EMBO/sd-character-level-ner huggingface dataset:
β‐Catenin directly interacts with the autophagy protein LC3.Figure 8(A) Co‐immunoprecipitation of YFP-LC3 or negative control YFP in HT29 cells. Binding of endogenous β‐catenin to YFP-LC3 was detected after 8 h of autophagy induction by starvation and starvation in the presence of lysosomal autophagy flux inhibitor chloroquine (10 μM). Input and immunodepleted lysates are shown. (B) Immunoprecipitation of endogenous LC3 in HT29 cells. IgG was used as a negative control. (C)β‐Catenin contains a W/YXXI/L motif (putative LC3‐interacting region) at amino‐acid positions 504-507. β‐Catenin wild‐type (green highlight) and W504A/I507A double point mutant (blue highlight) sequences used in D-F are shown. (D) Pulldown assays using recombinant GST or GST-LC3B and lysates from HEK293 cells expressing HA-tagged wild‐type β‐catenin (HA-β‐cateninWT) or W504A/I507A β‐catenin (HA-β‐cateninW504A/I507A). HA-β‐cateninW504A/I507A exhibited reduced GST-LC3B binding compared to HA-β‐cateninWT. (E) Recombinant myc-tagged His-LC3 (His-LC3-myc) interacted with recombinant GST-β‐cateninWTin vitro, but binding to GST-β‐cateninW504A/I507A was reduced (upper panel). Input recombinant proteins visualised with Coomassie blue are shown (lower panels). (F) Co‐immunoprecipitation experiments using lysates from HEK293 cells transiently expressing YFP-LC3 with β‐cateninWT or β‐cateninW504A/I507A starved for 8 h with chloroquine (10 μM). YFP-LC3 immunoprecipitated β‐cateninWT but not β‐cateninW504A/I507A. Input lysates are shown and immunoprecipitated p62 and YFP-LC3 served as positive controls. (G) HA-β‐cateninS33A/S37A/T41A/S45A/W504A/I507A (HA-β‐catenin6A) was more resistant to the starvation‐induced reduction in β‐catenin protein expression than HA-β‐cateninS33Y. (H) Relative cell death in HT29 cells subject to nutrient starvation for 24 h. Atg7 knockdown increased cell survival. The increase in cell survival following Atg7 knockdown was significantly reversed by simultaneously depleting β‐catenin (double knockdown of Atg7 and β‐catenin). Data are the mean±s.e.m. of three independent experiments performed in triplicate, ***P0.001; **P0.01.Source data for this figure is available on the online supplementary information page.
Residues of the pilin localized towards the distal end of pili are involved in early adhesion(A) The ratio between the adhesion and aggregation mutation scores for each amino acid in the PilE sequence is represented as dots.Figure 4The ratio between the adhesion and aggregation mutation scores for each amino acid in the PilE sequence is represented as dots. Mutation type is color-coded as indicated in the legend. The superimposed magenta line represents the average ratio averaged over a moving window of 5 amino acids (right axis). Shaded areas highlight the two regions identified as important for adhesion. The yellow to red heat map along the pilin sequence indicates the axial position of the amino acid in PilE as shown on the structure of PilE (distal tip in red, proximal tip in yellow). Early adhesion of pilE mutants as a function of aggregation. Values were normalized to that of pilESB. Each dot represents a pilE mutant and the pilV, pilD, pilX and pilC1 reference mutants. Mutants in the region surrounding A131 are in green and those surrounding Y50 in purple. A blown-up view of the zone of interest (lower left quarter) is presented on the right panel. The shaded area highlights the absence of significant adhesion. Early adhesion of PilE mutants as a function of piliation per bacterium normalized to that of pilESB. Piliation length of a selection of mutants determined by immunofluorescence. Average pilus length ± SEM is indicated. N=6 experiments with at least 50 bacteria analyzed in each experiment. Paired t-test. Number of pili per bacteria ± SEM determined as in (E). Paired t-test. p<0.05 (*). Amino acids from the two regions identified in panel A are highlighted in purple and green. The SM1 epitope is colored in light purple.
A schematic overview of automated single-pot solid-phase-enhanced sample preparation (autoSP3) workflows.Figure 1A) The overview shows the different steps of the autoSP3 protocol from protein input to enzymatic digestion. The setup of the Bravo deck is shown for the core clean-up protocol. B) The overview shows the steps and Bravo deck setup of the autoSP3 peptide acidification and recovery protocol. The protocol ends with MS injection-ready peptide samples. C) A schematic overview of all available autoSP3 protocol versions. The autoSP3 procedure is provided with three options for reduction and alkylation and with post-digestion peptide recovery as described in the Appendix Protocols (A to D). Protocol A: one-step reduction and alkylation using a TCEP/CAA mixture for 5 minutes at 95°C, followed by autoSP3. Protocol B: two-step reduction and alkylation using DTT and CAA consecutively with 30 minutes incubation at 60°C and 23°C, respectively, followed by autoSP3. Protocol C: the core autoSP3 protocol omitting reduction and alkylation such that the user can flexibly pre-treat manually prepared samples. Protocol D: post-digestion acidification and recovery, delivering MS injection-ready peptides to a new sample plate.
Mechanisms of autophagy induced by depletion of TAB2 or TAB3.Figure 4(A-D) Impact of bafilomycin A1 (BafA1) on the induction of GFP-LC3+ puncta by TAB2 and TAB3 depletion. HeLa cells stably expressing GFP-LC3 were transfected with a control siRNA (siUNR) or with siRNAs targeting TAB2 and TAB3 for 24 h. During the last 12 h of this period, BafA1 was optionally added. After fixation and permeabilization, LAMP2 was detected by immunofluorescence. Representative confocal microphotographs for the TAB2 siRNA are shown (A), together with the profiles of colocalization of fluorescent signals (B) along the indicated direction (α-ω). Columns in (C) represent the percentage of colocalization of GFP-LC3 and LAMP2 (mean values±s.d.; *P0.01 versus siUNR‐transfected cells), as quantified in at least 50 cells for each condition. The frequency (mean±s.d.) of cells with >5 GFP-LC3+cytosolic puncta (GFP-LC3VACcells) is plotted in (D). (E) Impact of BafA1 on LC3 lipidation. MEFs with the indicated genotypes were cultured in complete medium supplemented with BafA1 for 12 h and the proportion of LC3‐I/LC3‐II was determined by immunoblotting. GAPDH levels were monitored to ensure equal loading. (F, G) Impact of autophagy‐relevant proteins and of the TAK1‐IKK signalling axis on GFP-LC3 aggregation induced by the depletion of TAB2 or TAB3. HeLa cells stably expressing GFP-LC3 were transfected with siUNR or with siRNAs targeting the indicated proteins, alone or in combination, and 48 h later GFP-LC3VAC cells were quantified (mean values±s.d., n=4; *P0.01 versus siUNR‐transfected cells). (H) Inhibition of autophagy by dominant‐negative (DN) TAK1. HeLa cells stably expressing GFP-LC3 were co‐transfected with pcDNA3.1 (empty vector) or with plasmids encoding WT (TAK1WT) or a DN TAK1 variant (TAK1K63W) together with the indicated siRNAs for 24 h, followed by the quantification of GFP-LC3VAC cells (mean values±s.d., n=3, *P0.01 versus siUNR‐, pcDNA3.1‐transfected cells)
Figure 5A CD81 knockout of 293T cells. The knockout cell line was generated by CRISPR-Cas9. The immunoblots using CD81 antibody and actin antibody were conducted on the same film. B CD81 facilitates CD19 maturation. CD81 and CD19 (300 ng DNA) were transfected into the CD81-knockout 293T cells. Anti-FLAG immunoblot was performed for CD19 with a C-FLAG tag. The deglycosylation was performed by digesting the cell lysate with PNGase F or Endo H, following the manufacturers' instructions. The deglycosylation pattern of CD19 species (p: precursor, m and m': two mature forms) differs because the glycosylation content of these species is different C Trypsin digestion of CD19. The digestion was performed at 37 °C with intact cells. The mature CD19 forms (m and m') can be digested because they are surface expressed, whereas the intracellular precursor form (p) is protected from the digestion. D EC1-EC2 interaction in the CD81 open conformation (modeled as in Fig 3E, right). The EC2 hydrophobic groove is shown in surface representation, and the interacting EC1 residues are indicated. E EC1 mutants of CD81 interfere with the accurate glycosylation of CD19. Chol: mutations at the cholesterol-binding site of CD81. CD81 constructs and CD19 (300 ng DNA) were co-transfected into the CD81-knockout 293T cells. Top, Anti-FLAG immunoblot was performed for CD19 with a C-FLAG tag. In presence of wildtype CD81 (wt), most CD19 is in the properly glycosylated mature form (m), and the overglycosylated m' form is not observed. With the EC1 mutants (A4 in particular), however, m' emerges and m shifts to high MW, indicating an alteration in the CD19 glycosylation. The level of intracellular p form is low with the wildtype CD81 or with the mutants. Bottom, Control experiment showing similar expression level of wild-type CD81 and CD81 mutants. Anti-V5 immunoblotting was conducted for the CD81 constructs with a C-V5 tag, and anti-actin immunoblotting was conducted on the same film.
CAFs express the somatostatin receptor subtype 1 sst1 that mediates SOM230 inhibitory effect on CAF-induced chemoprotection.Figure 3A Expression of the somatostatin receptor subtype 1 (sst1) analysed by RT-qPCR in the human pancreatic endocrine cell line BON and in CAFs isolated from 11 different patients (mean ± SD). B Immunoblotting of protein extracts from PaSCs and CAFs, using anti-sst1, anti-α-SMA, anti-P-S6 or anti-GAPDH (loading control) antibody (representative of n = 3). C Immunoblotting of protein extracts from BON, CAFs and indicated pancreatic cancer cell lines, using anti-sst1 or anti-GAPDH (loading control) antibody (representative of n = 3). D Immunofluorescence confocal microscopy analyses of α-SMA and sst1 in CAFs. Co-localization of α-SMA and sst1 is shown (merge) (one representative field of n = 3) (scale bar = 100 μm). E-G Representative immunofluorescence confocal microscopy analyses of sst1 co-localization with cytokeratin-19 (n = 42 PDAC samples) (E), α-SMA (n = 42 PDAC samples) (F) or with P-4E-BP1 (n = 25 PDAC samples) (G) in serial slides (scale bar = 100 μm). Dashed white lines represent the limits between stroma and tumour epithelial glands. H, I Immunoblotting of protein extracts from siCTR- or sisst1-transfected CAFs treated (+) or not with SOM230 for 48 h (sst1, puromycin and GAPDH as loading control, H) or for 30 min (P-Akt, P-S6, 4E-BP1 and GAPDH as loading control, I) (representative of n = 3). J Panc-1 cell viability was assessed by MTT. Panc-1 cells were incubated with gemcitabine in the presence of CM from untreated or SOM230-treated CAFs transfected with siCTR or si-sst1. Results (mean ± SD) are presented as the percentage of the untreated cells (= 100%) (n = 3; **P = 0.003, §§P = 0.002).
Figure 5(A, B) (A) Analysis of the three candidate markers in EV pellets isolated from CCM of control and infected (red boxes) Jurkat cells. The successive pellets (2K, 10K, 100K) were analyzed by Western blot, using antibodies against SPN, MOV10, SERINC3 and p24 (HIV-1 protein). The 2K, 10K, and 100K pellets obtained from 20 x 106 cells were loaded on the gel. Representative images (A) and quantifications in three to five independent experiments (B) are shown. For each pellet, arbitrary units (AU) represent the ratio of signal intensity for the analysed protein in the given pellet to the total amount of proteins in the same pellet. In cell lysates from the same experiments, AU (given protein/total amount of protein) for SPN and MOV10 were 1.29±0.15 and 0.84±0.33 times the levels in control cells, respectively (Mean±SD). (C) WB showing the distribution of the same proteins in six fractions recovered after velocity gradient separation of vesicles contained in the 100K pellets of control or HIV-1 infected (red boxes) cells. Viral p24 protein was recovered in the bottom fractions of infected samples together with MOV10 and SPN. SERINC3 was recovered in the middle fractions. (T = top; B = bottom). (D, E) Quantification of MOV10, SPN, SERINC3 and p24 signals in 4 to 7 independent Western blots from HIV-1 infected samples. AU = band intensity in a given pellet / sum (band intensity in all six pellets). (F) Quantification of SERINC3 signals in the different fractions from control and infected samples. AU = band intensity in a given pellet / sum (band intensity in all 12 pellets). Lines represent the medians of three independent experiments. (G, H) EVs from supernatant of HIV-infected Jurkat cells were subjected to immuno-isolation with beads coupled to antibody against SPN. Bead-associated (Pull-down: PD) vesicles and those left behind (Flow-Through: FT) were loaded on a gel for Western blot analysis with antibodies specific for SPN, MOV10, CD63, SERINC3 and viral p24. A representative image (G) and quantification (H) Mean ± SD of the proportion of signal in PD as compared with total (PD+FT) in three independent experiments are shown. (I) EVs remaining in the FT, or EVs secreted by the same number of cells but not subjected to immuno-isolation (input) were used to infect a reporter cell line. Mean± SD of six independent experiments is shown.
Figure 4(A) Representative images of F-actin in Lifeact-mCherry Venus expressing cells with treatment as indicated (n = 3 independent experiments). WT-actin-NLS, expression vector encoding NLS-tagged wild type actin (WT); R62D-actin-NLS, expression vector encoding NLS-tagged non-polymerizable actin mutant (R62D). Scale bars, 20μm. (B Transwell for HCC cells with treatment as indicated (n = 3 independent experiments). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01. C) wound-healing migration analysis for HCC cells with treatment as indicated (n = 3 independent experiments). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01. (D) SNU-368 shTFAM cells with R62D-actin-NLS or WT-actin-NLS were infected with firefly luciferase expressing lentivirus and transplanted into livers of nude mice. Representative bioluminescent imaging for each group (n =10) (E) Representative H&E images of lung metastasis in mice used in (D). Scale bars, 100μm. (F and G) Number of metastatic nodules per lung in mice used in (F) and the incidence of lung metastasis (G) in mice used in (D). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01. (H) Kaplan-Meier survival curves for mice inoculated with orthotopic HCC cells as indicated. n=10 per group.
Figure 4Figure 4. HDA14 protein localizes to the chloroplasts and mitochondria in Arabidopsis, its activity is dependent on cofactors and can be inhibited by deacetylase inhibitors. (A) GFP localization (green) of the HDA14-GFP fusion construct in Arabidopsis protoplasts (35S:HDA14:GFP) from stable transformants. The mitochondrial marker TMRM is depicted in purple. GFP+TMRM shows the overlay image of 35S:HDA14:GFP and TMRM, AF indicates the chlorophyll autofluorescence and BF the (B) Deacetylase activity of the recombinant 6xHis-HDA14 protein using a colorimetric assay. Co2+ and Zn2+ were used as cofactors, apicidin (100 µM) and trichostatin A (5 µM) as deacetylase-inhibitors (n = 5, ±SD).
Fig.Figure 3(A) Essential dynamics during the shift from growth on glucose to growth on acetate: Protein synthesis by ribosomes depends on the availability of biosynthetic precursors which itself depends on the availability of metabolic enzymes which utilize acetate to provide novel precursors. When ribosomes synthesize more of these required enzymes (red arrow 1) instead of other proteins (gray arrow 2) novel precursors are generated from acetate (black arrow) faster and growth thus resumes faster (detailed considerations and full model introduction in Figs. EV3 and EV4 and Materials and Methods). (B) Lag-times fall reciprocally with the allocation towards required proteins (the fraction of mRNA encoding for required proteins) during the shift. Allocation of translation activity towards different proteins are represented by the weight of red and gray arrows (top). (C) Comparison of model prediction with observed changes in lag-time when overexpressing the non-required gene lacZ (Fig. 2). The model has one major free parameter, the metabolic rate describing precursor influx. We determined this parameter by comparing predicted and observed lag-times in the absence of induction (Materials and Methods). The model then predicts the change in lag-time without further fitting when lacZ expression was induced and the fraction of required genes fell as a consequence. Data and error bars represent mean ± SD of N=3-5 biological replicates, as in Fig. 2D. All model parameters are provided in Appendix Table S3. Results and figures can be regenerated using the Jupyter notebook available on GitHub.
Figure 6A, B, C Concentration of ceramide species as measured by LC/MS from DX and REL pair whole cell pellets. Data information: show mean of 3-4 biological replicates plotted; 3 technical replicates each D, E Cumulative ceramides and sphingomyelins (of C32-C36 chain length) for DX/REL pairs. Data information: show mean of 3-4 biological replicates plotted; 3 technical replicates each error bars are +/- SD. F Ratio of total C32-C36 sphingomyelins:ceramides for DX/REL pairs. Data information: show mean of 3-4 biological replicates plotted; 3 technical replicates each error bars are +/- SD. G Cytochrome c release for CHLA15 and SKNBE1 mitochondria following exposure to tBid, pre-incubated for 30min with or without GW4869, and CHLA20 and SKNBE2C preincubated without GW4869 (left panel); same experiment but with GW4869 added with tBid, after the 30min pre-incubation (right panel). Data information: data points are mean of duplicate wells (SD<0.05 at all points) in a representative experiment from at least two biological replicates. H Summary data for CHLA15/CHLA20 and SKNBE1/SKNBE2C showing relative cytochrome c release when pre-incubated with GW4869 to inhibit ceramide generation, compared with untreated cells. Data information: All data points from two (CHLA15) or three (SKNBE1) biological replicates are depicted in H. Statistical analyses were performed using an unpaired two-sided Student's t-test, with significance p<0.05 (trend, p<0.10).
Figure 2(A) Table showing the proteins binding to Cyclin B1 and Cyclin B2 identified by immunoprecipitation and subsequent mass spectrometry. In brief, Flag-Cyclin B1 and Flag-Cyclin B2 were expressed in 293T cells and immunoprecipitated with anti-Flag M2 beads. Then Cyclin B1/B2-bound beads were incubated with pre-cleared mitotic HeLa cell lysates. The immunoprecipitates were analyzed by mass spectrometry (B) Immunoprecipitation assay performed with two different rabbit Cyclin B2 antibodies. Nocodazole-arrested mitotic cells were collected and lysed. Immunoprecipitation was accomplished using control IgG, anti-Cyclin B2-1 (Abcam, ab185622), and anti-Cyclin B2-2 (Proteintech, 21644-1-AP). Immunoprecipitation samples were resolved by Western blotting using a mouse anti-Cyclin B2 antibody ( Santa Cruz, sc-28303) and a mouse anti-Mad2 antibody (Santa Cruz, sc-47747) (C and D) Flag immunoprecipitation assay. 293T cells were co-transfected with Flag-C-Mad2 and GFP-Cyclin B1/B2 (C), and with Flag-Mad1-1-215 and GFP-Cyclin B1/B2 (D). After 36 h, the cells were collected and lysed, and immunoprecipitation was accomplished with anti-Flag M2 beads. Immunoprecipitates were resolved by SDS-PAGE and analyzed by Western blotting using anti-Flag antibody and anti-GFP antibody (E) GST pulldown assay. Purified GST, GST-Cyclin B1-165-433, and GST-Cyclin B2-130-398-bound agarose beads were used as affinity matrices to absorb purified Trx-6×His-tagged C-Mad2 recombinant protein. Absorbed proteins were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining (F-I) Representative immunofluorescence images of HeLa cells stably expressing GFP-Cyclin B2 (F) or GFP-Cyclin B1 (H). The cells were transfected with siControl or siMad2 for 36 h followed by treatment with nocodazole plus MG132 for 2 h. Then cells were fixed and co-stained for Mad2 (red) and DNA (blue). Scale bar, 10 µm. Scatter graphs illustrating kinetochore intensity of GFP-Cyclin B2 (G) or GFP-Cyclin B1 (I) in cells treated as in F and H, respectively. Bars represent the mean kinetochore intensity (±SD) normalized to values of the siControl group. Each dot represents one cell (≥30 cells from three independent experiments)
a) FBXL2 binds p85α and p85β.figf1a) FBXL2 binds p85α and p85β. HEK293T cells were transfected with either an empty vector (EV) or the indicated FLAG-tagged F-box proteins (FBPs). 24 h post-transfection, cells were treated with MG132 for 3 h before collection for immunoprecipitation (IP) and immunoblotting as indicated. WCL, whole-cell lysate. (b) FBXL2 does not bind p110α and p110β. HEK293T and NIH3T3 cells were transfected with either an empty vector (EV) or the indicated FLAG-tagged proteins. The experiment was performed as described in a. (c) The CaaX motif of FBXL2 is required to bind p85α and p85β. HEK293T cells were transfected with either an empty vector (EV) or the indicated FLAG-tagged F-box proteins. The experiment was performed as described in a.
during the formation of chicken PGCs.Figure 1A. Seven LncRNAs targeted for BMP4 were identified by RNA-seq, and the relative positions of these 7 LncRNAs and BMP4 are shown. B. FPKM of the 7 LncRNAs identified in the RNA-seq with high expression in PGCs. The solid lines at each end of the box plot represent the maximum and minimum values, respectively. The three dotted lines in the middle of the violin diagram represent the 75% percentile, the mean, and the 25% percentile in turn. C. LncRNA classification. According to the positions of the LncRNA and the targeted mRNA, LncRNA-mRNA pairs were divided into 12 biotypes (see methods and Appendix Fig S1B for details), and the 7 LncRNAs were distributed in the XT, SU, and SD biotypes. D. GO analysis showed that the LncRNA-mRNA in the XT biotype was primarily involved in cell development and differentiation, the LncRNA-mRNA in the SU biotype was primarily involved in cell migration and cell development, and the LncRNA-mRNA in the SD biotype was primarily involved in gonadal development and cell migration. E. Correlation analysis between BMP4 and the SD biotype LncRNA showed a significant correlation between TCONS_00874170 (LncBMP4) expression and BMP4 expression F. The existence of LncBMP4 was confirmed by Northern blot, the results showed that LncBMP4 was significantly expressed in PGCs, but weakly expressed in CEF and ESCs.H2O was used as a negative control (n = 3 independent experiments).
Figure 5(A) Ultrastructure of mitochondria and insulin granules in typical β-cells of 3 mouse lines on BKS and Leprdb/db backgrounds. Arrows indicate the mitochondria whereas arrowheads indicate insulin granules. Scale bar = 1 μm. Data information: A total of 15-23 images per group were analyzed. Data from each group are presented as the mean ± SD. (B) Average number (left), average area (middle), and swelling percentage (right) of the β-cell mitochondria from the mouse pancreatic islets (A) in microscopic sections. Data information: A total of 15-23 images per group were analyzed. Data from each group are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant. (C) The average number of insulin granules in β-cells from the mouse pancreatic islets (A) in microscopic sections. Data information: A total of 15-23 images per group were analyzed. Data from each group are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.