EMBO/sd-character-level-ner huggingface dataset:
Figure 2Representation of protocol for the establishment of Ren-PDOXs. Ren-PDOXs were obtained by implantation of a total of 56 ccRCC human specimens on left mouse kidney. Representative images of Ren-PDOX tumor and lung metastases (mets, arrows) are shown. Total number of established and metastatic Ren-PDOXs is reported. Graph describes the proportion of metastatic Ren-PDOXs generated by tumor from patients with negative (pN0 and/or pM0) or positive (pN1 and/or pM1 and/or metachronous) metastatic status. *, one Ren-PDOX has not been evaluated for lung metastasis. Overall mass architecture of paired human specimens and Ren-PDOX tumors observed by HE staining (20X). TNM staging and Fuhrman grade (F) of implanted tumors are reported. For Ren-PDOXs, lung micrometastases in HE and vimentin stained sections are shown (40X). Human to mouse stroma substitution. CD34/CD31 staining for respectively human (h) and mouse (m) blood vessels in original human specimens and paired Ren13 and 28-PDOX are shown (20X). Ren-PDOX OT referred to tumor generated from first implantation of human specimen in mice. Insets show human and mouse kidney as positive/negative controls of staining (20X). Frequency histogram of SNV and/or indels found in Ren-PDOX by high-throughput sequencing in Ren13, Ren28, Ren36, Ren86 (targeted-NGS analysis by Haloplex) and Ren50-PDOX (WES). Only non-synonymous/stop gain SNVs and frameshift indels novel or with MAF<0.01 are included. Genetic comparison of original human specimen (patient #50) and its derived PDOX (Ren50-PDOX) by WES. Venn diagram shows number and percentage of variants called in human specimen (yellow), in its PDOX (blue) and overlapped variants (green).
Fbxw5 interacts with kinesin-13 family members A.Figure 2A. Co-IP of HA-MCAK with Flag-Fbxw5. Indicated proteins were expressed in Hek293T cells, extracted and subjected to anti-Flag IP followed by western blot analysis. B. In vitro pull-down experiment. Indicated proteins (purified from Sf21 cells, 5-fold molar excess of MCAK over Fbxw5/Skp1) were mixed with competing E.coli lysate and precipitated via Ni-NTA agarose. Proteins were washed, eluted in SDS sample buffer and analysed by SDS-PAGE. C. NanoBRET™ (Nano-bioluminescence resonance energy transfer) assay. Mdm2, p53 and Fbxw7 serve as controls. Mdm2 or MCAK were tagged with NanoLuc®-luciferase (Nluc), p53, Fbxw7 and Fbxw5 with HaloTag® (HT) and expressed in HeLa cells. After incubation with ligand overnight, substrate was added and plates were directly measured. Left: Quantification of 4 independent experiments. Error bars indicate standard deviation, asterisks the p-value of a two-tailed unpaired Student's t-test comparing each control with the MCAK/Fbxw5 pair (***P < 0.001). Right: Cartoon showing the NanoBRET™ principal. D. Endogenous co-IP of MCAK with Fbxw5. RPE-1 cells were either grown asynchronously or arrested for 24 hours with 2 mM HU (S phase), 10 µM RO-3306 (G2) or 75 ng/ml nocodazole (M). Protein extracts were subjected to unspecific rabbit IgG (-) or anti-Fbxw5 (+) IP and analysed by western blotting. Asterisk indicates an unspecific band detected by the Fbxw5 antibody. E. Co-IP of HA-Kif2a, HA-Kif2b or HA-MCAK with Flag-Fbxw5 F. In vitro pull-down experiment as in B. Asterisk indicates an unspecific protein from E.coli. Note: Kif2b purification from Sf21 cells was much less efficient and it was therefore not included in the experiment.
a) Subcellular localization of ceramide, generated by wild-type CerS1 induction (+ tet), was visualized in mitochondria by colocalization of ceramides and mitochondria with anti-ceramide and anti-Tom20, respectively, using confocal microscopy.figf4(a) Subcellular localization of ceramide, generated by wild-type CerS1 induction (+ tet), was visualized in mitochondria by colocalization of ceramides and mitochondria with anti-ceramide and anti-Tom20, respectively, using confocal microscopy. (b,c) Mitochondrial (Mito) targeting of LC3B-II and C18-ceramide, generated by induction of wild-type CerS1 (b), and that of the products of the catalytically inactive mutant CerS1 (+ tet) (c) was visualized by colocalization of ceramide (Cer; green), CFP-LC3B (blue) and MTR using anti-ceramide and confocal microscopy. Noninduced cells (− tet) were used as controls. Scale bars, 10 μm.
Figure 3Schematic representation of SHLD2 and the different mutants used in this study. Each putative structural domain of SHLD2 is represented. U2OS cells stably expressing HA-REV7 (Top) or HA-SHLD2 (Bottom) were pre-sensitized with 10ug/mL Hoescht 33342 before exposed to UV micro-irradiation. Immunofluorescence against endogenous HA and γ-H2AX epitope was subsequently performed to monitor REV7 and SHLD2 accumulation at sites of damage. Shown are representative micrographs. Scale bar = 5µm. U2OS LacR-Fok1 cells were transfected with GFP or GFP-SHLD2 and 24 h later DNA damage was induced using Shield-1 and 4-OHT. The cells were then processed for GFP and mCherry immunofluorescence. Shown are representative micrographs. Scale bar = 5µm. Quantification of the experiments shown in (C). Data are represented as the mean ± SD (n=3). At least 100 cells per condition were counted. Quantification of the experiments shown in (C). Shown is the quantification of the GFP signal at the mCherry-LacR-Fok1 focus. 293T cell lines expressing ER-AsiSI with Flag-SHLD2 and treated with 1 µM of 4-OHT. 6h later the cells were processed and immunoprecipitated with Anti-FLAG Magnetic Beads and anti-γ-H2AX.x/Protein A/G magnetic beads. DNA was purified and subjected to qPCR detection. Shown is the quantification of IP efficiency as the percentage of DNA precipitated from input (Bottom). U2OS cells stably expressing HA-SHLD2∆720-904 (Left) or HA-SHLD2∆1-680 (Right) were processed as in (B). Immunofluorescence against endogenous HA and RPA32 epitope was subsequently performed to monitor RPA32 and SHLD2 accumulation at sites of damage. Shown are representative micrographs. Scale bar = 5µm.
The CMA receptor LAMP2A is mislocalized in CTNS-deficient cells.Figure 5Immunofluorescence analyses of endogenous LAMP1, LAMP2A (A, B), LAMP2B (C) and total LAMP2 (D) localization in wild-type (WT) and Ctns−/− mouse fibroblasts. (A) Confocal microscopy analysis. White arrows represent LAMP1-positive circular structures (lysosomes) which were visible in both WT and Ctns−/− cells. Colocalization of LAMP1 and LAMP2A was observed in WT fibroblasts (white arrows, merged panel) but rarely in Ctns−/− cells (red arrow). Most LAMP2A showed a punctate pattern and lack of colocalization with LAMP1 in Ctns−/− cells (arrowheads, lower panels). Scale bars: 5 μm. Inset scale bars: 2 μm. Immunofluorescence analyses of endogenous LAMP1, LAMP2A (A, B), LAMP2B (C) and total LAMP2 (D) localization in wild-type (WT) and Ctns−/− mouse fibroblasts.(B) High-resolution stochastic optical reconstruction microscopy (STORM) analysis of the localization of LAMP1 and LAMP2A in wild-type and Ctns−/− cells. In Ctns−/− cells, LAMP2A was detected near (arrowheads, estimated distance > 50 nm) but not always adjacent (10-50 nm) to LAMP1 or at LAMP1-negative structures (arrows). Scale bar: 0.5 μm. Immunofluorescence analyses of endogenous LAMP1, LAMP2A (A, B), LAMP2B (C) and total LAMP2 (D) localization in wild-type (WT) and Ctns−/− mouse fibroblasts. (C) Immunofluorescence showing colocalization of endogenous LAMP1 and LAMP2B in both wild-type and CTNS-deficient fibroblasts (arrows). Scale bar: 20 μm. Immunofluorescence analyses of endogenous LAMP1, LAMP2A (A, B), LAMP2B (C) and total LAMP2 (D) localization in wild-type (WT) and Ctns−/− mouse fibroblasts. (D) Immunofluorescence analysis of LAMP1 and total LAMP2 (antibody ABL93 DSHB). Arrows show colocalization. Arrowheads show LAMP2-positive LAMP1-negative vesicles.
Figure 7A. Schematic representation of the SMP domains of Mdm12 and E-SYT2 showing different head structures depending on the presence of helix or strand. Based on our structural and biochemical data, we modeled Mdm12-Mmm1-Mdm34 ternary complex according to the organization of their respective SMP domains using head or tail regions. Two major contact regions among SMP domains are highlighted: H-H (head-to-head) and T-T (tail-to-tail) contacts. B. Sequence alignment between Mdm12 and Mdm34 along the N-terminal region (residues 1-7) critical for self-association or hetero-interaction. Each of the Mdm12 and Mdm34 sequences is displayed as a weblogo [33] representation to highlight sequence conservation. The sequences for Mdm12 and Mdm34 were analyzed using 34 and 60 orthologs, respectively. C. Schematic diagram showing the putative organization of the Mdm12 (yellow)-Mmm1 (red)-Mdm34 (blue)-Mdm10 (green) tetramer. Mmm1 forms a homo-dimer with a head-to-head contact in the center, capped on each end by a Mdm12 monomer through a tail-to-tail contact. Mdm12 associates with Mdm34 through a head-to-head contact. The hexameric SMP model was derived from the structure of six ΔMdm12 molecules within the asymmetric unit.
Figure 3(A) Principal component analysis of RNA-Seq data of genes from single Tet-TKD (purple), Tet3-KD (coral) and control (grey) 2-cell embryos. Each dot represents a single 2-cell embryo (control n=11, Tet3-KD n=11, Tet-TKD n=11). (B) MA plots of RNA-seq data from Tet3-KD/control (left) and Tet-TKD/control (right) 2-cell embryos. Significant differentially expressed genes are colored. The amount of up- and downregulated genes is indicated. Differentially expressed genes were determined using DEseq2 (padj<0.05, |log2FC|>0.7). (C) Venn diagrams visualizing the overlap of differentially expressed genes in Tet3-TKD, Tet-TKD and embryonically activated genes (EGA) in 2-cell embryos. EGA genes were calculated with DEseq2 comparing alpha-Amanitin treated and control 2-cell embryos (padj<0.05, log2FC>0.7). (D) Comparison of transcriptional profiles of Tet3-KD and Tet-TKD 2-cell embryos to a published database of early embryonic transcripts (DBTMEE, see (Park et al., 2015)). Shown are overlaps of DBTMEE categorized genes with the differentially expressed genes in Tet3-KD or Tet-TKD embryos compared to control 2-cell embryos.
a, Detection of wild-type (WT) and targeted alleles.figf2a, Detection of wild-type (WT) and targeted alleles. Genomic DNA was prepared from mouse tails and analysed by Southern blot analysis. Restriction enzymes used were HpaI for the 5′ probe and SpeI for the 3′ probe (see Supplementary Fig.S2 for the restriction map). KO, knockout (targeted) allele. b, Photograph of a representative Atg5-/- neonate compared to a wild-type littermate. c, Immunoblot analysis of Atg5 and LC3. Heart homogenates were prepared from neonates 10 h after birth. MEFs were cultured in Dulbeccos modified Eagles medium supplemented with 10% FBS (unstarved) or Hanks' solution for 2 h (starved), and total cell lysates were prepared. Immunoblot analysis was performed using anti-Atg5 and anti-LC3 antibodies. The position of the Atg12-Atg5 conjugate on the blot is indicated. d, Absence of GFP-LC3 dot formation in Atg5-/- mouse heart. Various tissues from Atg5-/- mice expressing GFP-LC3 were analysed for dot formation by fluorescence microscopy. A representative result from a neonatal heart at 3 h after birth is shown.
Figure 6(A-D) Changes in mRNA transcript levels of a series of adhesion molecules (A), chemokines (B, C) and cytokine receptors (D) in injured retinas compared with contralateral noninjured retinas, assessed 1 day after GT intoxication. n = 6 per group. (E) Representative micrographs showing the expression of ICAM-1 and VCAM-1 (red) by the RPE (green) 1 day after GT intoxication. Bar graphs throughout the figure show mean ± SE of each group. *, P < 0.05; **, P < 0.01; *** P < 0.001. GT - glutamate; noninj - noninjured control.
Quantification of starvation-induced autophagy in D.Figure 1.(A) Flow cytometry analysis of l(2) mbn cells starved for 2 h (2h S) or 4 h (4h S) showed an increase in LTG fluorescence levels (x axis) compared with control cells in full-nutrient medium (C). The gate shown on the histogram represents the LTGhigh population. (B) Representative images of GFP-LC3 puncta in control and 2-h starved l(2) mbn cells. An increase in GFP-LC3 puncta was observed in the starved cells (left). Bar, 10 μm. (C) Flow cytometry analysis of 4-h starved cells were incubated with 3MA (4h S + 3MA) and Baf (4h S + Baf). Both autophagy inhibitors reduced the LTG fluorescence levels compared with starved cells (4h S). Control cells in nutrient-full medium (C) are represented by the brown line. (D) Both autophagy inhibitors, 3MA and Baf, reduced the LTGhigh population significantly. (3MA, P = 0.00001; and Baf, P = 0.00006). (E) RNAi of representative Atg genes decreased the LTR fluorescence levels compared with control. (Atg1, P = 0.01; Atg5, P = 0.01; Atg7, P = 0.002; Atg8a, P = 0.008; Atg8b, P = 0.006; and Atg12, P = 0.02). (F) RNAi of all tested genes in the TOR-PI3K pathways had a statistically significant effect on LTG fluorescence levels. Known negative regulators of autophagy are shown with gray bars; positive regulators are shown with white bars. (Pten, P = 0.007; Tsc1, P = 0.027; Tsc2, P = 0.025; RheB, P = 0.005; Tor, P = 0.016; and S6k, P = 0.012). dsRNA corresponding to a human gene (Hs) was used as a negative control in E and F. Results represent the mean value ± SD from at least three independent experiments.
Ctcflos stands out as strongest regulated lncRNA in brite adipogenesis in vitro and during iBAT activation in vivo.Figure 2(A) Venn diagram of lncRNAs positively regulated in brite adipogenesis in vitro (see Figure 1 E) and induced in cold-mediated activation of interscapular brown adipose tissue (iBAT) in vivo. Seven lncRNAs are upregulated in both conditions. (B) Table of co-regulated lncRNAs. Three Ctcflos transcripts stand out as strongest regulated lncRNA candidates. (C-E) Ctcflos meets the three selection criteria for positively regulated lncRNAs in brite adipogenesis. (C) Correlation of Ctcflos (mean of transcript 1; 2; 3) with Ucp1 transcript levels across differentiated brite adipocytes derived from the five mouse strains (transcript levels in RPKM). Mean values, n=3 (biological replicates), pearson correlation, *p<0.05. (D) Ctcflos transcript levels (mean of transcript 1; 2; 3) in differentiated compared to undifferentiated brite adipocytes of 129S6 mice (transcript levels in RPKM). Mean and individual values, n=3 (biological replicates), unpaired t test, ****p< 0.0001. (E) Ctcflos transcript levels (mean of transcript 1; 2; 3) in differentiated brite (+rosi) compared to differentiated white (-rosi) adipocytes of 129S6 mice (transcript levels in RPKM). Mean and individual values, n=3 (biological replicates), unpaired t test, *p< 0.05. (F, G) Induction of Ctcflos along with Ucp1 in vivo in cold-activated iBAT Data ref: (Maurer S, 2018). Time course of (F) Ctcflos and (G) Ucp1 transcript levels in iBAT of 0, 6, 24 or 48 hours cold (4°C) exposed C57BL/6J mice (transcript levels in RPKM). Mean and individual values, n=3-4 (biological replicates), one-way ANOVA (Šídák-test), *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001. (H, I) Induction of Ctcflos and Ucp1 in vivo during cold-induced iWAT browning. Relative expression levels of (H) Ctcflos transcript 1 and (I) Ucp1 in iWAT of C57BL/6J mice held at 5°C for one week compared to age and weight matched mice held at 30°C or 23°C, assessed by quantitative PCR (qPCR). Mean and individual values, n=7 (biological replicates), one-way ANOVA (Šídák-test), **p< 0.01, ****p<0.0001.
Morphological changes induced by DAPk family proteins.Figure 1.(A) Quantification of cell death in 293 cells24 h after transfection with luciferase, p55/TNFR1, DRP-1 Δ73 or DAPk ΔCaM, and GFP. Graphs show the percentage of GFP-positive cells with altered cell morphology. (B) Photographs of GFP-positive cells cotransfected with luciferase (1), p55/TNFR1 (2), DRP-1 Δ73 (3), or DAPk ΔCaM (4). (C) Photographs showing the overlay of GFP and phase-contrast images of 293 cells24 h after transfection with GFP and luciferase (1) or with GFP- DAPk (2 and 3); both a blebbing GFP-DAPk-expressing cell (2, arrows indicate blebs) and spread nontransfected cells are shown (3). (D) Hoechst staining (4-6) of GFP-positive 293 cells (1-3) 60 h after cotransfection with GFP and DAPk ΔCaM (1 and 4), DRP-1 Δ73 (2 and 5), or p55/TNFR1 (3 and 6). Arrows point to condensed nuclei resulting from DRP-1 Δ73 or DAPk ΔCaM transfections (4 and 5).
Figure 2A-C The NMR structure of AMSIN. A. Ensemble of the 20 lowest-energy conformers superimposed over the backbone atoms of residues 3-36. B. A ribbon representation with disulfide connectivities shown in sticks. C. Electrostatic potential map. Red, blue, and gray represent negatively charged, positively charged, and electrostatically neutral zones, respectively. Data information: All structure images are displayed by MOLMOL. D Backbone-RMSDs for AMSIN-1 and CBSIN-2, shown as a function of time (30 ns simulations). The box marks the time for extracting the snapshots from the trajectories (49.9-50 ns) in (E) and (H). A total of 11 snapshots were taken from an MD simulation. Simulated 3D structures of AMSIN-1 and CBSIN-2. Their presentations are in the same pattern with that of AMSIN for AMSIN-1 The interchain disulfide bridge in AMSIN-1 and the Cys1-Cys2 vicinal disulfide ring (VDR) in CBSIN-2 (shown as sticks) are denoted by arrows. The N- and C-termini are labeled. Data information: All structure images are displayed by MOLMOL. Simulated 3D structures of AMSIN-1 and CBSIN-2. Their presentations are in the same pattern with that of AMSIN CBSIN-2). The interchain disulfide bridge in AMSIN-1 and the Cys1-Cys2 vicinal disulfide ring (VDR) in CBSIN-2 (shown as sticks) are denoted by arrows. The N- and C-termini are labeled. Data information: All structure images are displayed by MOLMOL.
Interactions of yNap1 with the basic DNA binding surface of the H2A–H2B heterodimer.Figure 3A Overview of the yNap1c2-H2AΔ14-H2BΔ28 complex.B Residues R17, R20, R29, R32, R35 and K36 of H2A make contacts with the acidic HBR1 in yNap1 comprising residues D201, D205 and E310.C Residues K75, R77 and R81 of H2A make polar contacts with the HBR2 comprising residues E332, D333 and E339 in α8 of yNap1. D In vitro GST-pulldown assay showing the role of residues in the HBR interfaces in H2A-H2B binding. The mutant labeled HBR1 (lane 6) contains the triple mutation D201R, D205R, E310R and HBR2 (lane 12) the triple mutations E332, D333, E336. HBR1+2 (lane 13) contains all six mutations. The mutant labeled HBR1+2A (lane 14) has Alanine replacements in the six HBR residues. Top panel: Bound. Bottom panel: Input. E Disruption of the Nap1-histone interaction by H2A mutation. Bottom panel: Inputs of the coexpressed proteins (soluble extract) and top panel the material bound to the glutathione affinity column were analyzed by SDS-PAGE and Coomassie staining. F Controls showing input (bottom) and bound (top) fractions. See also Fig EV2.
Figure 4A. A heatmap showing changes in indicated cytokines in response to NTD from indicated SARS-Cov-2 variants and inhibitors. Data are shown as fold change between NTD treatment and PBS control. Dex; dexamethasone. B. Dose-response curves of ponatinib treatment on SARS-Cov2 NTD (Wuhan)-mediated cytokine release in PBMCs. Right, a table showing EC50 (nM) values of ponatinib-mediated inhibition of cytokine release in response to indicated SARS-Cov2 variant NTD treatments in PBMCs.
Figure 3A,B EDN phosphoproteomics data was analysed in IPA. Significance of term enrichment is indicated through shading as -log (p-value). Regulated pathways belonging to the classes "Canonical Pathways" (A) and "Disease and Biofunction" (B) with significant enrichment (p < 10-3) are shown for UACC257 and A2058. Regulated terms were grouped according to highest order ontology terms, which are shown on the left.
Loss of either Eiger or Grindelwald, but not Wengen, leads to abnormal LD accumulation and age-associated retinal degeneration.Figure 4A. Alkaline phosphatase (AP)-shedding assays performed for Eiger in S2R+ cells expressing either GFP, Drosophila ADAM17, or activity dead mutants of Drosophila ADAM17, in the presence of either PMA, or PMA and TAPI-1 (DMSO is used as control; see methods); n=5. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01. B-C. Fluorescent images of retinas from 1 day old flies grown on either DMSO or TAPI-1, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively. Scale bars: 10μm. D. Integrated total lipid counts from retinas corresponding to flies reared either on DMSO or TAPI-1 starting from larval stages. n=10 fly retinas. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01. E-H. Fluorescent images of 1week old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (E) wild type; (F) eiger-/-; (G) grindelwald-/-; (H) wengen-/-. Scale bars: 10μm. I. Quantitation of the BODIPY signal shown as integrated total lipid measurements for the genotypes mentioned above. n=10 fly retinas. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean and the diamond an outlier. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01. J-K. mRNA level transcripts of ACC and FASN measured from head lysates of one week old wild type, eiger-/- and grindelwald-/- . n=3 biological replicates, with 3 technical replicates for each genotype. The box end points represent the maximum and minimum values respectively, the central band is the median and the square is the mean Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01. L-O. TEM images of two week old retinas corresponding to (L) control; (M) eiger-/-; (N) grindelwald-/-; (O) wengen-/-. n=180 ommatidia from 3 different fly retinas for each. Scale bars: 10μm. P. Quantitation of the percentage of normal, abnormal and missing rhabdomeres from 2 week old retinas observed in TEM, corresponding to the genotypes mentioned above; n=180 ommatidia from 3 different fly retinas for each. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.
Figure 6  Figure 6. Refined transport mechanism of ABC exporter McjD based on structural, biochemical and biophysical data. A, B) Binding of ATP alone (futile cycle) is not coupled to an opening of the TMD that remains occluded. NBDs can close in the presence of ATP and do not require substrate for closure. On the other hand, the NBDs are more dynamic and they can sample a nearly closed dimer that is driven to full closure in the presence of ATP. Binding of both ATP and MccJ25 are coupled to open up the TMD, which reverts back to an occluded conformation upon substrate release. ATP hydrolysis and ADP release resets the transporter in an inward conformation for a new transport cycle. Expanded View Figure Legends