EMBO/sd-character-level-ner huggingface dataset:
FFAT-motif-dependent recruitment of MOSPD2 in ER-endosome contacts by STARD3NL A-C: GFP-MOSPD2 (A and C) and GFP-MOSPD2 RD/LD (B) expressing cells were transfected with Flag-STARD3NL (A and B), or Flag-STARD3NL ∆FFAT (C), and labelled using anti-Flag (magenta) and anti-Lamp1 (late endosomes/lysosomes marker; red) antibodies.Figure 7 A-C: GFP-MOSPD2 (A and C expressing cells were transfected with Flag-STARD3NL (A and B and labelled using anti-Flag (magenta) and anti-Lamp1 (late endosomes/lysosomes marker; red) antibodies. The subpanels on the right are higher magnification (3.5x) images of the area outlined in white. The Overlay panel shows merged green and magenta images. Scale bars: 10 µm  A-C GFP-MOSPD2 RD/LD (B) expressing cells were transfected with Flag-STARD3NL (A and B and labelled using anti-Flag (magenta) and anti-Lamp1 (late endosomes/lysosomes marker; red) antibodies. The subpanels on the right are higher magnification (3.5x) images of the area outlined in white. The Overlay panel shows merged green and magenta images. Scale bars: 10 µm  A-C: GFP-MOSPD2 (A and C expressing cells were transfected wit Flag-STARD3NL ∆FFAT (C), and labelled using anti-Flag (magenta) and anti-Lamp1 (late endosomes/lysosomes marker; red) antibodies. The subpanels on the right are higher magnification (3.5x) images of the area outlined in white. The Overlay panel shows merged green and magenta images. Scale bars: 10 µm  D: Pearson correlation coefficients between MOSPD2 (WT or RD/LD mutant) and STARD3NL (WT or ΔFFAT) staining are shown. Each dot represents a single cell (number of cells: MOSPD2-STARD3NL: 20; MOSPD2-STARD3NL ΔFFAT: 18; MOSPD2 RD/LD-STARD3NL: 13, from three independent experiments). Means and error bars (SD) are shown. Kruskal-Wallis with Dunn"s multiple comparison test (***: P< 0,001)
Animals with Low TOR-Pathway Activity Exhibit Increased Autophagy and Require the Autophagy-associated Gene bec-1 during Adulthood to Live Long.Figure 4(A) Average number of LGG-1::GFP-containing puncta in let-363/TOR RNAi-arrested animals compared to N2 wild-type animals (WT) grown on control (vector-only) bacteria, p < 0.0001, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. We were not able to detect increased LGG-1::GFP puncta in long-lived let-363(RNAi) adults; however, one generation of daf-2 RNAi, our positive control, did not significantly increase the number of foci in adults either (data not shown). Please see Table S1 for quantification of all data.(B) Average number of LGG-1::GFP-containing puncta in daf-15(m81)/unc-24(e138) heterozygotes (daf-15/+) compared to N2 wild-type animals (WT), p < 0.0001, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. Please see Table S1 for quantification of all data. daf-15 encodes the TOR-binding partner Raptor. (C) Survival curves of daf-15(m81)/unc-24(e138) heterozygotes (daf-15/+, strain DR412) fed either control bacteria or bacteria expressing bec-1 dsRNA during adulthood at 20 °C. Mean lifespan: daf-15/+ animals grown on control RNAi-bacteria: 25.1 d, daf-15/+animals on bec-1 RNAi: 20.8 d, p = 0.0008, Log-rank (Mantel-Cox) test. The lifespan of daf-15/+ animals grown on bec-1 RNAi-bacteria during adulthood was measured again, yielding similar results; the lifespan of daf-15/+ animals was also measured three times following whole-life RNAi exposure. In these experiments, bec-1 RNAi generally shortened the mean lifespan of daf-15/+ animals to a greater extent than it shortened the lifespan of wild type. (We also attempted to perform double-RNAi experiments, in which animals were cultured on a 50:50 mixture of let-363/TOR and bec-1 [or control] RNAi bacteria. Although the trends we saw were in the expected direction, the effects produced by half-strength RNAi were small and not statistically significant [data not shown].) Please see Table 2 for additional data.(D) Survival curves of wild-type animals derived from strain DR412 (WT) fed either control bacteria or bacteria expressing bec-1 dsRNA throughout their whole life at 20 °C. These assays were performed concurrently with the daf-15/+ lifespan analysis shown in Figure 4C. WT grown on control RNAi-bacteria: 21.4 d, WT on bec-1 RNAi-bacteria: 21.1 d (p = 0.34), p between daf-15/+ and WT grown on control RNAi-bacteria, p < 0.0001, Log-rank (Mantel-Cox) test. Please see Table 2 for additional data.
RAD54 localizes to ALT telomeres in response to DNA damage A Combined IF and DNA FISH analysis of RAD54 (IF) and telomeres (FISH) in ALT and non-ALT cell lines.Figure 1A Combined IF and DNA FISH analysis of RAD54 (IF) and telomeres (FISH) in ALT and non-ALT cell lines. White arrows indicate RAD54 foci that colocalize with telomeres. Scale bars = 10 µm B Quantification of data in A. A cell was counted positive if it contained 1 or more colocalization event between RAD54 and the telomere. At least 100 cells were counted per cell line per repeat. For SaOS2, NOS, SJSA1, HeLa LT n = 3. For Cal72, U2OS, HuO9 n = 4. Values shown are mean±sd Combined IF and DNA FISH analysis of RAD54 (IF), PML (IF) and telomeres (FISH) in ALT cell lines SaOS2 and HuO9. Scale bars = 10 µm Quantification of HuO9 data from C. RAD54 foci were detected using particle analysis in Fiji. Total RAD54 foci were counted. RAD54 foci were considered in APBs if they colocalized with both PML and telomere. At least 100 cells counted per repeat, n = 3. Values shown are mean±sd Western blot showing expression of TRF1-FOK1-mCherry fusion protein following doxycycline induction, both WT and D450A (nuclease dead). Cells were treated with 40 ng/mL doxycycline for 16 hrs and then 1µM Shield-1 and 1µM 4-OHT for 4 hrs Combined IF and DNA FISH for RAD54 (IF) and telomeres (FISH) in U2OS-TRF1-FOK1-WT or U2OS-TRF1-FOK1-D450A (nuclease dead) cells. Cells were treated as in E immediately prior to staining. Scale bars = 10 µm Quantification of data from F. RAD54 foci were detected using particle analysis in Fiji. At least a total of 300 cells were counted from 3 repeated experiments. Values shown are mean±sem. Values were compared using Kruskal-Wallis test followed by Dunn's multiple comparison test. Quantification of data from F. RAD54 foci were detected using particle analysis in Fiji. Cells were counted as positive if they counted 8 or more RAD54 foci that colocalized with telomeres. Values shown are mean±sem of n=3 biological replicates. Values were compared using standard two-way ANOVA followed by Sidak's multiple comparison test.
Figure 4Correspondence between the Nuc/Cyto (left) or hnRNPK IP/Input (right) ratios of sequences carrying a double mutation and the minimum Nuc/Cyto ratio of the two sequences which carry only one of the two mutations. Spearman's R and P values are indicated. Color indicates point density. Boxplots compare the difference between the values on the Y and the X axis for sequences with the indicated number of X→C mutations. Ratios are based on two replicates for Nuc/Cyto and three replicates for RIP. Boxplots are as in Figure 1C. Spearman's correlation R and P-value are indicated above the scatter plots. Pairwise comparison P-values shown above the boxplots were computed using two-sided Wilcoxon rank-sum test. Top: effect of single point mutations on Nuc/Cyto ratios, from Figure 2B. Bottom: the number of sequences with double mutations where the difference between the double mutant and the minimum of the two single mutants was larger than 0.3 log2 units, separating counting the cases when both mutations at the indicated positions are X→C.
Expression levels of the transgenic DIA1(R1204X) mutant in the inner ear of TG mice.Figure 7(A) Illustration of the linearized expression cassette containing the CAG promoter, 3xFLAG-tagged DIA1(R1204X), and SV40 poly(A) sequence used for injection into fertilized eggs. (B) Expression of FLAG-tagged DIA1(R1204X) in the inner ear and heart of TG, but not WT, mice is confirmed by immunoblotting using a FLAG Ab. Representative of 3 experiments. (C) Expression levels of FLAG-tagged DIA1(R1204X) relative to endogenous Dia1 in the inner ear, thymus, and heart were determined by immunoblotting using a Dia1/DIA1 Ab (inner ear; n = 4, thymus and heart; n = 3), and quantified in the inner ear and thymus (*P= 0.0226 and **P= 0.0016 by two-tailed Student's t-test). Lysates of HEK293 cells overexpressing equal amounts of GFP-Dia1 and GFP-DIA1 (determined using a GFP Ab: right end) are detected with equal affinity by the Dia1/DIA1 antibody. Comparable loading of proteins is confirmed using an α−tubulin or GAPDH Ab.
Figure 5(A) Localization patterns of ASY1T142V;T184V:GFP together with ASY3-RFP in the wildtype and pch2 mutants. Please note that images of ASY1T142V;T184V:GFP in pch2 mutants were taken with increased sensitivity for a better visibility. Scale bar: 5 μm. (B) Seed sets (mean ± SD, n = 5) of WT, asy1, pch2, asy1 pch2, ASY1T142V;T184V:GFP (asy1) and ASY1T142V;T184V:GFP (asy1 pch2) plants. Asteriks indicate significant differences (two-tailed t-test, P < 0.01) and ns depicts no significant difference. Scale bar: 2 mm. (C) Localization patterns of ASY1:GFP, ASY1T142V;T184V:GFP, and ASY11-570:GFP in the wildtype (WT) and/or in pch2 mutant plants at early prophase I. Scale bars: 5 μm. (D) Localization pattern of PCH2:GFP together with ASY1:RFP in the male meiocytes of wildtype. Arrowheads indicate the chromosomal regions where the ASY1 removal was concomitant with the localization of PCH2. Scale bar: 10 μm.
Mfn2 ablation prevents caspase activation during ER stress and promotes paraptosis‐like cell death.Figure 2(A) WT and Mfn2 KO cells were treated with 1 μM Tg for 12 or 24 h. Total and cleaved caspase 3 levels were detected by western blot. Data are mean±s.e.m. (n=3). *P0.05 versus WT group. (B, C) WT and Mfn2 KO cells were treated with 0.5 μg/ml tunicamycin (Tm), 100 ng/ml brefeldin A (Bref), or 1 μM Tg for 24 h. Total and cleaved caspase 3 levels were detected by western blot (B) and caspase activity (C) by measurement of DEVD‐AFC substrate processing. Data are mean±s.e.m. (n=3). *P0.05 versus WT group. (D) Flow‐cytometry analysis of the Sub G1 DNA fragmentation in methanol‐fixed WT and Mfn2 KO cells after incubation with or without 1 μM Tg for 24 h. Data are given as mean±s.e.m. (n=3). *P0.05 versus WT group. (E) Scr (stably expressing scrambled shRNA) and Mfn2 knockdown (KD) (stably expressing shRNA directed against Mfn2) 3T3‐L1 fibroblasts were incubated in the presence or absence of 1 μM Tg for 24 h. Total and cleaved caspase 3 were detected by western blot. Data are mean±s.e.m. (n=3). *P0.05 versus WT group. (F) WT and Mfn2 KO cells were treated with 1 μM Tg for 24 h. Lactate dehydrogenase (LDH) release was analyzed by flow cytometry to assess necrotic cell death. Data are mean±s.e.m. (n=3). *P0.05 versus WT group. (G) WT and Mfn2 KO cells were incubated for 24 h with or without 1 μM Tg in the presence or absence of z‐VAD‐fmk and stained for annexin V/PI. Data are mean±s.e.m. (n=4). *P0.05 versus WT+Tg group. (H) Mfn2 KO cells transfected with the pEGFP plasmid were incubated with 1 μM Tg alone or in combination with 2 μM CHX for 24 h (3 h of pre‐incubation with CHX). Florescence microscopy images show that CHX prevents cytoplasmic vacuolization. Scale bar: 10 μm. (I) WT (black circles) and Mfn2 KO cells (white circles) were incubated with 1 μM Tg for varying times, and ALIX protein was detected by western blot. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.Source data for this figure is available on the online supplementary information page.
Figure 1A. Genes involved in innate immunity are significantly upregulated in nuo-6 and isp-1 worms. Gene expression changes in the mitochondrial mutants were determined by RNA sequencing and compared to wild-type N2 worms. Results represent counts per million (CPM) expressed as a percentage of wild-type of six biological replicates per strain. Data information: Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001. Statistical significance was determined using a one-way ANOVA with Dunnett's multiple comparison test B. Upregulation of innate immunity genes is dependent on the p38-mediated innate immune signaling pathway including NSY-1, SEK-1, PMK-1 and ATF-7. Gene expression changes were examined by quantitative RT-PCR of three biological replicates per strain. Data information: Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001. Statistical significance was determined using a one-way ANOVA with Dunnett's multiple comparison test C. Using a fluorescent reporter strain for the innate immunity gene T24B8.5 confirms that innate immunity genes are upregulated in the long-lived mitochondrial mutants and that components of the p38-mediated innate immune signaling pathway are required for their upregulation. Scale bar indicates 250 µM. Three biological replicates per strain were quantified. Data information: Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001. Statistical significance was determined using a two-way ANOVA with Bonferroni post-test D. Differentially expressed genes in long-lived mitochondrial mutants show significant overlap with genetic targets of the p38-mediated innate immune signaling pathway. Significantly modulated genes in long-lived mitochondrial mutants nuo-6 and isp-1 (six biological replicates per strain) were compared to genes that are modulated in response to exposure to the bacterial pathogen P. aeruginosa strain PA14 in a PMK-1- and ATF-7-dependent manner There is a highly significant degree of overlap between genes upregulated by activation of the p38-mediated innate immune pathway and genes upregulated in nuo-6 and isp-1 mutants. Similarly, there is a highly significantly degree of overlap between genes downregulated by activation of the p38-mediated innate immune pathway and genes downregulated in nuo-6 and isp-1 mutants. The p-values indicate the significance of the difference between the observed number of overlapping genes between the two gene sets and the expected number of overlapping genes if the genes were picked at random.
Autophagy is decreased at later time points post-infection in human macrophages.Fig 3Macrophages were infected with HIV and cells harvested at 1, 3, 5, 7, and 10 days post-infection. (A) Cells were analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 6. (B) Cells were harvested then subjected to flow cytometry analysis of saponin-resistant LC3B-II in macrophages. Representative histograms of cells displaying saponin-resistant LC3B-II are shown. (C) Cells were harvested, lysed then analyzed for CYP27B1 and VDR by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 6.
Figure 1Left: Fluorescence changes of IRDye700 labelled dsDNA (at 125 nM) when incubated at increasing ID2 (I+D2, IUb+D2Ub, I+D2Ub) or ubiquitinated FANCD2 (D2Ub) concentrations (ranging from 1.3 nM to 5.9 µM). Measurement of fluorescence enhancement for each ID2 complex was conducted for two separately prepared complexes (two technical repeats) and all data points for each protein-combination were used in fitting of a one-site binding model. Right: Bar graph showing mean apparent Kd values calculated from model fitting. Error bars: Asymmetric 95% confidence intervals from non-linear regression (23-24 data points each). Assessment of protein-DNA interactions using Electro-mobility shift assays (EMSA). IRDye700 labelled dsDNA (at 2 nM) was incubated with indicated amounts of non/single/double-ubiquitinated ID2 (His6-TEV-V5-FANCI and FLAG-FANCD2) protein complexes (I+D2, IUb+D2Ub, I+D2Ub­) or ubiquitinated FLAG-FANCD2 (D2Ub). Mixes were ran on non-denaturing gels, and the resolved free- and protein-bound DNA bands were visualized using an infrared scanner. EMSA gels of ID2 complexes are representative of 3 replicate experiments.
Figure 7(A-J) Embryos injected dorsally with wt Wip1 (1 ng), wt Smad4 (1 ng), Smad4(T277A) (1 ng) and Smad4 (T277E) (1 ng) as denoted were subjected to in situ hybridization against Chordin or Xbra at stage 10.25. Control, control embryo injected with LacZ mRNA only. Scale bar, 150 μm. (K) Quantification of the results from in situ hybridization analysis in (A-J). (L) Wip1-mediated neural induction is inhibited by Smad4 with a phosphorylatable or phospho-mimetic amino acid at the MAPK site. (-), no co-injection of wt or mutated Smad4. (M) MDA-MB231 and MDA-MB468 cells were transfected with wt Wip1 alone or with wt Smad4 or Smad4(T277A) and then subjected to CCK-8 assays to measure cell growth. Data are presented as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test. (N) Anchorage-independent colony-formation assays were performed in MDA-MB231 and MDA-MB468 cells transfected with Wip1 or negative control constructs. Scale bar, 100 μm. (O) Quantification of the assays shown in (N). The number of colonies larger than 50 μm in diameter was scored. Data are expressed as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test. (P) Proposed model of the mechanism by which Wip1 controls the transcriptional activity and stability of Smad4. See the discussion for details.
Figure 1(A) Comparison of migration and invasion abilities between T- and B- lines. PC3 T- and B-line cells were incubated for migration (16h) and invasion (24h) assay. Data shown represent the means ± SD (n=3 biological replicates). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01). (B) Comparison of cell proliferation rate between T- and B- lines. Cell proliferation assay was performed by MTS reagents. Data are the means ± SD (n=3 biological replicates). (C) Western blot analysis of the endogenous CTH in PC3 orthotopically implanted primary tumor-derived (T2 and T3-lines) and bone-metastases-derived cancer cells (B2 and B3-lines). (D) Top: H2S catalyzing activity of cell lysates from PC3 T2, B2, T3, and B3 lines. Lead acetate-soaked paper strips show a PbS brown stain as a result of reaction with H2S. Bottom: The level of H2S production was quantified by densitometry, and the histograms represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis (*P<0.05). (E, F) 1x106 PC3-T2, B2, T3, and B3 cells were orthotopically injected into mouse prostate for 15-20 days. Incidences of paraaortic lymph nodes metastasis (E) and bone metastasis (F) are shown (n=6-8 mice per group). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01). (G) Top: expression of CTH in prostate tumors by immunocytochemistry analysis of commercial tissue arrays. Samples were sub-grouped by TNM stages. The representative images from different stages are shown. S: Stroma; Scale bars: 250μm. Bottom: The statistical significance was determined using the χ2 test The RNA-seq data of CTH was downloaded from the TCGA database (H) The mRNA expression of CTH in PC specimens and normal tissues. Data are presented as means ± SD (n=50 cases for adjancent non-tumor parts and 469 cases for PC tumors). Student's t-test was used for the statistical analysis (**P<0.01). (I) Kaplan-Meier survival analysis of PC patients with high or low CTH expression. The statistical significance was determined using the χ2 test.
Figure 1(A) Consecutive in vivo imaging of the same apical dendrites from layer V pyramidal neurons in the somatosensory cortex over 7 weeks reveals formation and elimination of dendritic spines (white and empty arrowheads, respectively) in WT and APP-KO mice. All mice were housed in standard conditions. Scale bar - 10 μm. (B-E) Graphical representations of the relative spine density, spine turnover rate (TOR), elimination and formation over the imaging period. Non-linear regression (F test) has been used for fitting the data points and comparing the difference between groups. N=4 mice per group ** p<0.01.
Figure 1A Electron micrograph of unstained UMOD filament sheets in human urine. The inset highlights a double helix-like structure resulting from juxtaposition of two individual filaments. Scale bars: 50 nm and 10 nm (inset).F Orthogonal views of the sharpened cryo-EM map of UMODfl (3.8 Å resolution), oriented as in panels B and C, respectively. The map is fitted with an atomic model that consists of a complete EGF IV+ZP module (chain A; blue), the ZP-C domain of a second molecule (chain B; teal) and the EGF IV+ZP-N domain of a third one (chain C; magenta). G-J Details of the UMODfl map shown in panel F: ZP-N N396 glycan (G); interdomain linker α1β (H) and β1 (I); ZP-C αEFβ/ZP-N βF' intermolecular interface (J). K Sharpened cryo-EM map of UMODe (4.0 Å resolution) in two orthogonal views oriented as in panels D, E. Comparison of this map with that of UMODfl identifies density belonging to the N-terminal half of UMOD (salmon contour in the front view of panel F), which is lost upon site-specific cleavage by elastase (orange arrow). L Goodsell-style depiction of a complete UMODfl filament model, with protein subunits shown in different colors.
Figure 3Autosharpened Cryo-EM density of the full TE complex composed of TssJ, TssL and TssM. The inner pillars are coloured in green and the outer pillars in blue. The unstructured tip and base are in grey, while the top of the core is in orange Vertical cross section of the cryo-EM density. Colouring is according to (A). The position of the slices for (C-E) are indicated. The density of the base appears to be composed of two leaflets, indicated by 2 arrows Cross sections of (A), sliced at positions indicated in (B) Subtracted, masked and unsharpened cryo-EM density of the, mostly unstructured, base Locally sharpened cryo-EM density of the subtracted and masked core. Three different views are shown. The colouring is according to (A).