EMBO/sd-character-level-ner huggingface dataset:
Miro1 is promiscuously ubiquitinated during the mitophagic process.Figure 2A) Schematic representation of hMiro1 sequence highlighting domains and lysine residues (Lys) in its sequence. Key lysine residues reported to be ubiquitinated upon mitochondria damage and mutated to generate the mycMiro15R mutant construct are highlighted. B) Representative images showing full length control and selected Miro1 lysine mutants (Miro15R, Miro1allR and Miro1R572K) expressing in MiroDKO MEF cells. All constructs (myc-tag, green) localise to mitochondria (Tom20, red). C) Immunoblot showing efficient expression of Miro1 lysine mutants in FlagParkin expressing SH-SY5Y cells. D) Ubiquitination assay showing that Miro1 lysine mutants have reduced ubiquitination upon FCCP treatment (1h, 10 μM) in FlagParkin overexpressing SH-SY5Y. The effect is quantified in (E) (n=4 independent experiments for Miro1WT and Miro15R and n=3 for Miro1allR and Miro1R572K, ANOVA with Sidak's post hoc test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001. F) Representative western blot showing a degradation assay in FlagParkin overexpressing SH-SY5Y cells transfected with Miro1WT, Miro15R or Miro1allR constructs and treated with FCCP (10 μM) for 3 or 6 hours. Quantification of Miro1 levels (G) the matrix protein PDH E1α in (H) and Parkin levels (I) (n=4 independent experiments; ANOVA with Sidak's post hoc test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.
Figure 1(a-c) Low-magnification cryo-EM projection images of pig (a), horse (b), and mouse (c) sperm. Different regions of the flagellum are annotated as follows: green - neck, yellow - midpiece, coral - principal piece, pink - endpiece. (d-f) Tomographic slices through cryo-FIB milled lamellae of pig (d), horse (e), and mouse (f) sperm. Transverse slices (d'-f') show complete triplets in the pig (d') and the horse (e'), but not in the mouse (f'). Complete triplets are indicated by green arrowheads with black outlines, while degenerated triplets are indicated by white arrowheads with green outlines. Note the electron-dense material in the lumen of the pig sperm proximal centriole that is continuous with the connecting piece (asterisks in d and d'). (g) In situ structure of triplet microtubules from the proximal region of the pig sperm PC with the tubulin backbone in grey and microtubule inner protein densities colored individually. A-tubule MIPs are colored: MIP1 - green, MIP2 - yellow, MIP3 - orange, MIP4 - red, MIP5 - purple, MIP6 - blue. B-tubule MIPs are colored: MIP7 - magenta. C-tubule MIPs are colored: MIP8 - light pink, MIP9 - pink. The A-C linker is colored gold and the putative A-link is colored olive green. (h) Reconstruction of the proximal region of the pig sperm PC generated by plotting the average back into the original particle positions and orientations in the tomogram. This plotback only contains four triplets as only part of the centriole was captured in the lamella. The A-C linker is colored in gold and the putative A-link in olive green.
Figure 5Graph reporting cell viability of control (shCTR) and antimiR-9 FaDu cells treated with increasing concentration of the indicated drugs for 72 hours and analyzed using the MTS cell viability assay. Data represent the mean (±SD) of three independent experiments each performed in sextuplicate and unpaired t test was used to calculate the statistical significance per each dose. Clonogenic assay of control (shCTR) and antimiR-9 FaDu cells not irradiated (NIR) or treated with 2 or 5Gy IR. On the left, typical images of cell clones are shown. On the right the graph reports the percentage (±SD) of survived cells respect to not irradiate cells in three independent experiments performed in triplicate. Unpaired t test was used to calculate the statistical significance per each dose. Left, WB analyses of the indicated protein expression in control (shCTR) and antimiR-9 FaDu cells not irradiated (NIR) or treated with 5Gy IR and allowed to repair for the indicated hours (h). Tubulin was used as loading control. On the right, protein quantification analyses of KLF5 and Sp1 expression normalized on tubulin loading control. On the right, graphs report the quantification of the indicated proteins normalized on tubulin expression. Data are expressed as mean (±SD) of three independent experiments and unpaired t test was used to calculate the statistical significance at time point. Left panel reports typical immunofluorescence images of control (shCTR) or antimiR-9 FaDu cells, not irradiated (NIR) and analyzed 1, 4 or 8 hours after 2Gy IR (γH2AX green, pS10-H3 red, nuclei in blue). Graphs report the percentage of γH2AX (middle) and pS10-H3 (right) positive cells. Data represent the mean (±SD) of three independent experiments in which at least 10 randomly selected fields were evaluated. Unpaired t test was used to calculate the statistical significance at each time point. Graphs report the qRT-PCR analyses evaluating the expression of miR-9 (left) or SP1 (right) in cells treated as in C. Data are the mean (±SD) of three independent experiment performed in duplicate. Two-way ANOVA with Sidak's multiple comparison test was used to calculate the statistical significance. Graph reporting cell viability of control (shCTR) and antimiR-9 FaDu cells, overexpressing or not SP1, and treated with increasing concentration of Bleomycin (BLEO) for 72 hours and analyzed using the MTS cell viability assay. Data represent the mean (±SD) of three independent experiments each performed in sextuplicate. Two-way ANOVA with Sidak's multiple comparison test was used to verify the statistical significance. *p<0.05; **p<0.01; ***p<0.001.
Figure 6A. Flag-AMOTL2 constructs were designed to express regions spanning from the N-terminus to the coiled-coil domain (AA 1-306), the coiled-coil domain (AA 307-581), and the C-terminal portion (AA 582-781). These constructs were co-transfected into 293T cells with Myc-Ub, and an in vivo ubiquitination assay was conducted. *, non-specific band. B. The SBP-AMOTL2 coiled-coil domain was expressed in 293T cells, subjected to tandem affinity purification, fractionated by SDS-PAGE and stained with Coomassie blue. Bands corresponding to the AMOTL2 coiled-coil domain were excised and analyzed by mass spectrometry for identification of ubiquitinated sites (K-G-G linkages). Shown are MS/MS spectra recoded using an LTQ-Orbitrap mass spectrometer for the doubly charged peptide, IEK*LESEIQR (MH+ = 1358.72, z = 2+; upper), and the triply charged peptide, LASK*TQEAQAGSQDMVAK (MH+ = 1992.96, z = 3+; lower). These peptides contain the K347 and K408 ubiquitination sites, respectively (marked by asterisks). Fragmented ions are annotated according to the nomenclature for peptide fragmentation in mass spectrometry. C. The indicated Flag-AMOTL2 WT or mutant proteins were subjected to an in vivo ubiquitination assay in 293T cells. D. RPE or MCF10A cells were transduced with Flag-AMOTL2 WT or Flag-AMOTL2 K347/408R, and immunoprecipitated with an anti-Flag antibody. Immunoprecipitated and WCL samples were analyzed by Western blotting for the indicated proteins. E. RPE or MCF10A cells were transduced with Flag-AMOTL2 WT or Flag-AMOTL2 Y213A, and processed as (D).
Figure 7Analysis of correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 during fasting-feeding Western blot analysis of hepatic Sel1L-Hrd1 ERAD and Crebh of nuclear (Nuc) and cytosolic (Cyto) fractions from 10-week-old Sel1Lf/f and Sel1LAlb mice under overnight fasted or fed states. Analysis of correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 during fasting-feeding Quantitation of levels of Sel1L/Hrd1/Crebh proteins and Fgf21 mRNA in the livers of 10-week-old Sel1Lf/f mice. during fasting-feeding Quantitation of total Crebh (Crebh+Crebh-N) protein levels in the livers of 10-week-old Sel1Lf/f and Sel1LAlb mice. during fasting-feeding Serum Fgf21 levels in 10-week-old Sel1Lf/f and Sel1LAlb mice. Analysis of correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 during growth Western blot analysis of hepatic Sel1L-Hrd1 ERAD and Crebh of nuclear (Nuc) and cytosolic (Cyto) fractions from the livers from Sel1Lf/f and Sel1LAlb mice at 3, 9 and 24 weeks of age. Analysis of correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 during growth Quantitation of levels of Sel1L/Hrd1/Crebh proteins and Fgf21 mRNA with the livers from Sel1Lf/f and Sel1LAlb mice at 3, 9 and 24 weeks of age. Quantitation of total Crebh (Crebh+Crebh-N) protein levels in the livers from Sel1Lf/f and Sel1LAlb mice at 3, 9 and 24 weeks of age. Serum Fgf21 levels in the livers from Sel1Lf/f and Sel1LAlb mice at 3, 9 and 24 weeks of age.
SARS-CoV-2 infects human organoid-derived 2D bronchioalveolar-like cultures.Figure 5A, B) Infectious virus titers can be observed by virus titrations on VeroE6 cells of apical washes at 2, 24, 48, and 72h after infection of bronchioalveolar-like cells from donor 1 (A) and donor 2 (B) at MOI 0.01 (red) or 0.1 (black) with SARS-CoV-2. The dotted line indicates the lower limit of detection. Error bars represent SEM. N=4 for all except donor 2 MOI 0.1, which is N=3. H p.i. = hours post infection. Immunofluorescent staining of SARS-CoV-2 infected differentiated bronchioalveolar-like cultures. Nucleoprotein (NP) stains viral capsid (red), which colocalized with HOPX Scale bars indicate 50 μm. Immunofluorescent staining of SARS-CoV-2 infected differentiated bronchioalveolar-like cultures. Nucleoprotein (NP) stains viral capsid (red), which colocalized with HTII-280 Scale bars indicate 50 μm. Immunofluorescent staining of SARS-CoV-2 infected differentiated bronchioalveolar-like cultures. Nucleoprotein (NP) stains viral capsid (red), which colocalized with SFTPC Scale bars indicate 50 μm.
ROCK inhibition induces the collective invasion of Caco-2 cysts A) Representative images of Caco-2 cysts transfected with control siRNA (siGlo) or siRNAs targeting ROCK1 and ROCK2, fixed and stained for prominin-1, F-actin (Phalloidin) and Nuclei (Hoechst).Figure 3A) Representative images of Caco-2 cysts transfected with control siRNA (siGlo) or siRNAs targeting ROCK1 and ROCK2, fixed and stained for prominin-1, F-actin (Phalloidin) and Nuclei (Hoechst). Confocal Z-sections of cysts are displayed. Arrowheads show non-protruding cells. Arrows point to protruding cells. White stars mark nuclei that are off-centered relative to the cyst's monolayer. Scale bars: 20 μm. B) Representative confocal images of Caco-2 cysts treated with ROCK inhibitors Y27632 and H1152, non-treated (NT) or transduced with ROCK1 and ROCK2 shRNAs (shROCK1+2). The cysts were fixed 2 days after invasion and stained for E-cadherin, F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i and ii are displayed at high magnification. White arrowheads point to non-protruding cells, green arrowheads point to the apical pole, arrows point to protruding cells and white stars show off-centered nuclei. Scale bar: 20 μm. C) Bar graph representing the percentage of protrusive Caco-2 cysts from three independent experiments. Caco-2 cysts non-treated (NT), incubated with ROCK inhibitors Y27632 and H1152 or infected with lentiviruses encoding shRNA control and targeting ROCK1 and ROCK2. The cysts were fixed 2 days after invasion, over 100 cysts were counted for each condition. Error bar represents the Standard Error of the Mean (Means ± SEM). P values were calculated using unpaired t-test (***p<0.001, **p<0.01). D) (Left panel) Schematic representation of the 3D Boyden chamber invasion assay. The Caco-2 were grown as cysts in 3D matrigel on the top chamber of the Boyden for 3 days, then the serum gradient was induced. Two days after, the nuclei of the cells that have reached the bottom side of the membrane were stained with Hoechst and quantified by automated acquisition and segmentation. (Right panel) DIC image of the membrane facing the top chamber reveals the cysts and the immunofluorescence image of the membrane facing the bottom chamber stained with Hoechst reveals the cells that have invaded. E) The number of cells that have invaded from the top to the bottom chamber of the Boyden chamber was quantified by automated segmentation in non-treated (NT), in ROCK inhibited conditions (Y27632, H1152) or in cells transfected with siRNA against ROCK1 and ROCK2 (siROCK1+2). The number of cells that have invaded was normalized to control conditions and reported as the average invasion rate from at least three independent experiments (Means ± SEM). P values were calculated using unpaired t-test (****p<0.0001, **p<0.01). F) High magnification of boxed region from (D). The top (i) and the bottom (ii) side of the Boyden chamber membrane were stained for F-actin, E-cadherin, ZO-1, Ezrin and Hoechst. Arrow points to the protrusive cells of an invasive cyst and the white stars show the nuclei that engage in the protrusion. Scale bars: 40 μm.
Fig.Figure 2A) Heat map analysis of the expression of circRNAs (both human circRNAs and ebv-circRNAs) in the fourth passage xenografts treated with 5-Fu or PBS. Red colour represents upregulated circRNAs, and green colour represents downregulated circRNAs. (B) Volcano plot representing the expression fold changes in circRNAs. The red dots represent upregulated circRNAs, and green dots represent downregulated circRNAs. (C) The back-spliced read distribution of ebv-circRNAs (n=261). (D) The length distribution of ebv-circRNAs. (E) Genomic origin of ebv-circRNAs. (F) Number of ebv-circRNAs produced from one gene.
Figure 5Cell cycle profile for HEK293 cells released from synchronization after double thymidine block. Cells were released and harvested at the indicated time points (AS= asynchronous). Analysis of cell cycle arrest and release was performed using propidium iodide (PI) staining and flow cytometry. A representative experiment of three performed is shown. Expression of the indicated proteins in HEK293 cells was examined by western blotting, and β-Actin was used as loading control. This summary is a representation of three independent experiments. Graph of flow cytometry data shows the percentage of cells in given cell‐cycle phases 48h after transfection with plasmids encoding scrambled siRNA or siRNA specific for CDK2. Graph represents the mean of three independent experiments. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with **p≤0.01 (ns=not significant). Graph of flow cytometry data shows the percentage of cells in given cell‐cycle phases 48h after transfection with plasmids encoding scrambled siRNA or siRNA specific for CDK1. Graph represents the mean of three independent experiments. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with *p≤0.05 and **p≤0.01.
Characterization of the human Airway Chip and its infection with influenza virus.Fig. 1.(A) Schematic diagram of a cross-section through the Airway Chip. (B) Immunofluorescence micrographs showing the distribution of ZO1-containing tight junctions and cilia in the epithelium and VE-cadherin-containing adherens junctions in the endothelium of the Airway Chip in the absence (Control) or presence (+ Virus) of infection with GFP-labeled influenza PR8 (H1N1) virus (MOI = 0.1) for 48 h (blue, DAPI-stained nuclei; bar, 50 μm). (C) Fold changes in gene expression levels of 4 different epithelial cell serine proteases (TMPRSS4, TMPRSS11D, TMPRSS2, DESC1) in the well-differentiated Airway Chip versus MDCK cells (one of the most commonly used cell lines in influenza studies) or undifferentiated primary human lung airway epithelial cells. Immunofluorescence micrographs showing the expression of ACE2 receptor (D) in the well-differentiated primary human lung airway epithelim on-chip (Post) versus the same cells prior to differentiation (Pre). fold changes in mRNA levels of ACE2 (E) and TMPRSS2 (F) in the well-differentiated primary human lung airway epithelim on-chip (Post) versus the same cells prior to differentiation (Pre). (G) Increase in barrier permeability as measured by apparent permeability (log Papp) within the human Airway chip 48 h post-infection with PR8 (H1N1) or HK/68 (H3N2) virus (MOI = 0.1) compared to no infection (Control).
Figure 2(A) Flow cytometry profiles of peritoneal cells from WT and KO mice stained for CD19 and F4/80 (left). FACS profile of CD19- subset showing CD11b+ and F4/80+ subsets of cells from WT and KO macrophage populations (middle). Cell numbers (right) represent the average of 4 WT and 4 Brd4 KO mice +/- S.D. Note a reduction in F4/80+ cells from Brd4 KO peritoneum. (B) Flow cytometry profiles of peritoneal cells from IL-4C injected WT and KO mice (left). Cell numbers (right) represent the average of 4 WT and 4 Brd4 KO mice +/- S.D. (C) Flow cytometry profiles of BrdU incorporation and Ki67, ARG1 expression in peritoneal cells (gated for F4/80+, CD19- macrophages) from IL-4C treated WT and KO mice (left). The numbers of cells positive for BrdU, Ki67 and ARG1 (right). The numbers represent the average of three experiments +/- S.D. (D) Microarray analysis of macrophage gene expression in IL-4C treated WT or Brd4 KO mice. Macrophages were isolated from peritoneal cells by the MACS system. Differentially expressed genes were identified with a cut-off line of log 2fold change >2 difference with p value of < 0.05. Red dots indicate differentially expressed genes (KO vs WT) and gray dots are those below the cut-off line. (E) Hierarchical clustering of differentially expressed genes. Representative downregulated genes in KO macrophages are shown by gene symbols. (F) qRT-PCR confirmation of representative genes downregulated in KO macrophages from IL-4C treated mice. Values represent the average of three independent experiments +/- S.D.
Figure 710-fold dilutions of diploid and tetraploid cells were spot inoculated onto SCD medium with or without the DNA-damaging agents hydroxyurea (HU, 20 mM) or methyl methanesulfonate (MMS, 0.01%). Cells were grown for 48 h at 30°C or 37°C and the plates imaged. Assay to monitor for loss of GAL1 (2-DOG resistant cells) arising from tetraploid strain RBY18 after culture at 37°C on SCD medium alone, SCD supplemented with HU (20 mM) or MMS (0.01%), or SCD medium with exposure to UV light for five seconds a day for 7 days.
Figure 6A Cartoons of ds/ss/ds oligonucleotides having either pyrimidines (T or C) or purines (A) in the ss-region. B Proportion of protein aggregation following renaturing with the ds/ss/ds oligonucleotides shown in (A). C Renaturing capacity of structurally different oligonucleotides. The diagrams on the right show a theoretical structure of each oligonucleotide. All oligonucleotides, except the 3x-loop and 3x-bulges, contain a stretch of 30 Ts in the single-stranded region and the same sequences (15 nucleotides each) in the double stranded regions. The 3x-loops and bulges oligonucleotides have 3 stretches of 9 Ts and the same sequence in the ds-regions. D Proportion of protein aggregation in Jurkat cell lysate supplemented with various amounts and configurations of the M1x4 or M2x4 DNA oligonucleotides. e Insoluble proteins from two ALS brain tissues were chemically denatured in guanidine hydrochloride and treated with either buffer (Vehicle), total RNA from Jurkat cells or the complementary strands of the M1x4 DNA oligonucleotides (M1 F/R) (Pellet 1, top panel). After removal of GuHCl, the soluble fraction from these samples was treated with RNase and any aggregated proteins (Pellet 2), analysed by western blot (middle panel). Remaining supernatants were then treated with Benzonase to degrade any remaining nucleic acids and aggregated proteins collected by centrifugation and analysed by western blot (bottom panel). Proteins aggregated by enzymatic RNA degradation in Jurkat cell lysates, which do not contain NF-H, were used as a control.
Figure 6Confocal whole-mount immunofluorescence (WM-IF) analysis of Kitl-tdTomato expression in the E8.5 (6-7sp) conceptus (maximum intensity 3D projection from a 500μm-thick Z-stack). Kitl-tdTomato expression (red) is seen in the YS, allantois (a; from which the umbilical artery derives), and para-aortic splanchnopleura (PAS) of Kitl-tdTomato::23GFP transgenic concepti (the embryo proper, from which the head has been removed, is outlined with a purple dashed line). The 23GFP transgene (green) labels the YS blood islands (bi; yellow dashed line); this GFP reporter is transcribed from a heterologous hsp68 promoter under the spatiotemporal control of the Runx1 hematopoietic +23 enhancer, resulting in GFP expression in all primitive erythroid(-fated) cells, hemogenic endothelium, and emerging HSPCs[2,74]. Endothelial cells (CD31+) are in white. Arrowheads indicate the paired aortae (pa). Purple boxed area is magnified in (B); yellow boxed area is magnified in (C). Scale bar: 100μm. N=2 embryos analyzed. Single 2.5μm-thick Z slice from the region highlighted in the purple box in (A). Arrowheads indicate Kitl-tdTomato+ CD31+ endothelial cells in one of the paired aortae (pa). Scale bar: 25μm. Single 2.5μm-thick Z slice from the region highlighted in the yellow box in (A), showing Kitl-tdTomato expression in the E8.5 YS BI. Arrowheads indicate Kitl-tdTomato+ CD31+ endothelial cells; the arrow indicates a Kitl-tdTomato+ perivascular cell. Scale bar: 25μm. Single 2.5μm-thick Z slice of 10.5 (35sp) YS (WM-IF) showing Kitl-tdTomato (red), Kit (white) and CD31 (green) expression. Arrowheads indicate Kitl-tdTomato+ CD31+ endothelial cells. N=3 embryos analyzed. Scale bar: 50μm. Confocal WM-IF analysis of Kitl-tdTomato expression (red) in a E10.5 (36sp) AGM region. Top panels (3D view) show a maximum intensity 3D projection from a 400μm-thick Z stack. Bottom panels (slice view) show a single 2.5μm-thick Z slice of the dorsal aorta, from the boxed region in the top panels. CD31 (vasculature; green); Kit (hematopoietic progenitors; white). Scale bars: 100μm (top); 25μm (bottom). fl: fetal liver; va: vitelline artery plexus; da: dorsal aorta; fp: floor plate; nt: neural tube. N=6 embryos analyzed. Confocal immunofluorescence analysis of cryosections through the AGM region of E10.5 (34-36sp) Kitl-tdTomato transgenic embryos. Arrowheads indicate Kitl-tdTomato+ CD31+ endothelial cells (top), Kitl-tdTomato+ PDGFR-β+ (middle) or Kitl-tdTomato+ α-SMA+ (bottom) perivascular smooth muscle cells. Scale bars: 25μm or 50μm (PDGFR-β panel). N>6 embryos analyzed. Confocal immunofluorescence analysis of Kitl-tdTomato expression in the E10.5 FL. A cryosection through a E10.5 (35-36sp) Kitl-tdTomato transgenic FL is shown. Arrowheads indicate Kitl-tdTomato+ CD31+ Kit- endothelial cells. Asterisks indicate Kitl-tdTomato+ CD31- Kit- mesenchymal cells or hepatoblasts. Note proximity of Kitl-tdTomato+ cells and Kit+ hematopoietic cells. Scale bars: 50μm (left panel), 25μm (magnified inset). N>5 embryos analyzed.
a) During a 72-h serum starvation, NHFs were transfected with either siRNAs targeting FBXL2, p85β, both FBXL2 and p85β, or a non-silencing siRNA (NS).figf6a) During a 72-h serum starvation, NHFs were transfected with either siRNAs targeting FBXL2, p85β, both FBXL2 and p85β, or a non-silencing siRNA (NS). Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. The graph at the right shows FBXL2 mRNA levels in the different samples analysed using real-time PCR in triplicate measurements. The values represent the ratios between FBXL2 and GAPDH mRNAs. SR, serum re-addition. (b) Cells were treated as in a, except that 3 h after serum re-addition, cells were lysed, immunoprecipitated (IP) with an anti-IRS1 antibody and immunoblotted as indicated. Uncropped images of blots are shown in Supplementary Fig. S8.