EMBO/sd-character-level-ner huggingface dataset:
Figure 3A. Depletion of Ccdc108 causes disorganized basal body distribution in MCCs. Representative 3D-SIM images (x-y) and 3D-reconstructions (x-z) of MCCs of embryos show a failure of apical trafficking of basal bodies in Ccdc108 depleted cells. Cell membranes (mGFP, blue), basal bodies (Cetn1, green) and distal appendages (Cep164, purple) were labeled with indicated antibodies. Select area of Cep164 channel was zoomed to show centriolar Cep164 accumulations. Orange arrowheads and numbers mark individual centriole. > 70 MCCs from 6 embryos for each condition were counted. Data information: Quantitative data were from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001 ; ** p<0.01; * p<0.05). Mean ± s.d. values are presented. B. Transmission electron microscopy images of Xenopus embryos displaying aberrant apical trafficking and docking of centrioles in Ccdc108 depleted MCCs. Orange arrowheads mark centrioles. Percentage of centrioles docked to plasma membrane in each field were scored. At least 7 cells for each sample were counted. C. Representative 3D-SIM images reveal that depletion of Ccdc108 has no effect on apical expansion in MCCs. Embryos were fixed and stained with phalloidin (purple). Cilia (Ac-tub, gray) were also presented. Apical area of MCCs in each condition were measured ; 80 MCCs from 6 embryos for each condition. D. Representative confocal images show a significant reduction in apical actin following depletion of Ccdc108. Cell boundaries and apical actin network were marked with mGFP (green) and phalloidin (purple) and phalloidin intensity levels measured and plotted. > 80 MCCs from 6 embryos for each condition. Data information: Quantitative data were from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001 ; ** p<0.01; * p<0.05). Mean ± s.d. values are presented.
A PI3Kα specific transcriptomic signature predicts pancreatic cancer aggressiveness.Figure 1A, List of 19 HALLMARK pathways (out of 50) significantly altered (ranked with p-value - top: low, bottom: high) in a cohort of 9 metastatic pancreatic adenocarcinoma patients (PDACmet), 9 localised PDAC (PDACloc), 8 chronic pancreatitis (CP) compared to 9 normal pancreas samples (normal); corrected p-values correspond to the comparison indicated. ANOVA test corrected using the Benjamini & Hochberg method (BH) on SES (sample enrichment score). Significantly altered corrected p-values are highlighted in colour. The signature in red corresponds to the signature with the lowest p-value for discriminating PDAC from CP. B, List of PI3K related pathway gene signatures (Reactome) altered in the same cohorte; corrected p-values are shown. ANOVA test corrected using the Benjamini & Hochberg method (BH) on SES (sample enrichment score). Significantly altered corrected p-values are highlighted in colour. C C, Transcriptomic signature indicative of PI3Kα activation in the same cohorte. Genes whose expression is increased only in metastatic patients are underlined. The unsupervised hierarchy of each sample is shown in the top part of the figure; the unsupervised hierarchy of genes is shown on the left; the list of genes is detailed on the right. Blue=low expression, Red=high expression. D, Scoring of the PI3Kα activation transcriptomic signature was used to cluster patients with high, medium (both groups were combined, high+medium: red) and low (black) scoring levels in the primary tumours of confirmed PDAC patients from PAAD (TCGA) or PACA-AU (BH corrected p-values). The survival curves of each cluster were then plotted, and the statistical significance was calculated using the logrank test. E, PI3Kα activation scores from localised PDAC in the TCGA database according to their UICC staging were retrieved, clustered into two groups (low and med) (BH corrected p-values) and their overall survival was plotted, with logrank testing.
a) Cell extracts from DLD1 cells treated with DMSO or bafilomycin A1 overnight were resolved using SEC and each fraction was analysed for the presence of NCOA4 and FTH1 by western blot.figf6(a) Cell extracts from DLD1 cells treated with DMSO or bafilomycin A1 overnight were resolved using SEC and each fraction was analysed for the presence of NCOA4 and FTH1 by western blot. Proteins elute in high- to low-molecular-weight fractions from 18 to 33, respectively. Dashed lines are for visual comparison only. (b) DLD1 parental or NCOA4−/− cell extracts were resolved using SEC and each fraction was analysed for the presence of NCOA4, FTH1 and FTL by western blot. (c) NCOA4−/− cells were transfected with control or SBP-NCOA4 cDNA for 36 h and incubated overnight in the presence or absence of the indicated inhibitors, lysed and the occurrence of the faster-migrating FTH1 band was determined. (d) Endogenous NCOA4 immunoprecipitations from DLD1 parental, FTH1−/− or FTL−/− (clones 1 and 2 for each genotype) protein extracts. The presence of FTH1 and FTL was detected by western blot. Uncropped images of blots are shown in Supplementary Fig. 7.
Figure 4A. HEK293 cells were transfected with non-targeting (Ctrl) or C9orf72 siRNA. Cells were treated with rapamycin for 6 h to induce autophagy. Activation of ULK1 was determined on immunoblots using phospho-ULK1 (Ser757), total ULK1 and GAPDH Abs (loading control). B. HeLa cells treated with non-targeting (Ctrl) or C9orf72 siRNA were transfected with mCherry-FIP200. 24 h post transfection cells were treated for 3 h with Torin1 (250 nM) or vehicle (Ctrl). Translocation of the ULK1 complex was quantified as the number of mCherry-FIP200 positive puncta per cell from 3 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test, ns: not significant, **** P ≤ 0.0001; N (cells) = Ctrl/Ctrl: 65; Ctrl/Torin1: 60; C9orf72/Ctrl: 54; C9orf72/Torin1: 49). C9orf72 knockdown was determined by RT-qPCR (Appendix Fig S2). Scale bar = 10 µm. C. Primary cortical neurons (DIV5/6) were transfected with EmGFP non-targeting (Ctrl) or C9orf72 miRNA (green) and mCherry-FIP200 (red); for rescue experiments the cells were additionally transfected with mCerulean-tagged C9orf72s and C9orf72L (cyan). 3 days post transfection neurons were treated for 3 h with Torin1 (250 nM) or vehicle (Ctrl). Translocation of the ULK1 complex was quantified as the number of mCherry-FIP200 positive puncta per soma from 2 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test, ns: not significant, *** P ≤ 0.001, **** P ≤ 0.0001; N (cells) = Ctrl miRNA/Ctrl: 134; Ctrl miRNA/Torin1: 125; C9orf72 miRNA/Ctrl: 101; C9orf72 miRNA/Torin1: 78; C9orf72 miRNA+C9orf72L+C9orf72S: 41; C9orf72 miRNA+C9orf72L+C9orf72S/Torin1: 39). Scale bar = 5 µm. D. HeLa cells were co-transfected with mCherry-FIP200 (red) and empty vector (EV), FLAG-C9orf72L or FLAG-C9orf72S (green). As positive control EV transfected cells were treated for 3 h with Torin1 (250 nM). Translocation of the ULK1 complex was quantified as the number of mCherry-FIP200 positive puncta per cell from 3 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test, *** P ≤ 0.001, **** P ≤ 0.0001); N (cells) = EV: 47, EV + Torin1: 31, C9orf72L: 46, C9orf72S: 45). Scale bar = 10 µm.
Figure 4Endogenous METTL14 interacts with RNAPII. Endogenous co-immunoprecipitation (IP) was performed using the METTL14 antibody for IP and the RNAPII antibody for Western blot detection (left panel) and using the RNAPII antibody for IP and the METTL14 antibody for Western blot detection (right panel). Arginine methylation of the METTL14 C-terminal IDR enhances its interaction with RNAPII. HEK293 cells were transfected with HA-tagged WT or arginine methylation-deficient (RK) mutant METTL14. IP was performed using an anti-HA antibody, and Western blot analysis was performed using the indicated antibodies. Co-IP assays were performed to compare the interactions between METTL14 and RNAPII in control and MS023-treated HEK293 cells. Cells were treated with either DMSO or MS023 (1 μM) for 48 h before they were lysed. IP was performed using control IgG and METTL14 antibodies, respectively. Western blot analysis was performed using anti-RNAPII, anti-ADMA, anti-METTL14, and anti-METTL3 antibodies.   Co-IP assays were performed to examine the involvement of RNA in the METTL14-RNAPII interaction. Total cell lysates were either left untreated or treated with RNase A to remove the RNA component before IP. Western blot analysis was performed using anti-RNAPII, anti-METTL3, and anti-METTL14 antibodies.
Figure 7Quantification of intracellular amino acid levels in brown fat cells (day 6) treated with or without 1mM asparagine. n=4 independent experiments. Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test. B-D. mTORC1 pathway activity was analyzed by immunoblotting for the phosphorylation of 4E-BP1 and S6K in brown adipocytes (B), BAT from mice fed asparagine (C) or asparaginase (D). (B) Asparagine was added to cultured media from day 4 to day 6. A Click-iT AHA labeling assay to detect newly synthesized glycolytic enzymes. The level of HSP90 in total cell lysates was used as a loading control. Brown preadipocytes were induced to differentiation with or without 1mM asparagine. Rapamycin (75nM) was added to cells for 48h. Then western blot detected protein expression on day 6. Brown preadipocytes were knocked down Raptor following standard induction to mature adipocytes with or without asparagine supplementation. Protein expression analysis was performed on day 6.
a, MRE11::MYC cells were treated as in Fig.f2a, MRE11::MYC cells were treated as in Fig. 1a. Cell samples were processed for ChIP analysis and the fold enrichment of the 0.2-kb fragment after ChIP of Mre11-Myc without (-VPA) or with (+VPA) VPA was calculated. b, EXO1::FLAG SAE2::PK MRE11::MYC cells were grown as in a. Cell samples were taken and processed for western blot analysis using anti-Flag, PK and Myc antibodies.
Figure 3Decreased SREBP-1/2 and SCAP levels in Golgi apparatus in Cideb-/- mice. IB of organelle fractions of the indicated proteins and quantification of IB data from 3 independent experiments. Data information: Data represent the Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test. Cideb deficiency impairs the packaging of SCAP/SREBP into COPII vesicles. Protein present in the vesicle or microsome fractions obtained from WT or Cideb-/- liver were detected by IB. Quantification of IB data from 3 independent experiments. Data information: Data represent the Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test. Schematic of the BioID assay with Sar1A-BirA*. Cideb depletion decreases the recruitment of SCAP to the COPII machinery in cells. Primary hepatocytes transfected with control or Cideb siRNA were infected with adenovirus expressing Sar1A-Flag-BirA*. Cells were cultured in sterol-depleted medium and treated with 80 µM biotin, prior to pulldown with streptavidin-conjugated beads. The biotinylated proteins were detected by IB with the indicated antibodies following SDS-PAGE. Cleaved SREBP bypasses Cideb deficiency. WT and Cideb-/- mice were injected with adenovirus expressing GFP, SREBP-1c full length (FL) or SREBP-1c active form (N). Mice were sacrificed 7 days after injection, and liver TAG levels and hepatic expression SREBP-1 target genes in the indicated groups were determined (n=5 per group). Data information: Data represent the Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.
Figure 4 A) Levels of murine TIMP1, MMP7 and TSP2 in plasma of mice bearing PDAC-PDX (HuPa4, HuPa8 and HuPa11) growing orthotopically in the pancreas (mean ± SEM; n≥3 for each group), *p<0.05 (Mann-Whitney). The exact n and p-values are indicated in Appendix Table S4A  B) Correlations between the levels of the three selected biomarkers and the tumor volume in mice bearing HuPa8. Pearson coefficient (r)  C) Expression of murine TIMP1, MMP7, TSP2, CCN2, ICAM1, IGFBP2 and PLG analyzed in tumors from pancreas (HuPa4, HuPa8 and HuPa11) by RT-PCR. The expression level of target genes was normalized to the geometric median of βactin and GAPDH housekeeping genes and expressed as 2-ΔΔCT (mean ± SEM, *p<0.05; Healty n=7; HuPa4, HuPa8 and HuPa11 n=4) (Mann-Whitney). The exact p-values are indicated in Appendix Table S4B  D) Histological analysis of representative PDAC-PDX (HuPa4, HuPa8 and HuPa11). Hematoxylin-eosin, anti-TIMP1, anti-MMP7, anti-TSP2 and anti-CCN2 staining of PDAC-PDX tumors (200X, scale bars: 100μm)
Figure 5A Coimmunoprecipitation assay showing the physical interaction between OsPRR73 and HDAC10 in vivo. CoIP was conducted with GFP-Trap beads, and GFP and FLAG antibodies were used to detect OsPRR73 and HDAC10 in immunoprecipitants as indicated. B Split luciferase complementation (SLC) assay showing OsPRR73 interacts with HDAC10 in the co-infiltrated N.benthamiana leaves. C Bimolecular-fluorescence complementation (BiFC) assay shows the interaction between OsPRR73 and HDAC10 occurs in nucleus. BF represents bright field. Scale bar, 40 µm. D N-terminus of OsPRR73 containing PR domain is required and sufficient for interacting with HDAC10. Upper panel shows the schematic illustration of full-length and the truncated OsPRR73 proteins. CoIP was performed with GFP-Trap beads, and immunoprecipitants were detected by FLAG for HADC10 and GFP for truncated proteins of OsPRR73 respectively. E, F Chromatin Immunoprecipitation-qPCR (ChIP-qPCR) assay to detect the enriched promoter regions of OsHKT2;1 by using the H3K9ac (E) and H3 (F) antibodies. Two-week-old seedlings were harvested at ZT12. The locations of amplicons were described in Figure 4E. Data represent means ± SD. n = 3 technical replicates. The asterisk indicates the significant difference by student's t-test with (*) P ≤ 0.05. The experiments were repeated at least twice with similar result.
Figure 2A Experimental scheme. See text for details. B Fractions from the discontinuous gradient were blotted with anti-ApoE (WUE-4) antibody; the doublet is a characteristic feature on ApoE western blots due to ApoE sialylation [16]. C An ApoE-containing fraction (fraction 5 [+]), an ApoE-negative fraction (fraction 3 [-]), and recombinant lipid-free ApoE protein were applied to human fibroblasts. Note increase in phospholipid synthesis using an ApoE4-containing lipoprotein fraction, but not with either an ApoE4-negative fraction or with lipoprotein-free recombinant ApoE3 or ApoE4 protein (mean ± SE; n=3, with 3 replicates/experiment).
Figure 6Western blot analysis of MICU1 and MICU2 levels in lung cancer cells with low or high Akt activity Lung cancer cells were transfected with the GFP-tagged WT MICU1. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with a PAS (phospho-Akt substrate) antibody by western blotting Western blot analysis of MICU1 and MICU2 levels in melanoma cells with low or high Akt activity Melanoma cells were transfected with the GFP-tagged WT MICU1. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blotting Western blot analysis of MICU1 and MICU2 levels in glioblastoma cells with low or high Akt activity Glioblastoma cells were transfected with the GFP-tagged WT MICU1. MICU1-GFP immunocomplexes were precipitated with GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blotting Number of soft agar colonies (n=3 independent experiments formed by Rat2 cells stably expressing the indicated constructs xenograft tumor volumes (H) and tumor weights at day 21 (n=8 mice for each group) (I) formed by Rat2 cells stably expressing the indicated constructs Representative images (J) and analysis (K) of MitoSOX-based ROS measurements in ShRNA MICU1 HeLa stable cells stably expressing either MICU1 S124D (SD) or MICU1 S124A (SA) (n=3 independent experiments). Scale bar 10 μm. f.a.u.: fluorescence arbitrary unit ShRNA MICU1 HeLa stable cells stably expressing either MICU1 S124D (SD) or MICU1 S124A (SA) were treated with 500 μM H2O2 alone or in combination with the ROS scavenger Tempol and analyzed by western blotting as indicated. l. e.: long exposure. Speculative model of the mitochondrial Akt-MICU1 axis in the regulation of tumor growth
Figure 1A Western blot analysis of lysates from WT and MIC10 KO or MIC60 KO HAP1 cells. MIC10 KO cells show a drastic reduction of MIC13, MIC26 and MIC27 protein levels while protein levels of other MICOS components remain unchanged. MIC60 KO cells show a drastic reduction of protein levels of all MICOS components. B Representative electron micrographs of WT, MIC10 KO and MIC60 KO HAP1 cells. Scale bar 500 nm. C Quantification of CJs per mitochondrial section from different mitochondria in WT, MIC10 and MIC60 KO HAP1 cells using EM represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. Data from n = 55-69 mitochondria (from 2 independent experiments) are shown as data points in the boxplots. ****P < 0.0001 (using unpaired Student's t-test). D Quantification of cristae per mitochondrial section from different mitochondria in WT, MIC10 and MIC60 KO HAP1 cells using EM represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. Data from n = 55-69 mitochondria (from 2 independent experiments) are shown as data points in the boxplots. ****P < 0.0001 (using unpaired Student's t-test). E Comparison of oxygen consumption rates (pmol O2/s, normalized for cell numbers by Hoechst staining) of basal, proton leak, maximum and non-mitochondrial respiration in WT and MIC10 KO cells obtained from 3 independent experiments. The data is normalized to basal respiration from HAP1 WT. Bar and error bar represent mean ± SEM from 3 independent experiments. *P = 0.034 for basal (using one sample t-test) and *P = 0.018 for maximum respiration (using unpaired Student's t-test). F Comparison of oxygen consumption rates (pmol O2/s, normalized for cell numbers by Hoechst staining) of basal, proton leak, maximum and non-mitochondrial respiration in WT and MIC60 KO cells obtained from 3 independent experiments. The data is normalized to basal respiration from HAP1 WT. Bar and error bar represent mean ± SEM from 3 independent experiments. *P = 0.01 for basal (using one sample t-test), ***P = 0.0004 for maximum respiration and *P = 0.012 for non-mitochondrial respiration (using unpaired Student's t-test).
, Basal USP18 amounts determine cell-type specific desensitization threshold, while abundance of STAT2 predicts in vivo responsiveness to Roferon The relative antiviral response upon 608 pM Roferon stimulation (ΔAUC(24 - 28h) of OccGASbs+OccISREbs compared to untreated) is shown as a function of the prestimulation dose for the cell lines Huh7.Figure 7The relative antiviral response upon 608 pM Roferon stimulation (ΔAUC(24 - 28h) of OccGASbs+OccISREbs compared to untreated) is shown as a function of the prestimulation dose for the cell lines Huh7.5 and HepG2-hNTCP as well as for primary human hepatocytes derived from six patients (left). Desensitization thresholds (i.e. the presimulation dose beyond which the relative antiviral response is smaller than one) are indicated by symbols and plotted against the amount of USP18 in corresponding untreated cells (right). Spearman's rank order correlation coefficient (r) and p-value (p) are indicated. Plasma levels of IFNα were measured in a cohort of 36 patients with chronic HBV. A virtual cohort of 114 patients was simulated with the primary human hepatocyte-specific mathematical model. Distributions of IFNα, STAT2, STAT2, IRF9 and USP18 matching experimentally measured values as shown in Fig. 6B and 7B are shown. For each patient in the virtual patient cohort a stimulation with 608 pM Roferon was simulated and the relative antiviral response (ΔAUC(24 - 28 h) of OccGASbs+OccISREbs compared to untreated) was calculated. The relative antiviral response was plotted against the cellular abundance of STAT2. Spearman's rank order correlation coefficient (r) and p-value (p) are indicated.   For each patient in the virtual patient cohort, a stimulation with 608 pM Roferon was simulated and the absolute antiviral response (ΔAUC(24 - 28 h) of OccGASbs+OccISREbs) was calculated. The absolute antiviral response was plotted against the cellular abundance of STAT2. Spearman's rank order correlation coefficient (r) and p-value (p) are indicated.
Figure 1(A) Raji cells stably expressing HIV-1 YU-2 ENV (Raji-Env) were stained with bNAbs (10-1074 or 3BNC117) or isotype control (mGO53) and analyzed by flow cytometry. The numbers indicate the % of positive cells. One representative experiment is shown. (B) Binding of the 24 indicated antibodies to the surface of Raji-Env cells. Results are expressed as the median fluorescence intensity (MFI) of staining. n=6 independent experiments. Data information: Error bars indicate SEM. Significance was determined by comparing each antibody to mGO53. Only significant comparisons are depicted; *P<0.05, Mann-Whitney test. (C) Raji-Env cells were incubated with indicated antibodies and either normal (NHS) or heat-inactivated (HIHS) human serum. After 1h, surface levels of C3 were analyzed by flow cytometry. One representative experiment is shown. (D) C3 deposition by the 24 indicated antibodies. For each antibody, a normalized MFI is calculated by subtracting the MFI of the "no antibody" condition. (nd. not done). n=3 donors of serum. Data information: Error bars indicate SEM. Significance was determined by comparing each antibody to mGO53. Only significant comparisons are depicted; *P<0.05, Mann-Whitney test. (E) Raji-Env cells were incubated with indicated antibodies and either normal (NHS) or heat-inactivated (HIHS) human serum. After 24h, cell death was determined by flow cytometry. The numbers indicate the percentage of dead cells. One representative experiment is shown. (F) CDC induction by the 24 indicated antibodies. The % of CDC was calculated as the relative percentage of dead cells compared to the "no antibody" condition. n=3 donors of serum. Data information: Error bars indicate SEM. Significance was determined by comparing each antibody to mGO53. Only significant comparisons are depicted; *P<0.05, Mann-Whitney test. (G) Raji-Env cells were incubated for 24h with NHS and indicated concentrations of WT or E430G mutant bNAbs (10-1074, 3BNC117 or PGDM1400) or isotype control (mGO53). Cell death was measured by flow cytometry and the % of CDC was calculated as the relative % of dead cells compared to the "no antibody" condition. n=3 donors of serum.