microglia expressing IL-6, TNF-α, TGF-β, and IL-10 in the brains of MCAO mice receiving indicated treatment. n = 6 mice per group. (F) Representative images of immunofluorescent staining for ly6G in peri-infarct areas of MCAO mice receiving indicated treatment. (G) Bar graph shows the number of ly6G+ neutrophils. n = 6 mice per group. (H) Representative images of immunofluorescent staining for Iba-1 in peri-infarct areas of MCAO mice receiving indicated treatment. (I) Bar graph shows the number of Iba-1+ microglia. n = 6 mice per group.
Concerted action of exosites and active site in substrate binding and cleavage.Figure 6A Schematic representation of the substrate-binding chase experiment assaying whether C99 crosslinked at exosites (Exo) can be cleaved during chase incubation. Note that crosslink product of substrates with Bpa placed in the Aβ region cannot be recovered by Ni-NTA affinity pulldown after γ-secretase cleavage in the 37°C chase. B Substrates bound to exosites in the PS1 NTF at 4°C can be cleaved in the 37°C chase incubation in a L-685,458-dependent manner. Note that, although many of these substrates crosslinked only weakly at the exosites, their cleavage in the 37°C chase proved that these reflected functional substrate-binding states.C Substrates crosslinking in the exosites to NCT and PEN-2 cannot be cleaved. D, E Substrates can be cleaved when crosslinked to PS1 NTF but not when crosslinked to NCT (D) or PEN-2 (E). F Excess amounts of Notch1 substrate N102-FmH compete with exosite-binding of C99. See Fig EV4 for competition of C99 binding in the active site region.
Figure 1(A) Heatmap representing the differentially expressed genes in ALSC9orf72 (ALSC9orf72 I and ALSC9orf72 II lines) vs Healthy (Healthy I and CorrectedC9orf72 lines) MN transcriptomes. Colour scale represents gene expression relative to the Healthy genotype. GO (biological processes) terms of the top significant down- or up-regulated pathways in ALSC9orf72 MN (hypergeometric test). GO (biological processes) terms of the top significant up-regulated pathways in ALSC9orf72 MN (hypergeometric test). (D) Reactome representation of clustering and distribution of the significantly up- and down-regulated pathways in ALSC9orf72 MN. (E) PASTAA ranking of the top 3 transcription factors involved in the regulation of the differentially expressed genes in ALSC9orf72 cultures. Significance was set with the algorithm published in Roider et al. (2009). (F) Single tube qPCR experiments showing the down-regulation of the synaptic genes NLGN3, SLC6A, TRIM9, SYT1, SYNGR1, and SNAP91 in ALSC9orf72 MN (Welch's t-test). n=3 independent cultures for each hiPSC line. Data information: *p<0.05; **p<0.01; ***p<0.001. Error bars represent SEM. Arrow indicates the structure displayed at higher magnification. Exact p-values are reported in Appendix Table S1. (G-H) Time course analysis of nuclear pCREBS133 levels and MN survival in ALSC9orf72 and Healthy cultures (two-way ANOVA). n=3 independent cultures for each hiPSC line (for each time point). Scale bar: 25 µm. Data information: *p<0.05; **p<0.01; ***p<0.001. Error bars represent SEM. Arrow indicates the structure displayed at higher magnification. Exact p-values are reported in Appendix Table S1. (I) Representative confocal image showing colocalization of the CREB interactor CBP within cytotoxic aggresomes. Scale bar: 5 µm. Dashed line represents the cell soma.
Figure 1Overexpression of USP36 promotes SUMOylation in cells. Whole cell lysates (WCL) from H1299 (A), U2OS and HeLa (B) cells transfected with control empty vector or Flag-USP36 were assayed by IB. Knockdown of USP36 reduces SUMOylation in cells. H1299 (C) cells infected with scrambled (scr) or USP36 shRNA encoding lentiviruses were assayed by IB. Knockdown of USP36 reduces SUMOylation in cells. HeLa (D) and U2OS (E) cells infected with scrambled (scr) or USP36 shRNA encoding lentiviruses were assayed by IB. USP36 promotes nucleolar SUMOylation. H1299 cells transfected with control or V5-USP36 were fractionated to the cytoplasm (Cyto), nucleoplasm (Np) and nucleolus (No) fractions and assayed by IB.
Figure 8NADH-dependent respiration (either aerobic or anaerobic/nitrate respiration) allows cells to keep and active TCA cycle and proper acetate catabolism by oxidizing NADH. Free fatty acids accumulate under NADH-deficient respiration and repress the expression of SaeRSPQ, which regulates the production of virulence factors and biofilm formation.
Impact of gene mutations on overall survival across treatment groupsA-E) Overall survival (OS) of patients with tumors containing mutations in the genes A) TP53, B) PIK3CA, C) ERBB2, and D) PTEN.Figure 5A-E) Overall survival (OS) of patients with tumors containing mutations in the genes A) TP53, B) PIK3CA, C) ERBB2, and D) PTEN. E) OS by PTEN-MutExp genotype ("low" defined as PTEN mutation or PTEN expression in the lower quartile across the cohort, "normal" otherwise) stratified by groups receiving no systemic treatment (n=336), endocrine therapy only (Endo only; n=1,579), endocrine and chemotherapy (Endo + Chemo ± any; n=914), as well as HER2 treatment with any other treatment or none (HER2 ± any; n=348). Specific treatments in these groups are detailed in Table EV5. In each Kaplan-Meier plot, wildtype (wt) and normal cases are plotted in blue, mutated (mut) and low cases are plotted in red, the log-rank P value is given, and the hazard ratio (HR) for mutation/low is given with a 95% CI and after uni-variable and multi-variable (MV) Cox regression adjustment. Covariables included in the MV analysis were age at diagnosis, lymph node status, tumor size, and the variables denoted by the following symbols: ¶, ER, PgR, HER2, and NHG; ¤, ER, PgR, and NHG; $, HER2 and NHG. Abbreviations: ER, estrogen receptor; PgR, progesterone receptor; HER2, human epidermal growth factor receptor 2; NHG, Nottingham histological grade.
F accelerates RNAPII transcription in vivo.Figure 2Schematic drawing of the experimental design to investigate RNAPII transcription speed in vivo. In brief, Arabidopsis seedlings of NRPB2WT-FLAG Col-0 and NRPB2Y732F-FLAG Col-0 were grown on MS media for 12 days and then were transferred to MS liquid media for 2 days. Flagellin peptides (flagellin 22) were added into media and treated samples were collected in a 0 minute (no treatment), 2 minutes and 4 minutes time course. The nascent RNA was isolated and used for reverse transcription and qPCR analyses to reveal RNAPII accumulation at different region in genes. See Methods for technical details. Nascent RNA profile of AT5G41750. Nascent RNA RT-qPCR assay measuring RNAPII signal at 3 positions (dark red bars: probe 1, 2 and 3) on the gene upon flagellin 22 treatment in a 0 minute, 2 minutes, 3 minutes and 4 minutes time course. Nascent RNA signal values were normalized to reference gene ACT2. Error bars represent SEM from 3 independent replicates. The statistical significance of differences between NRPB2Y732F and NRPB2WT at the same time point were assessed by the two-sided Student's t-test. n.s. denotes not significant; * denotes p<0.05 and ** denotes p<0.01. Scale bar (black) represent 0.5 kb. Nascent RNA profile of AT4G19520. Nascent RNA RT-qPCR assay measuring RNAPII signal at 3 positions (dark red bars: probe 1, 2 and 3) on the gene upon flagellin 22 treatment in a 0 minute, 2 minutes, 3 minutes and 4 minutes time course. Nascent RNA signal values were normalized to reference gene ACT2. Error bars represent SEM from 3 independent replicates. The statistical significance of differences between NRPB2Y732F and NRPB2WT at the same time point were assessed by a two-sided Student's t-test. n.s. denotes not significant; * denotes p<0.05 and ** denotes p<0.01. Scale bar (black) represents 0.5 kb.
a, b) Depletion of Beclin 1 by shRNA promotes growth in soft agar.figf7(a, b) Depletion of Beclin 1 by shRNA promotes growth in soft agar. SW480 cells stably expressing Beclin 1 shRNA or control shRNA were seeded in soft agar and treated with or without Dkk (a); the colonies were then scored after 10 days (b). Numbers in bars indicate the numbers of colonies. Results are means ± s.d. (n = 4). (Asterisk, P 0.05). Scale bar, 200 μm. (c) Representative images from colon cancer tissues by immunohistochemistry staining. Negative correlation between Beclin1 and Dvl was observed in late stages of tumours, whereas positive correlation between p62 and Dvl in late stages of tumours. Scale bar, 25 μm. (d) Immunoblotting of human colon tumour samples versus normal tissues using indicated antibodies. N, normal tissue; T, tumour. (e) Working model of autophagic degradation of Dvl2.
Figure 1Coomassie- stained proteins from bovine retina that were co-immunoprecipitated by the anti-RIBEYE antibody 2D9 (lane 3). Lane 2, control precipitation by irrelevant mouse IgGs; lane 1, input (10% of the total loaded in each lane) Western blot of the immunoprecipitated proteins (as in A, 1% of the total protein was loaded per lane) probed with anti-RIBEYE (Ba), anti-CASPR1 (Bb) and anti-CNTN1 (Bc). The 2D9 antibody immunoprecipitated RIBEYE (Ba, lane 3), CASPR1 (Bb, lane 3) and CNTN1 (Bc, lane 3); these proteins were not detectable in the control immunoprecipitations. Bd) As a loading control, heavy chains of the precipitating antibody (IgG hc) were visualized by the goat anti-mouse IgG antibody conjugated to peroxidase
CMA is abnormally activated in PPCA‐deficient fibroblasts.Figure 3(A) Rates of protein degradation in skin fibroblasts from wild‐type (control) or PPCA(−/−) mice maintained in the presence (serum +) or absence of serum (serum −). Values are expressed as the percentage of initially radiolabeled proteins remaining at each time and are means ± SE of four different experiments. (B) Degradation of [14C]GAPDH after 30 min incubation with lysosomes isolated from wild‐type and PPCA(−/−) mice skin fibroblasts supplemented or deprived of serum was measured as described in Material and methods. Where indicated, 25 μg of RNase A were added. Values are means ± SE of four different experiments. Differences from the control value were significant to P 0.001 (**). (C) Immunofluorescent staining for lamp2a and lamp1 of skin fibroblasts from wild‐type and PPCA(−/−) mice. Gain for lamp2a images (top) was 1/8 of the one for lamp1 (bottom) to visualize the punctate pattern of lamp2a in PPCA(−/−) mice. Inset: detail of the vesicular distribution of lamp2a. The gain in wild type was eight times the gain in PPCA(−/−) mice). Bars: 100 μm (top), 50 μm (bottom), 10 μm (inset). (D) Immunoblot analysis for lamp2a, all lamp2s, lamp1 or cathepsin D (cath D) of homogenates (75 μg of protein), lysosomes (10 μg of protein) and lysosomal membranes (L.MB) and matrices (L.MTX) (5 μg of protein) isolated from wild‐type (WT) and PPCA(−/−) skin fibroblasts. The bottom chart shows the densitometric quantification of the membrane proteins (means + SE) from four immunoblots similar to those shown here. Differences from the control value were significant to P 0.001 (**) and P 0.05 (*).
Figure 1a-c, Expression of CD45RO and CCR7 (a), CD45RO and CD27 (b), CD27 and CD28 (c) on peripheral blood CD8 T cells of three participants [P1 (heterozygous mutation), P2 (homozygous mutation) and P3 (heterozygous) for the c.192G>C mutation in the DJ-1 gene]. d-g, Expression of CD57 (d), PD-1 (e), EOMES (f) and T-bet (g) on peripheral blood CD8 T cells of three participants. The enlarged number in the corresponding gate represents the corresponding percentage out of the parent population. h h-k, mRNA expression of senescence related genes (h), exhaustion related genes (i), KIR or KLR genes (j) and cyclin-dependent kinase inhibitor genes (k) in sorted CD8 T cells of the peripheral blood from three participants. l-m, Comparison of the lower bound of TCR-beta repertoire (l) and richness (m) of naïve CD8 T cells among three participants. n, The sample clonality index of TCR repertoire of naïve CD8 T cells among three participants. o, Serology detection of CMV infection for three participants. The threshold of 11 was decided according to the manufacture instruction. The data in panel o represents as mean of technical replicates ± standard deviation (s.d.).
Figure 1(A, B) BMDCs were infected with Pru parasites (at a MOI of 4 parasites/BMDC) or treated with tunicamycin (TN) for 16h and the expression of UPR target genes were analyzed by RT-qPCR. The results are expressed as (Log2) Fold change (A) or relative mRNA level (B) compared to non-infected (NI) conditions. IRE1α/XBP1s pathway: Ern1 (IRE1α), Xbp1s, Dnajb9 (ERdj4), Sec24D, Bloc1s1 and Sec61a1; PERK/ATF4 pathway: Atf4, Ddit3 (CHOP) and Ppp1r15a (GADD34); ATF6 pathway: Atf6 and Herpud1; classical ER chaperons: Hspa5 (BIP) and Hsp90b1 (GRP94). Results are normalized to the housekeeping gene Gapdh. Unpaired Student T-test, ns: p>0.05; **: p<0.01; ***: p<0.001; mean ± S.E.M (n=2-5 independent experiments depending of the studied gene, 3 mice / independent experiment). (C, D) Immunoblot analysis of IRE1α, PERK, Phospho-PERK (detected by the presence of a second upper band after migration on a 6% acrylamide gel), Phospho-eIF2α, CHOP and XBP1s protein expression in non-infected BMDCs, BMDCs treated with TN and Pru infected BMDCs (C) and in non-infected and Pru infected Flt3/Notch-DCs (D) for the indicated times. GAPDH and tubulin were used as loading controls. The same blots are shown in Figure EV3B-C.
Distribution of γ-TuRC-binding factors containing complexes A-B WB of AKAP450 (A) and PCNT (B) co-immunoprecipitation assays revealed with the indicated antibodies.Figure 3WB of AKAP450 co-immunoprecipitation assays revealed with the indicated antibodies. Asterisks mark immunoglobulin heavy chains. Cntrl: non-specific Igs immunoprecipitates; IP: specific Igs immunoprecipitates; SP: supernatant. WB of AKAP450 and PCNT (B) co-immunoprecipitation assays revealed with the indicated antibodies. Asterisks mark immunoglobulin heavy chains. Cntrl: non-specific Igs immunoprecipitates; IP: specific Igs immunoprecipitates; SP: supernatant. Centrosomes from WT, single and double KO cells, as indicated, were labelled for AKAP450 and PCNT Right panels show quantifications. A region of interest of 1,5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean. Centrosomes from WT, single and double KO cells, as indicated, were labelled for AKAP450 and PCNT Right panels show quantifications. A region of interest of 1,5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean. Centrosomes from WT, single and double KO cells, as indicated, were labelled for AKAP450 and PCNT Right panels show quantifications. A region of interest of 1,5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean. Centrosomes from WT, single and double KO cells, as indicated, were labelled for CDK5Rap2 Right panels show quantifications. A region of interest of 1,5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean. Centrosomes from WT, single and double KO cells, as indicated, were labelled for γ-tubulin (G). Right panels show quantifications. A region of interest of 1,5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean. WT and pc-ak-2KO cells were transfected with either scramble or CEP192 siRNAs, fixed and stained for γ-tubulin. Quantification of the fluorescence intensity is show at right. IF images of WT and pcnt KO RPE-1 cells treated with NZ for 3h and triple stained for AKAP450 (green), CDK5Rap2 (red) and GMAP210 as a Golgi marker (blue). At right, quantification of fluorescence co-localization of either AKAP450 or CDK5Rap2 with the Golgi marker GMAP210 in WT and pcnt KO cells­. Scheme summing up the changes in AKAP450-based and PCNT-based complexes distribution in WT, akap KO and pcnt KO RPE-1 cells and their impact in γ-tubulin recruitment. GA, Golgi apparatus. CTR, centrosome.
Figure 6(A) Schematic graph of ASY1 full length protein (aa 1 to 596). The HORMA domain is depicted in orange, the presumptive closure motif is highlighted in blue. The lines below indicate the constructs used for yeast two-hybrid interaction assays. (B) Interaction assays of different ASY1 fragments (with and without the closure motif). (C) Interaction analysis of the ASY1 closure motif (ASY1571-596) with different ASY1 HORMA domain variants.
fdsc-αDEC induces IL-6 and IFN-α production mediated by MYD88 and TLR9.Figure 3IL-6 was evaluated by ELISA in supernatants of BMDCs obtained from C57BL6, MYD88, TLR9 or TLR4 KO mice and incubated for 20 h with wild-type or fdsc-αDEC phage particles. LPS or CpG-ODN were used as controls. IL-6 release from DCs derived from MyD88−/−mice was totally abolished and dramatically reduced in DCs derived from Tlr9−/−mice, but not affected in Tlr4−/− DCs. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples. IFN-α release evaluated by ELISA in supernatants of BMDCs obtained from C57BL/6 or KO mice and incubated for 20 h with wild-type or fdsc-αDEC phage particles. LPS or CpG-ODN were used as controls. MyD88−/− and Tlr9−/− but not Tlr4−/− BMDCs were unable to produce IFN-α after fdsc-αDEC stimulation. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples. IL-6 mRNA expression by DC cells. C57BL/6, MyD88−/− and Tlr9−/− mice were inoculated intraperitoneally with fdWT or fdsc-αDEC bacteriophages or, as a control, with LPS. Mice were sacrificed 2 h later, and purified spleen dendritic cells were analyzed for IL-6 mRNA levels by quantitative real-time PCR. Bars represent the mean fold increase ± SD. The experiments were performed three times (n = 2 per group). Comparative analyses were performed using Student's t-test for unpaired samples. Proliferation of OT-ICD8+T cells after 72 h of co-culture with 1 × 104DCs isolated from C57BL/6mice or from MyD88−/− or Tlr9−/− transgenic mice previously injected with fd/sc-αDECbacteriophage particles. The panel shows the percentage of divided, CFSE-low OT-ICD8+T cells. As a control, the proliferation of OT-ICD8+T cells co-cultured with DCs isolated from non-immunized (NI) C57BL/6mice is reported. The mean ± SD of two independent experiments with n = 3 per group is reported. Comparative analyses were performed using Student's t-test for unpaired samples. IFN-γ production by OT-I CD8+ T cells co-cultured for 12 h in the presence of 3.3 × 103 DCs isolated from C57BL/6 mice or MyD88−/− and Tlr9−/− transgenic mice injected as in (D).
Figure 6A schematic cartoon model of ZBD-relocation mechanism during σ28-mediated transcription initiation is presented. The ZBD-relocation shows an overall effect of facilitating the isomerization of closed RNAP-promoter complex (RPc) to open RNAP-promoter complex (RPo) by contributing to RNAP-promoter interaction. The RPc with the relocated ZBD (state 2) has a higher efficiency in isomerizing to RPo or is more stabilized than the one with the state 1 ZBD, consequently advancing transcription efficiency. The inset is the zoom-in view of ZBD relocation from state 1 to state 2.
Figure 6A Schematic of Pah1 phospho-regulation. Phosphorylated Pah1 is cytosolic and inactive. Interaction of Pah1 and the ER-localized Nem1-Spo7 complex results in Pah1 dephosphorylation and activation, promoting conversion of PA into DAG. B Western blot of HA from WT, Δice2, Δnem1 and Δnem1 Δice2 cells expressing endogenously tagged Pah1-HA (SSY2592, 2593, 2594, 2718). Blots of SDS-PAGE and Phos-tag PAGE gels are shown. C Schematic of Pah1 dephosphorylation assay with phosphorylated Pah1 from ∆nem1 mutants and microsomes from different strains. D Western blot of HA from Pah1 dephosphorylation reactions that contained phosphorylated Pah1-HA from ∆nem1 mutants (SSY3065) and microsomes from cells of the indicated genotypes (SSY3053, 3074, 3075, 3095). Phosphorylated Pah1 and dephosphorylated Pah1 resulting from treatment with recombinant alkaline phosphatase (PPase) are shown for reference. E Western blot of HA, Sec61 and Pgk1 from cell lysates and microsomes prepared from WT and ∆ice2 cells expressing Nem1-HA (SSY3140, 3141). Nem1 is undetectable in cell lysates due to its low abundance. F Western blot of HA from Pah1 phosphorylation reaction that contained hypophosphorylated Pah1-HA from ∆ice2 cells (SSY3096) and microsomes from ∆nem1 cells (SSY3075). Phosphorylated Pah1 from ∆nem1 cells (SSY3065) and hypophosphorylated Pah1 from ∆ice2 cells are shown for reference.