EMBO/sd-character-level-ner huggingface dataset:
The WD β-propellers of Atg16L1 recruit LC3 in the absence of FIP200.Figure 4.(A) Yeast two-hybrid interactions of the ULK1 complexes with other human Atg proteins. ULK1, ULK2, Atg13, FIP200, and Atg101-AD fusions (rows 2-6) or control AD constructs (row 1, Empty) were coexpressed with control DBD constructs (column 1, Empty) or Atg protein-DBD fusions (columns 2-35) and tested for positive yeast two-hybrid interactions (top) or cotransformation (control, bottom). White lines divide images derived from the same plate, and red lines divide images from different plates. Atg16L1 positively interacted with FIP200 in this assay. (B) FIP200 binds to exogenously expressed Atg16L1. Myc-Atg16L1 coprecipitations with an empty vector control (lane 1) or One-STrEP-FLAG (OSF)-FIP200 (lane 2). (C) FIP200 binds endogenous Atg16L1. Atg16L1 coprecipitations with an empty vector control (lane 1) or OSF-FIP200 (lane 2). (D) Wild-type or FIP200-KO MEFs stably expressing the empty vector, full-length Atg16L1, or ΔWD mutant were incubated in growth medium (−) or Earle's balanced salt solution (EBSS) (+) for 1 h and examined by Western blotting using the indicated antibodies. We previously reported that stable expression of an exogenous Atg16L1 construct can be used to replace the endogenous Atg16L1 protein with an exogenous Atg16L1 protein (Fujita et al., 2009) because free Atg16L1 molecules not complexed with the Atg12-Atg5 conjugate are preferentially degraded by the ubiquitin-proteasome system. In control cells (Empty), two Atg16L1 splicing variants, the α- and β-forms, were detected. In full-length β-form-replaced cells (Full), the α-form was not detected, whereas in ΔWD-replaced cells (ΔWD) both the α- and β-forms were minimally detected. Thus, we successfully obtained cells that express only endogenous levels of the full-length or ΔWD mutant. (E-L) Atg16L1-replaced wild-type or FIP200-KO MEFs were infected with Salmonella (MOI = 10) for 1 h and subjected to immunocytochemistry for Atg16L1 and Ub (E and F) or LC3 and Ub (I and J). The percentages of Atg16L1-positive (G and H) or LC3-positive per Ub-positive bacteria were enumerated (K and L). At least 50 Salmonella were counted. The average ± SD is shown for three independent experiments. Statistical analysis was performed by Student's t test. *, P < 0.05; NS, not significant. Bar, 5 µm.
Figure 3(A) The back skins of WT (Tsc2+/+) and Tsc2+/- mice were intradermally injected with LL37 or control vehicle (n=5 for each group). Pictures were taken on day 0.5, 2, 5, 8, 12 after the first LL37 injection. The mouse experiments were repeated for three times, and 5 mice were included in each group for each time. The results of a representative mouse experiment were displayed. Scale bar: 2 mm. (B, C) The severity of the rosacea-like phenotypes was evaluated with the redness area (B) and score (C) (n = 5 for each group). **P <0.01, comparison between Tsc2+/- (LL37) and Tsc2+/+ (LL37) group. ##P <0.01, comparison between Tsc2+/+ (LL37) and Tsc2+/+ (Control). (D) HE staining of lesional skin sections from Tsc2+/+ and Tsc2+/- mice injected with LL37 or control vehicle (n = 5 for each group). Scale bar: 50 μm. (E) Dermal infiltrating cells were quantified (n = 5 for each group). Data represent the mean ± SEM. **P < 0.01. 1-way ANOVA with Bonferroni's post hoc test was used.
a) Bif-1+/+ and Bif-1−/− MEFs were transfected with Flag–PI(3)KC3, then incubated in complete medium or EBSS for 2 h and subjected to an in vitro PI(3)KC3 kinase assay.figf7(a) Bif-1+/+ and Bif-1−/− MEFs were transfected with Flag-PI(3) KC3, then incubated in complete medium or EBSS for 2 h and subjected to an in vitro PI(3) KC3 kinase assay. The expression of Flag-PI(3) KC3 was confirmed by immunoblot analysis (left panel). A representative autoradiograph is shown (middle). The results were quantified by phosphoimaging with a Typhoon scanner and shown as fold activation (right). The data are presented as means ± s.d. of five independent experiments. (b) Bif-1+/+ and Bif-1−/− MEFs were transfected with p40(phox) PX-EGFP, cultured in complete medium or EBSS for 2 h and analysed using a fluorescence microscope. The number of p40(phox) PX-EGFP-positive vesicles per cell was counted (mean ± s.d.; n = 35). Statistical significance in a and b was determined by Student's t-test. (c) Stable Bif-1 shRNA clones or control HeLa cells were transfected with Flag-PI(3) KC3 alone or in combination with Flag-tagged Beclin 1 and UVRAG for 48 h and subjected to an in vitro PI(3) KC3 lipid kinase assay. The results were quantified and are shown as relative activities (mean ± s.d.; n = 3). (d) 293T cells were cotransfected with Flag-PI(3) KC3 and either wild-type Bif-1, Bif-1ΔSH3 or control empty vector for 24 h and subjected to an in vitro PI(3) KC3 kinase assay (mean ± s.d.; n = 3). (e) Schematic representation of the Bif-1-UVRAG-Beclin 1-PI(3) KC3 protein complex.
Fig.Figure 2Fig. 2 Identification of protein complexes and communities in the cellular extracts. (A) Elution of selected protein complexes as a function of their retention times (see Appendix Fig. S4 for their corresponding subunit elutions). (B) Scatter plot indicating discrepancies in the expected and measured molecular weights of 102 protein complexes that elute as a single peak; 50% of protein complexes are observed to have higher molecular weights than structurally characterized, indicating that they are organized in higher-order assemblies.
Figure 7(A-E) Wild-type and Cd22R130E primary B cells were labelled with Atto 633-conjugated Fab fragments against CD22 or IgM, settled onto nonstimulatory coverslips and imaged. Single particle trajectories were then analysed.(A) Trajectories of CD22 in Wild-type (left) and Cd22R130E cells (right) showing diffusion of single particles over 6 s.B) Diffusion coefficients of 300 representative CD22 and IgM particles. Bars and numbers indicate the median.(C) Confinement analysis. Bars and numbers indicate the median.(D and E) Two-population analysis of CD22 diffusion. Analysis of CD22 diffusion plotted on a logarithmic scale and fitted to two Gaussian-shaped curves to account for slower and faster diffusing populations.(D) Wild-type (blue line and circles) and Cd22R130E (blue dotted line and squares middle) primary B cells were compared in an overlay (left) and the proportions of the faster diffusing populations plotted (right).(E) Proportions of faster-diffusing populations. (F and G) Wild-type and Cd22R130E primary B cells were stained with Alexa 647-conjugated antibody against CD22 and settled onto nonstimulatory coverslips. Cells were then fixed, imaged with dSTORM and analysed.(F) 2D (top) and pseudo 3D (bottom) dSTORM images were reconstructed from single molecule localizations. The white dashed square is shown in pseudo 3D (bottom).(G) Quantification of the distribution of CD22 with Hopkins index and H function. Error bars (Hopkins index) and thin lines (H function) denote mean ± SEM. Data are pooled from three experiments. (H) Wild-type and Cd22R130E primary B cells were treated with 1 µM LatA and intracellular calcium flux was measured by flow cytometry. (I-K) Wild-type and Cd22R130E primary B cells were treated with vehicle control (-) or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-CD19 (p-CD19), phospho-Akt (pAkt), phospho-ERK (pERK) and actin and total ERK as loading controls. The intensity of phosphorylated proteins, normalized to actin or ERK, was referred to the unstimulated sample of the wild type cells, set as 1.Data are representative of at least two experiments. (L) Computer simulation of diffusing CD22 nanoclusters with the nanocluster size from Cd22R130E primary B cells. (M-O) Area covered by diffusing CD22 with the nanocluster parameters or the diffusion coefficient of Cd22R130E. Data are from seven independent simulation runs.(M) Computer simulation of diffusing CD22 with the nanocluster parameters of Cd22R130E cells.(N) Computer simulation of diffusing CD22 with the median diffusion coefficient of Cd22R130E.(O) Computer simulation of diffusing CD22 nanoclusters with the real diffusion coefficient and nanocluster parameters for CD22 observed in wild-type and Cd22R130E cells. The graph shows the area covered.Data are from seven independent simulation runs.*P <0.01, **P <0.001, ***P <0.0001 (Student's t-test).
Figure 4(A) Overlap of active regions identified in CLL and NBCs by ChromHMM, ATAC peaks or bidirectionally expressed RNA loci labeled as "Bidi". Venn diagram showing the total number of overlapping regions (not area-proportional). (B) Chromatin transitions within differential active states between NBCs and CLL. Heatmap representation of the amount of chromatin (log2 Mb+1) transitioning from a particular state in NBC (rows) to CLL (columns). Transitions were considered for all recurrent active chromatin state regions (states 1, 8 ,9 and 11) present in a minimum of 3 samples even if the consensus state was not an active state. Accordingly, the matrix includes transitions between non-active states at low frequencies. (C) Chromatin states at bidirectionally transcribed predicted enhancers loci. All Bidi loci identified in NBC samples (n = 961) and CLL samples (n = 8530) are shown. The Bidi loci show an enrichment of the states "Active 2 (predicted active enhancer)" and "Active 1 (predicted transcription start sites)" (D) Clustering of samples via expression of bidirectionally regions that are differential between NBCs and CLL and quantified using DESeq2. (E) Volcano plot of differential super-enhancers targeting known leukemia and cancer genes. Examples include SE loss at CDKN1A, PI3KC2B and KMT2B (MLL2) and SE gain at FMOD, CREB3L2, CTLA4, TCF4, LEF1 and BCL2. Points represent non-differential SEs (grey), and differential SEs (FDR < 0.01) with fold change greater than 1 (orange). (F) RNA expression changes of selected genes associated with differential SEs. Top: genes significantly (FDR < 0.05) up-regulated by SEs in CLL. Bottom: Genes significantly down-regulated by SEs in CLL. (G) Comparison of normalized gene expression of histone deacetylases between CLL and NBCs. Histone deacetylases significantly up-regulated in CLL are shown in bold. (H) HDAC activity and its inhibition by panobinostat in B cells from CLL patients (red) in comparison to healthy donors (grey). Error bars indicate standard deviation measured in four biological replicates. (I) Genome browser view of H3K27ac tracks (in grey) at exemplary genes for NBCs and CLL cells 24 hours after mock and after panobinostat treatment. At genes such as CDKN1A (cell cycle control) and KLF13, reduced H3K27ac signal in CLL was increased upon HDAC inhibition to the level found in NBCs. WNT11 is shown as an example of a de novo gain of an active enhancer due to treatment with panobinostat. (J) Heatmap displaying changes in H3K27ac read occupancy in CLL upon panobinostat treatment for 24 h. A general gain of H3K27ac in enhancers upon panobinostat treatment was observed.
Difference in β7-neck interaction between the WT and the β7 mutant E239K.Figure 6(A) The positions of the β6-L10-β7 and neck-coil peptides are shown in brown and green. The positions of the neck-linker peripheral peptides (α1-β3-P loop, α4-L12 and L12-α5) are shown in light blue. (B-D) Quantitation of site-specific intramolecular interactions. Representative MS/MS spectra of the indicated peptides, including the mutated amino acid (β6-L10-β7) (B and C) and neck-coil (D). In the inserted graphs, the intensities in the ADP-BeFx and ADP-AlFx states were measured with respect to the nucleotide-free state as 1.0. Grey bar graphs, WT; black bar graphs, E239K; (-), nucleotide-free. (E) Schematic diagram of the β7 mutation and motor activity. KIF1A moves on microtubules by repeating dynamic structural changes in the neck domain in response to the ATP binding and hydrolysis cycle. The β7 mutation exhibits enhanced interaction with the neck-linker and neck-coil and affects motor movement. MT, microtubule; α, α-tubulin; β, β-tubulin; NL, neck-linker; NC, neck-coil.
a) Microscopy of hemocytes from third instar larvae cultured ex vivo and infected with wild-type L.figf2(a) Microscopy of hemocytes from third instar larvae cultured ex vivo and infected with wild-type L. monocytogenes. Hemocyte nuclei (filled arrowhead) and DNA of L. monocytogenes (open arrowhead) are visualized by DAPI staining (blue or white); the actin cytoskeleton is stained with rhodamine-labeled phalloidin (red). hml-Gal4>Atg5IR, expression of an inverted repeat sequence of Atg5 to induce RNAi against Atg5. Scale bars, 10 μm. (b) Intracellular wild-type L. monocytogenes (WT Lm) or Δhly L. monocytogenes (Δhly Lm) counted manually in DAPI-stained infected hemocytes. LE112,hml>LE, hml-Gal4-driven expression of PGRP-LE in PGRP-LE112 hemocytes; imd1, imd; RelE20, RelishE20; hml>Atg5IR, inverted repeat sequence described in a; w, hml>Atg1, hml-Gal4-driven expression of Atg1 in w hemocytes; LE112,hml>Atg1, hml-Gal4-driven expression of Atg1 in PGRP-LE112 hemocytes; PGRP-LE112+rap, PGRP-LE112 hemocytes plus rapamycin. *, P 0.001, compared with wild type (t-test). Data are representative of four experiments (a) or at least three independent experiments (b; error bars, s.d. of triplicate measurements).
Ca2+ oscillations in immature DCs migrating in micro‐channels A Left, kymograph from sequential epifluorescence (20×) images showing cytosolic Ca2+ oscillations in speed‐fluctuating immature DC after slow motility phases (upper and lower panel) and a change in direction (lower panel).Figure 1A Left, kymograph from sequential epifluorescence (20×) images showing cytosolic Ca2+ oscillations in speed‐fluctuating immature DC after slow motility phases (upper and lower panel) and a change in direction (lower panel). DCs were loaded with the Ca2+ dye Oregon GreenBAPTA 1‐AM, introduced in micro‐channels, and imaged every 10 s. Right, sequential epifluorescence (20×) images (1 image/20 s is shown). Instantaneous velocities and intracellular calcium oscillations were measured as described in Materials and Methods. Scale bar: 10 μm. B, C Mean cell velocity of immature DCs (n > 125 cells from two independent experiments) (B) or CD8+T lymphocytes (n > 40 cells from one experiment) (C) migrating in micro‐channels with or without BAPTA. Boxes illustrate 10-90 percentiles of values, and whiskers represent the range of values. P‐values were calculated using a Kruskal-Wallis test. D Mean intracellular calcium concentration in speed‐fluctuating (ΔV/V0 > 0.3; ΔV/V0 relative speed variation divided by the global median velocity) and non‐speed‐fluctuating (ΔV/V0 0.3) immature DCs (n = 187 cells from more than three independent experiments). P‐values were calculated using a Mann-Whitney test. E, F Velocity fluctuations (ΔV/V0) (E) or mean Ca2+ activities (F) displayed by DCs migrating in micro‐channels in the presence of 100 ng/ml CCL3. P‐values were calculated using a Mann-Whitney test, with respect to non‐treated DCs (NT). G Mean Ca2+ activities during phases of fast (> 50% of max velocity) and slow ( 50% of max velocity) motility in speed‐fluctuating DCs (Supplementary Fig S2F) (n = 64 cells, from three independent experiments). Activities were normalized to the mean Ca2+ activity shown by each cell during the entire movie. The P‐value was calculated using a Mann-Whitney test.
Figure 5(A) Real-time quantitative PCR of the indicated genes (M1-like and M2-like macrophages markers) of A375 xenograft treated with PLX4720, bevacizumab or COMBO. Data are presented as expression fold change (log2) compared with vehicle after normalization for housekeeping gene TBP (n=3 tumors), (*P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle (PLX4720: m-Arg1 P = 5.65E-06) (bevacizumab: m-Ccl5 P = 0.025) (COMBO: m-Ccl5 P = 0.0070; m-Cd40 P = 0.00099; m-Cxcl10 P = 0.043; m-Cxcl9 P = 0.046; m-Il1b P = 0.0367; m-Stat1 P = 0.049; m-Tlr2 = 0.00182; m-Arg-1 P = 0.0013). (B) Representative images of COMBO-treated tumor sections co-stained with an antibody against CD68 and an antibody anti-CCR7 (M1-like polarization) or anti-CD206 (M2-like polarization). (C) Graphs showing the quantification of FACS analysis of infiltrating F4/80+ tumor macrophages and average percentage of median fluorescence intensity (MFI) of TNFα+ or INOS+ on F4/80+ cells after ex vivo stimulation with PMA and ionomycin in vehicle (n=4 tumors) and COMBO (n=6 tumors), *P < 0.05 versus vehicle (TNFα P = 0.037; INOS = 0.038). (D) Graphs showing the quantification of FACS analysis of infiltrating CD11b+Ly6C+ tumor monocytes and average percentage of MFI of TNFα+ or INOS+ on CD11b+Ly6C+ cells after ex vivo stimulation with PMA and ionomycin in vehicle (n=4 tumors) and COMBO (n=6 tumors), *P < 0.05 versus vehicle (TNFα P = 0.048; INOS = 0.038). (E) Graph showing the quantification of circulating CD11b+Ly6C+ monocytes in peripheral blood analyzed by FACS in vehicle- (n=4 tumors) and COMBO- (n=6 tumors) treated tumors. (F) Cell viability measured as fluorescence intensity of ZsGreen-A375 cells co-cultured with cells isolated from xenograft treated with vehicle or COMBO with after 48h. Data are representative of one experiment out of two done in triplicate *P < 0.05 versus vehicle (P = 0.046).
XL-MS analyses of fully assembled Elongator.Figure 3Schematic overview of highly confident (LD score at least 30) crosslinks between Elp1 (orange), Elp2 (yellow), Elp3 (pink), Elp4 (green), Elp5 (blue) and Elp6 (sand). The size of the used squares is adjusted to the length of the individual proteins and also indicated the location of the respective crosslinks. Inter cross-links are drawn as lines colored according to one of the linked subunits, intra crosslinks as arcs colored according the subunit, whereas "dimeric" cross-links (linking same residues of a protein) are depicted as red loops. Structural domains are indicated. Protein regions not present in the crystal structures and homology models are colored black. Figure was drawn using xiNET [76].
T55delinsRT mutation in MEK1 confers resistance to BRAF inhibition and promotes local invasion in vivo.Figure 5A375 melanoma cells expressing GFP, MEK1WT, or MEK1T55delinsRT were injected into immune-deficient mice (n = 8 per group), and after the tumor size of ˜100 mm3 was reached, mice were treated with 30 mg/kg dabrafenib or vehicle. Graphs represent fold change in tumor volume normalized on the tumor volume on the day of the start of the treatment. Error bars indicate standard error of the mean. Average tumor weight of all groups at the end of the experiment described in (A). Error bars indicate standard deviation. Unpaired t-test was used to calculate statistical significance. P-values are indicated in the graph. Immunoblotting for MAPK pathway components performed on tumors of all groups. Immunohistochemistry for Sirius Red (staining for collagen) and H&amp;amp;E on tumors from all groups, to identify grade of demarcation and local invasion. Scale bar represents 500 (upper row) and 100 (lower row) μm.
Figure 1A. PCR analysis of the human H49K variant of the ATPIF1 and rtTA constructs in wild-type (wt), ACTA1-rtTA (T), ATPIF1H49K-TRE (H) or double transgenic (T/H) mice. (B WB expression of the human (h) ATPIF1 protein in Skm (B extracts. hATPIF1 is only expressed in Skm from ATPIF1H49K|T/H mice. HSP60 are shown as loading controls. n= 3 mice/genotype. (C) Immunofluorescent staining of wt and ATPIF1H49K|T/H hindlimb muscle slices with hATPIF1 and βF1 antibodies. Images are representative of 3 mice/genotype, 5 fields/mouse. hATPIF1 H49K is only expressed in T/H mice. D) WB expression of the human + mouse (m+h) ATPIF1 protein in Skm D), brain, liver, and WAT (D) extracts. hATPIF1 is only expressed in Skm from ATPIF1H49K|T/H mice. α-tubulin are shown as loading controls. n= 3 mice/genotype. E. Polarographic profiles of isolated mitochondria from wt (lower trace) and ATPIF1H49K|T/H (upper trace) animals. Histograms show a reduction in state 3 respiration consistent with the inhibition of ATP synthase in ATPIF1H49K|T/H mice. Bars are the mean ± s.e.m. of n= 3 mice/genotype, 3 traces/mouse; OL, oligomycin; Ant A, antimycin A. (F, G) Quantitative proteomic analysis (iTRAQ) of hindlimb Skm from wt and LowOXPHOS mice. The pie charts (F) and volcano plot (G) present the upregulated (log2[LowOXPHOS/wt]≥1, red) or downregulated (log2[LowOXPHOS/wt]≤-1, blue) proteins. A (-) log p value of >4 was considered statistically significant. Data information: Data are the mean ± s.e.m. of n= 12 animals/ genotype measured in 2 different iTRAQ analysis *p <0.05 when compared to wt by ANOVA and Student's t-test. H. Cytoscape representation of the most upregulated proteins in Skm from LowOXPHOS mice. Data information: Data are the mean ± s.e.m. of n= 12 animals/ genotype measured in 2 different iTRAQ analysis *p <0.05 when compared to wt by ANOVA and Student's t-test. I. Cytoscape representation of GSEA analysis. Fourteen significantly altered pathways and their normalized enrichment score (NES) are shown. 1. Hallmarks of adipogenesis; 2. Go_Mitocondrial signal; 3. Kegg_TCA Cycle 4; Pyruvate Metabolism Reactome; 5. Hallmarks of OXPHOS; 6. Go_ETC; 7. Go_NADPH metabolism; 8. Kegg_Propionate metabolism; 9. Redox system; 10. Kegg_BCAA catabolism; 11. Go_Lipid metabolism; 12. Kegg_Lipid oxidation; 13. NEFA, Tg and ketone bodies; 14. Go_FAD binding proteins. Data information Data are the mean ± s.e.m. of n= 12 animals/ genotype measured in 2 different iTRAQ analysis *p <0.05 when compared to wt by ANOVA and Student's t-test. J. Enrichment score (ES) graphs from GSEA analysis. Data information: Data are the mean ± s.e.m. of n= 12 animals/ genotype measured in 2 different iTRAQ analysis *p <0.05 when compared to wt by ANOVA and Student's t-test. K. Skm iTRAQ ratio of proteins from BCAA catabolism. Higher intensities of red or blue colors represent higher or lower LowOXPHOS/wt expression ratios, respectively. Data information: Data are the mean ± s.e.m. of n= 12 animals/ genotype measured in 2 different iTRAQ analysis *p <0.05 when compared to wt by ANOVA and Student's t-test.
Figure 2(A) Average plots of Mediator cleavages around the TSSs of SAGA- and TFIID-dependent genes. The distance of cleavage maximum to TSS is indicated by the dotted lines and corresponding numbers. (B) Signal tracks of Med8-MNase and free MNase cleavages at the previously characterized UASs of the CLB2 and RPS5 genes. TSSs are indicated by arrows.