EMBO/sd-character-level-ner huggingface dataset:
Figure 7(A) Primary hippocampal neurons (DIV6+4) were transduced with shRNA targeting TDP-43 (shTDP) or control (shCtrl) and surface expression levels of glycoproteins was analyzed by metabolic labeling followed by surface biotinylation and subsequent streptavidin purification. Volcano plot depicts proteins that are significantly (p < 0.01, t-test, Benjamini-Hochberg, FDR: 0.05) decreased or increased on the cell surface in three independent experiments with two technical replicates. For full dataset see Table EV2. (B-C) Primary cortical neurons (DIV6+5) were transduced with shCtrl or shTDP. Neurons were treated with the ErbB4 ligand NRG1 (10 nM) for 10 min right before cell lysis. Immunoblots with the indicated antibodies (B). Quantification of total and phosho-ERBB4 (Y1056) using densitometry 3-7 biological replicates (C). (D-E) Primary cortical neurons (DIV6+5) were transduced with shCtrl or shTDP. Neurons were treated with the FGFR1 ligand FGF1 (10 ng/ml) for 10 min right before cell lysis. Immunoblots with the indicated antibodies (D). Quantification of total and phosho-FGFR1 (Y654) using densitometry 3-7 biological replicates (E). (F) Primary hippocampal neurons (DIV6+4) were transduced with shTDP or shCtrl. Gene expression was analyzed by quantitative RT-PCR normalized to the housekeeping genes YWHAZ and GAPDH (n=3) (G) Hippocampal neurons were transfected with ErbB4 containing a luminal HA-tag (HA/T-ErbB4) and shCtrl or shTDP (DIV9+3). Surface HA/T-ErbB4 was stained in living cells and intracellular HA/T-ErbB4 was stained after fixation and permeabilization with HA-antibodies. (H) Quantification of HA/T-ErbB4 surface levels. At least 10 images per condition per experiment were analyzed in three independent experiments. Scale bar represents 50 µm.
Figure 3A. Western blot analysis of phosphorylated PKD (S916/S876) in small intestine of male C57BL/6 mice after overnight fasting, olive oil gavage (or PBS in controls) and dissection at different time-points. B. Lipids tolerance test after oral gavage of olive oil (10µL per gram of body weight). n=6. C. Lipids tolerance test 30 min after tyloxapol i.p. injection (250µg/g body weight). In this case, only 5µL of olive oil per gram of body weight were gavaged orally. n=6. D. Western blot analysis and quantification of phosphorylated PKD2 (S876) and phosphorylated PKD (S916/S876) in small intestine of Pkd2wt/wt and Pkd2ki/ki male mice. n=3. E. Western blot analysis and quantification of phosphorylated PKD (S916/S876) in Caco2 cells expressing shscramble or shPkd2. n=3. F. Representative light microscopy pictures of organoids derived from jejunum crypts of indicated male mice. G. Percentage of released, uptaken and retained 14C-labeled fatty acids from Caco2 cells shscramble or shPkd2 (sequence 1) in a transwell system. n=6. H. Percentage of released 14C-labeled fatty acids by Caco2 cells expressing shscramble* or shPkd2* (sequence 2) grown in a transwell system. n=6. I. Transepithelial electric resistance of the Caco2 cells shscramble or shPkd2 (sequence 1). n=6.
Figure 5A Immunoblot expression analysis of proteins implicated in IFNγ signal transduction in U2OS mock or polyclonal MLLT6 knockout cells treated with or without IFN-γ. Numbers indicate band intensities normalized to GAPDH. B Immunoblot showing total protein levels of STAT1 isoforms (α and β), IDO1, GBP5 and CD74 in wild type U2OS cells transfected with esiRNAs targeting renilla luciferase (Rluc, negative control), total STAT1, STAT1α and STAT1β treated with and without IFN-γ. C Schematic model of MLLT6 function in IFN-γ signaling.
Figure 1Flag-INVS interacted with HA-Akt (lanes 1-3), but not with HA-PDK1 (lanes 4-6) or HA-PrKA (lanes 7-9) in HEK293T cells. Expression levels of HA-Akt, PDK1, PrKA, and Flag-INVS were similar, as determined by immunoblotting (HA: anti-HA antibody; F: anti-Flag antibody; C: control antibody). The results presented (A-E) are representative of at least two independent experiments. B. Flag-tagged INVS interacted with HA-tagged Akt1 (lane 1-3), Akt2 (lane 4-6), and Akt3 (lane 7-9) in co-immunoprecipitationassays. Expression of each Akt isoform and INVS were similar. C. INVS was co-immunoprecipitated with endogenous Akt from HEK293 cells, in which INVS is also endogenously expressed (lane 2). D. To determine the Akt domain responsible for interacting with INVS, Akt subfragments were generated. The C-terminus of Akt is the primary domain for INVS interaction (lanes 7-9). Similar levels of each Akt fragment were immunoprecipitated using anti-HA antibody. E. Schematic showing the structure and the functional domains of Flag-tagged full-length and fragmented INVS in mammalian expression vectors (N-Term: 1-421, Intermediate domain: 421-678, and C-Term: 678-1065) used in the current study. Intermediate (lanes 6-9) and C-terminal (lanes 10-12) fragments of INVS were important for interaction with Akt.
Exon skipping-based rescue of sarcomeric defects in TTN Ser14450fsX4 iPSC-derived cardiomyocytes.Figure 3Experimental design. Percentage of iPSC-derived cardiomyocytes with cross-striated myofibrils occupying a fraction (partially organized, dark gray) or the whole cytoplasm (fully organized, light gray) from two control and two patient clones. Statistical difference was tested using the two-sided chi-squared test (CTR1 basal: n = 283, CTR2 basal: n = 250, DCM1 basal: n = 255, DCM2 basal: n = 236; ***P = 1.93E−16). Immunofluorescence images of titin (Z-disk portion) and cardiac troponin T (cTNT, A-band) in well-organized sarcomeres from CTR and DCM single cardiomyocytes under basal conditions. Scale bars, 5 μm. Immunofluorescence images (left) of α-actinin and cTNT in CTR and DCM single cardiomyocytes, illustrating 3 different levels of sarcomeric organization (perinuclear, fully and peripherally organized). Percentage of cells with different levels of sarcomeric organization (right) under basal and stress conditions. Statistical difference was tested using the chi-squared test (CTR1 basal: n = 283, CTR2 basal: n = 250, DCM1 basal: n = 255, DCM2 basal: n = 236, CTR1 Iso: n = 245, CTR2 Iso: n = 230; DCM1 Iso: n = 242 and DCM2 Iso: n = 269; ***P = 1.93E−16 CTR basal versus DCM basal; ***P = 9.61E−34 CTR Iso versus DCM Iso, **P = 0.001 DCM basal versus DCM Iso). No significant differences were observed comparing CTR basal and CTR Iso groups. Scale bars, 25 μm. Percentage of perinuclear, fully, and peripherally organized single cardiomyocytes from two CTR and two DCM iPSC clones after infection with the U7snRNA-ScrAONs-IRES-GFP and U7snRNA-TTNAONs-IRES-GFP lentiviruses. Statistical difference was tested using the two-sided chi-squared test (CTR1 Scr-AON: n = 190, CTR2 Scr-AON: n = 200, DCM1 Scr-AON: n = 221, DCM2 Scr-AON: n = 115, CTR1 TTN-AON: n = 223, CTR2 TTN-AON: n = 187, DCM1 TTN-AON: n = 171, DCM2 TTN-AON: n = 243; ***P = 4.22E−15 CTR Scr-AON versus DCM Scr-AON; ***P = 4.61E−02 DCM Scr-AON versus DCM TTN-AON). No significant differences were observed comparing CTR Scr-AON and CTR TTN-AON groups.
Validation of ATF6 Partner Proteins Schematic of the ATF6α constructs used in this study.Figure 1Schematic of the ATF6α constructs used in this study. The N- terminus of ATF6α is tagged with a HA epitope or GFP as indicated. The C-terminus is V5-epitope-tagged. The region N-terminal to the transmembrane domain is localised in the cytosol whereas the region C-terminal is localised to the ER (lumenal domain). The positions of the cysteine residues and mutations are as indicated as well as the S1P cleavage site mutation (R415A/R416A). TAD: transactivation domain. Immunofluorescence microscopy of HEK293T exogenously expressing ATF6α-V5 tagged. Cells were fixed and stained with anti-calnexin (green) or anti-V5 (red). An overlay of the cells is presented in the third panel. Bar = 10 µm. Whole cell lysates from HEK293T cells, with or without stable expression of ATF6αWhole cell lysates from HEK293T cells stably expressing ATF6α were either untreated (-) or treated (+) with 5 µM thapsigargin (TG) for 1 h. THBS4 was transiently transfected into cells in the experiment depicted in panel 2. ATF6α was either immunoisolated with anti-V5 (panel 1 and 3) or anti-HA (panel 2). For the experiment in panel 3 the cells were cross-linked prior to immunoisolation. Samples were separated by SDS-PAGE under reducing conditions followed by western blots to determine co-isolation of endogenous PDIR, ERp18 or exogenously expressed THBS4 with ATF6α. HC indicates immunoglobulin heavy chain.
Figure 1(A) Live-cell nanoscopy of HeLa cells. COX8A-SNAP was labeled with SNAP-cell SiR and imaged with STED nanoscopy. (B) 3D architecture of cristae in HeLa cells visualized by FIB-SEM. The OM and the IBM are together shown as a transparent surface. Cristae of two mitochondria are depicted in light and dark blue. A twisted crista is shown in grey. (C) Live-cell recording as in (A). In addition, mitochondrial DNA was stained with PicoGreen and imaged by confocal laser scanning microscopy. Arrows mark sites where nucleoids but no lamellar cristae are present. (D) STED nanoscopy of mitochondria immunolabeled for Mic60 and dsDNA. Arrows mark sites where nucleoids are present. (E) Dual-color STED nanoscopy of cells labeled for COX8A-SNAP and immunolabeled for Mic60. Arrows mark Mic60 clusters in the absence of fully developed lamellar cristae.
Figure 4SGK3 [S486E]-GST and SGK3 [R90A S486]-GST were purified from HEK293 cells transiently overexpressing these enzymes. One hour prior to lysis, cells were treated with 5 µM VPS34-IN1 and 5 µM GSK2334470 to ensure that the SGK3 was in its inactive dephosphorylated form. (A) SGK3[S486E]-GST was incubated with lipid vesicles comprising phosphatidylcholine (PC) and phosphatidylserine (PS) containing the indicted concentrations of PtdIns or PtdIns(3) P in the presence or absence of added recombinant PDK1 (50 ng) and kinase reactions were initiated by addition of MgATP. After 30 min at 30oC, PDK1 was inhibited by addition of the 1 µM GSK2334470 PDK1 inhibitor and reaction mixture was supplemented with 2 µg GST-NDRG1 SGK3 substrate. After another 30 min at 30oC the reaction was terminated by addition of SDS Sample Buffer. The reaction mixtures were subjected to immunoblot analysis with the indicated antibodies. SGK3 [S486E]-GST and SGK3 [R90A S486]-GST were purified from HEK293 cells transiently overexpressing these enzymes. One hour prior to lysis, cells were treated with 5 µM VPS34-IN1 and 5 µM GSK2334470 to ensure that the SGK3 was in its inactive dephosphorylated form. (B) As in (A) except that non PtdIns(3) P-binding mutant SGK3[R90A, S486E]-GST was utilised.
Figure 4(A,B) Three-dimensional representation of (A) CA2 and (B) CA7 structure. The amino acids 101-105 (SLDGQ in CA2 and KKHDV in CA7), 113, 115 and 237-242 (GEPEEL in CA2 and DDERIH in CA7) form a ridge at the protein surface (highlighted in yellow). R223 is located close to the ridge. (C) Superimposing the CA7 α-helix-6 (CA7 in blue, areas where mutations1-3 are located is in yellow, and the putative actin interacting CA7 helix in red) on the Twf-C/G-actin structure (grey) shows a sterically compatible structure. (D) Sequence alignment of human CA7 and CA2 protein sequences generated using the Clustal O (1.2.1) multiple sequence alignment. Residues forming a ridge at the CA7 protein surface in the CA7 3D structure are highlighted in the CA7 sequence (bold/underlined). R223 is marked in turquoise and H96 and H98 (mutated to gain a catalytically loss-of-function mutant) are highlighted in green. The row below sequence alignment indicates structural features of CA7 based on the UniProt database (B = beta strand, H = helix and, T = turn). (E) Schematic representation of four mutants with a full (mutant1) or partial (mutant2 and mutant3) replacement of the amino acids encoding the ridge in CA7 by the corresponding CA2 sequence. In the reversed mutant (CA2-revCA7) the amino acids replaced in mutant1 were introduced to CA2.
Absence of HMGB1 sustains Beclin1–Bcl-2 interactions.Figure 4.(A) MEK inhibitors block starvation-induced p-ERK and Bcl-2. HMGB1−/− and HMGB1+/+ MEFs were starved in the presence or absence of 10 µM U0126 and 20 µM PD98059 for 6 h. Protein expression levels were assessed as indicated by co-IP or Western blotting. (B) Knockout of HMGB1 limits the disassociation of the Bcl-2-Beclin1 complex during treatment with autophagic stimuli. HMGB1−/− and HMGB1+/+ MEFs were starved in the presence or absence of 10 µM U0126 for 3 h. Protein expression levels were then assayed as indicated by co-IP or Western blotting. (C) HMGB1 interacts directly with Beclin1 during autophagy. HCT116 and Panc02 cells were treated with HBSS for 3 h and then assayed for protein expression levels as indicated by co-IP or Western blotting. (D) HMGB1 direct interactions with Bcl-2 are dependent on Beclin1. Knockdown of Beclin1 and Bcl-2 by siRNA in HMGB1 wild-type MEFs was performed, and protein expression levels were then assayed as indicated by co-IP or Western blotting. Ctrl, control. (E and F) Quantitative data demonstrating the interaction between HMGB1-Beclin1 and Beclin1-HMGB1 using densitometry software to assay the relative protein band intensity in co-IP experiments as shown in A-D. **, P < 0.005 and ***, P < 0.0005; n = 3. UT, untreated. Data are means ± SEM.
Figure 2A Schematic of the FLC locus. Exons are indicated with black boxes; promoter and introns are shown as lines. P1, P2 and P3 refer to genomic regions examined by ChIP. B ChIP-qPCR assay of H3K4me2 levels of indicated regions at FLC chromatin. C ChIP-qPCR assay of H3K4me3 levels of indicated regions at FLC chromatin. D ChIP-qPCR assay of H3K36me3 levels of indicated regions at FLC chromatin. E ChIP-qPCR assay of H3K27me3 levels of indicated regions at FLC chromatin .Data information: For ChIP analysis, 12-d-old plants were collected and three independent experiments were conducted. Each bar represents the mean ± s.d. of three independent experiments, n=3. The relative abundance was normalized to the input.
Figure 5Mean ± SEM AEA levels in cerebellar extracts from WT and ASM-KO mice after two months of vehicle or PF treatment (**PWT Veh vs KO Veh = 0.0063; ****PWT Veh vs KO PF < 0.0001; ****PWT PF vs KO PF < 0.0001; ****PKO Veh vs KO PF < 0.0001, n = 4 mice per group, one - way ANOVA, Bonferroni post hoc). Mean ± SEM SM levels, expressed as percentage of vehicle values, in cerebellar extracts from WT and ASM-KO mice after two months of vehicle or PF treatment (***PWTVeh Vs KO Veh <0.0001, *PKO Veh Vs KO PF = 0.0465), n = 4 mice per group, one - way ANOVA, Bonferroni post hoc). Western blot against NSM and GAPDH (used as loading control) and graph showing mean ± SEM NSM protein levels in cerebellar extracts from WT and ASM-KO mice after two months of vehicle or PF treatment (n = 3 mice per group, one - way ANOVA, Bonferroni post hoc). Mean ± SEM CB1 mRNA levels in cerebellar extracts from WT and ASM-KO mice after two months of vehicle or PF treatment (**PWTVeh Vs KO Veh = 0.0026, *PKO Veh Vs KO PF = 0.0345), n = 4 mice per group, one - way ANOVA, Bonferroni post hoc). Immunofluorescence against CB1 in Purkinje cells of the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graph shows mean ± SEM intensity associated to CB1, expressed as percentage of values in vehicle-treated mice (n = 4 mice per group, > 20 Purkinje cells per mouse Immunofluorescence against CB1 and Lamp1 in the Purkinje cell layer of the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graphs show Mander´s coefficient for the colocalization of CB1 with Lamp1 (left) and mean ± SEM lysosomal size (right) (***PWT Veh vs. KO Veh = 0.0002; **PKO Veh vs. KO PF = 0.0052, n = 4 mice per group, >20 lysosomes per mouse, one - way ANOVA, Bonferroni post hoc). Scale bar, 5 μm. Immunofluorescence against the Purkinje cell marker Calbindin in the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graph shows mean ± SEM number of calbindin positive cells per area (***PWTVeh Vs KO Veh <0.0001, **PKO PF Vs KO Veh = 0.00642), n = 4 mice per group, one - way ANOVA, Bonferroni post hoc). Scale bar, 100 μm. Immunofluorescence against the astrocytic marker GFAP in the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graph shows mean ± SEM intensity associated to GFAP in arbitrary units (***PWTVeh Vs KO Veh < 0.0001, **PKO PF Vs KO Veh = 0.0083), n = 4 mice per group, one - way ANOVA, Bonferroni post hoc). Scale bar, 10 μm. Immunofluorescence against the microglia marker Iba1 in the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graph shows mean ± SEM number (left) and area (right) of Iba1-positive cells (Left ***PWTVeh Vs KO Veh < 0.0001, **PKO PF Vs KO Veh = 0.0034; Right ***PWTVeh Vs KO Veh < 0.0001, PKO PF Vs KO Veh = 0.00123), n = 4 mice per group, > 20 cells analyzed per mouse, one - way ANOVA, Bonferroni post hoc). Scale bar, 10 μm.
Figure 3(A) MG1655 WT, single mutants of pstA and ugpB, and a double mutant of pstA ugpB were transformed with pBR322 and various pBR322-based folding biosensor constructs. The transformants were examined by a spot titer assay on LB agar plates containing various ampicillin concentrations. Strains containing pBR322 bla::Im7 L53A I54A gain considerable ampicillin resistance in the presence of a pstA mutation (compare uppermost left and right panels). This resistance is completely lost upon transducing in a ugpB mutation (compare next panels down), showing that ugpB is responsible for the resistance gained by the pstA mutation. (B) Ampicillin sensitivity conferred by a ugpB deletion mutant (CL543) is complemented by UgpB expression from the arabinose inducible expression vector pBAD33 ugpB (pCL1). (C) UgpB expression is sufficient to lead to higher levels of Im7 L53A I54A protein. Im7 L53A I54A and UgpB were expressed in a ugpB mutant strain (CL543) from pCDFTrc and pTrc99a vectors, respectively, both of which have an IPTG-inducible promoter. Periplasmic fractions were isolated and examined by Western blotting. "-" indicates strains containing empty vector and "+" indicates strains containing a recombinant plasmid encoding either Im7 L53A I54A or UgpB. MBP was used as a loading control. Data information: These experiments were repeated twice with similar results and one representative is shown.
The WD β-propellers of Atg16L1 directly interact with Ub.Figure 3.(A) HeLa cells were lysed with 1% Triton X-100 lysis buffer and subjected to immunoprecipitation with FK2- or control IgG-immobilized beads. The coimmunoprecipitated molecules were examined by Western blotting using the indicated antibodies. (B) Schematic diagram of Atg16L1 (Full) and its deletion constructs. (C) Purified recombinant GST or GST-Ub-immobilized glutathione Sepharose beads were incubated with lysates from HEK293T cells transiently expressing FLAG-tagged Atg16L1 constructs for 1 h at 25°C with gentle agitation. The beads were washed three times with ice-cold PBS and the bound complexes were eluted with 50 mM reduced glutathione and then subjected to Western blotting for FLAG. (D) Purified GST or GST-Ub-immobilized glutathione Sepharose beads were incubated with lysates from bacteria expressing trigger factor (TF) or TF-FLAG-WD β-propellers for 1 h at 25°C with gentle agitation. Washing and elution were performed as described in C. The eluted samples were subjected to SDS-PAGE and Western blotting for FLAG.
Figure 5(A) Schematic overview of MAC assembly on convertase labeled bacteria by C5b6 that is locally generated by incubation with C5 and C6 (top) or by preassembled C5b6 (bottom). (B) Bacterial viability of convertase-labeled E. coli MG1655 exposed to Buffer (Conv), preassembled C5b6 (Conv + C5b6MAC) or a mixture of C5 and C6 (Conv-MAC), in the presence of C7, C8 and C9. Dotted line represents the detection limit of the assay. Data information: (B-D) Data represent mean ± SD of 3 independent experiments. (B) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as ** p≤0.01. (C) Inner membrane damage (% Sytox positive) of convertase-labeled perimCherry/cytoGFP E. coli exposed to a concentration range of preassembled C5b6 or a mixture of C5 and C6, in the presence of 100 nM C7. After washing, bacteria were exposed to 20 nM C8 and 100 nM C9. Data information: (B-D) Data represent mean ± SD of 3 independent experiments. (B) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as ** p≤0.01. (D) outer membrane damage (mCherry) of convertase-labeled perimCherry/cytoGFP E. coli exposed to a concentration range of preassembled C5b6 or a mixture of C5 and C6, in the presence of 100 nM C7. After washing, bacteria were exposed to 20 nM C8 and 100 nM C9. Data information: (B-D) Data represent mean ± SD of 3 independent experiments. (B) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as ** p≤0.01.
Figure 4(A) Strategy of conditional knockdown of COUP-TFII in adult mice. A LacZ knock-in allele is inserted into the genomic COUP-TFII locus after the second LoxP site. (B) Activation of Cre recombinase by tamoxifen (TAM) results in COUP-TFII deletion and the expression of LacZ reporter (detected by immunostaining of β-galactosidase (β-Gal, red) in the UUO mice model (n=2). Quantification by confocal micrographs in 200x hpf. **p<0.01; ***p<0.001 by paired t test; mean ± SD. (C) Using heterozygous mice (F/+; Cre/+), β-Gal+ cells (red) increased after TAM injection in UUO kidneys, and co-localize with COUP-TFII+ cells (green) (n=3). (D) COUP-TFII+ cells decrease significantly in the UUO kidney in TAM-treated homozygous (F/F;Cre/+) mice (KO group, n=6) compared to wild-type littermates (WT group, n=4). Expression of αSMA (red) and collagen1 (yellow) are also markedly reduced. Masson Trichrome staining shows less kidney fibrosis in KO compared to WT group. Quantification by confocal micrographs in 200x hpf. **p<0.01 by unpaired t test, mean ± SD.
Figure 3DNA and crRNA sequence used for experiments of a single target and multiple PAMs. DNA sequence is shown for the single-target single-PAM construct and schematic representation is shown for multiple-PAM containing constructs. FRET histogram of all binding events from the construct containing a single PAM next to the partial target. Y axis represents total number of counts. Example FRET traces from single-target multiple-PAM experiments. Top trace shows an example of a single-step binding event and a binding event that starts at a lower FRET state before transitioning to productive binding high-FRET state. Bottom trace shows an example of FRET fluctuations between the high-FRET state and lower FRET states. A histogram showing the percentage of dynamic. The percentage values were obtained from five experiments on five different days. Error bars represent standard error of the mean. The percentage of events showing dynamic binding behavior was calculated by dividing the number of binding events that showed such behavior by the total number of binding events. A scatter plot showing the time Cas9 spends in a lower FRET state before transitioning to a high-FRET state. The dwelltimes increase with the number of PAMs adjacent to the target. Such dwelltime could not be determined for a construct containing a single PAM. The values were obtained from five experiments on five different days. Error bars represent standard error of the mean.