EMBO/sd-character-level-ner huggingface dataset:
Figure 2A. Schematic representation of TAC-mediated (I) ER movement and ER sliding (II). B. Representative time-lapse spinning disk confocal images of n = 3 of live COS7 cells stably expressing mCherry-KDEL (green), transiently expressing EB3-GFP (yellow) and SiR-lysosome-stained LE/Lys (magenta). Zoom insets show regions of interest containing a mobile LE/Lys as denoted by the white arrow. First row shows ER (mCherry-KDEL) (green), the second row shows SiR-lysosome-stained LE/Lys (magenta), the third row shows EB3-GFP (yellow) and the fourth row shows the merge of the three preceding channels. Scale bar: 30µm and zoom insets 3µm. All images acquired for 90 frames at 1Hz. Images related to movie EV6. C. Representative time-lapse spinning disk confocal images of n = 3 of live COS7 cells stably expressing GFP-TMD (green) marked ER and SiR-lysosome-stained LE/Lys (orange) . Zoom insets highlight regions of interest containing a mobile endosome as denoted by the white arrow. First row represents ER (GFP-TMD) (green), the second row shows MDA generated ER movement (magenta), third row shows a merge of the two preceding channels and the fourth row shows a merge of ER (green) and LE/Lys (orange). Samples were treated with either DMSO or 2µm paclitaxel. Scale bar: 30µm and zoom insets 3µm. All images acquired for 90 frames at 1Hz.Images related to movie EV7 and 8 D. Kymograph of panel (C). E. MDA quantification of panel (C) normalized to control cells, showing the average ER movement surrounding LE/Lys under control or treated conditions and the overall average ER mobility under control or treatment conditions. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n = 20 cells per condition from 3 independent experiments). Significance two-tailed student t-test **** P<0.0001; ns = not significant. F. Representative images of n = 3 of COS7 cells stably expressing GFP-TMD (green) to mark the ER and SiR-lysosome stained LE/Lys (orange) merged. Zoom insets highlight region of interest containing a mobile endosome as denoted by the white arrow. First row represents ER (GFP-TMD) (green), the second row shows MDA generated ER movement (magenta), third row shows a merge of the two preceding channels and the fourth row shows a merge of ER (green) and LE/Lys (orange). Samples were treated with either siControl or siKTN1. Scale bar: 30µm and zoom insets 3µm. All images acquired for 90 frames at 1Hz. Images related to movie EV9 and 10 G. Kymograph of panel (F). H. MDA quantification of panel (F) normalized to control cells, showing the average ER movement surrounding LE/Lys under control or treated conditions and the overall average ER mobility under control or treatment conditions, respectively. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n=20 cells per condition from 3 independent experiments). Significance two-tailed student t-test. **** P<0.0001; ns = not significant
a) UVRAG knockdown impairs the interaction of the RINT-1–ZW10 complex with COPI in an autophagy-independent manner.figf5(a) UVRAG knockdown impairs the interaction of the RINT-1-ZW10 complex with COPI in an autophagy-independent manner. Cells (HEK293T) were transfected with control shRNA, UVRAG shRNA or Atg16L1 shRNA for 72 h. Whole-cell lysates (WCLs) were used for immunoprecipitation with anti-RINT-1 (first and third panels), anti-ZW10 (second panel), or anti-UVRAG (fourth panel), followed by immunoblotting with the indicated antibodies. Input represents 10% whole-cell lysates. Note that β′-COP interaction was suppressed by UVRAG knockdown but unaffected by Atg16L1knockdown. See Supplementary Fig. S9 for uncropped data. ( (b) Retrograde transport of tsO45-VSVG-KDELR-YFP from the Golgi to the ER is inhibited by UVRAG knockdown. HeLa cells serially transfected with UVRAG shRNA or control shRNA, then with VSVG-KDELR-YFP expression vector, were incubated at 32°C (permissive condition) overnight. On shifting to 40°C (non-permissive condition), the ER-like distribution pattern of VSVG-KDELR-YFP was registered. Representative confocal images of the distribution pattern of VSVG-KDELR at 40°C and its co-localization with PDI are shown (upper panel). Data are mean±s.d., n = 100 cells obtained from three independent experiments; **P0.01 (bottom panel). Scale bars, 10 μm. (c) UVRAG interaction with bothPtdIns(3) P and RINT-1 is required for COPI-dependent retrograde transport, as shown by the redistribution of VSVG-KDELR from the Golgi to the ER. HeLa cells were transfected with control shRNA or UVRAG shRNA. The UVRAG-depleted cells were then complemented with empty vector, Flag-UVRAG, UVRAGΔ270−442, UVRAGK78A/R82A or UVRAGΔC2, along with the transfection of VSVG-KDELR-YFP. The ER pattern for the chimaeric KDELR was quantified (left panel). Data are the mean±s.d.; n = 200 cells obtained from three independent experiments; *P0.05; **P0.01. Endogenous and reconstituted UVRAG expression was confirmed by immunoblotting (right panel). (d) PtdIns(3) Pdepletion inhibits Golgi-to-ER retrograde transport of VSVG-KDELR. HeLa cells were transfected with empty vector, wild-type MTMR3, the C413S mutant of MTMR3, or treated with wortmannin or rapamycin, and then the ER pattern of VSVG-KDELR was quantified. UT, untreated. Data are the mean±s.d.; n = 100 cells for each group obtained from three independent experiments; **P0.01. e) Interaction between endogenous UVRAG and the ER tethering complex under wortmannin treatment. WCLs of HEK293T cells treated with wortmannin were used for immunoprecipitation with anti-UVRAG or anti-RINT-1 antibody, followed by immunoblotting with the indicated antibodies. The input panel shows endogenous protein expression. See Supplementary Fig. S9 for uncropped data.
3-MA and chloroquine blocked sodium butyrate induced autophagy in colorectal cancer cells.Fig 4HCT-116 or HT-29 cells were treated with 5mM 3-MA or 5μM chloroquine (CQ) for 30 min, and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of LC3-I and LC3-II expression were quantified by densitometry and normalized to GAPDH (ratio of LC3:GAPDH). The fold change from control is indicated as the mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis. ** p<0.01, compared to the respective control group. (A) HCT-116 cells treated with 3-MA and NaB; HCT-116 or HT-29 cells were treated with 5mM 3-MA or 5μM chloroquine (CQ) for 30 min, and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of LC3-I and LC3-II expression were quantified by densitometry and normalized to GAPDH (ratio of LC3:GAPDH). The fold change from control is indicated as the mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis. ** p<0.01, compared to the respective control group. (B) HT-29 cells treated with 3-MA and NaB HCT-116 or HT-29 cells were treated with 5mM 3-MA or 5μM chloroquine (CQ) for 30 min, and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of null and LC3-II expression were quantified by densitometry and normalized to GAPDH (ratio of LC3:GAPDH). The fold change from control is indicated as the mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis. ** p<0.01, compared to the respective control group. (C) HCT-116 cells treated with CQ and NaB; HCT-116 or HT-29 cells were treated with 5mM 3-MA or 5μM chloroquine (CQ) for 30 min, and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of LC3-I and LC3-II expression were quantified by densitometry and normalized to GAPDH (ratio of LC3:GAPDH). The fold change from control is indicated as the mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis. ** p<0.01, compared to the respective control group.(D) HT-20 cells treated with CQ and NaB.
Figure 3(A) RNA bisulfite sequencing map for mitochondrial tRNA Met-CAU in wild type (top) and noNSUN (bottom) strains. Each row represents one sequence read and each column one cytosine. Data information: In (A), a representative map of the replicates is shown, n = 3 independent biological replicates. Similar effects were observed in all replicates analysed. (B) Percent identity matrix of human NSUN proteins according to the Clustal Omega multiple alignment tool. (C, D) Targeted bisulfite-sequencing heat map showing non-conversion rates of cytosines in mitochondrial tRNA Met-CAU (C) and mitochondrial 18S rRNA (D). Each row represents one genetic strain analysed and each column represents one cytosine. Data information: In (C, D), the average of two experiments is plotted, n = 2 independent biological replicates, 10 clones sequenced per strain, per replicate. Similar effects were observed in all replicates analysed. (E) Treefam phylogenetic tree based on sequence conservation of NSUN3 proteins in different model organisms. Bootstrap values are indicated on branches.
Figure 2(A) Immunoblot of protein lysates from wild-type, PERK-KO and GCN2-KO MEF cells incubated in the presence of DMSO (Control), Tunicamycin (0.1 µg/ml), or starved in EBSS for 16 h. Data are representative of three independent experiments. (B-D) Wild-type (B), GCN2-KO (C), and PERK-KO (D) MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (0.1 µg/ml) as indicated in (A). Cells were fixed, permeabilized and stained with antibodies against TFE3 (green) and CHOP (red). Scale bar, 10 µm. Data are representative of three independent experiments.(E-F) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO (Control), Tunicamycin treatments for 16 h (E), or starvation for 2 h (F) (mean ± SD of three independent experiments, one-way ANOVA analysis versus DMSO treated cells, ns= not significant, ***p < 0.001; n>400 cells per condition). (G) Quantification of phospho-4EBP1 protein levels in wild-type and GCN2-KO MEF cells upon starvation for 2 h (the mean ± SD of the fold increase of the phospho-4EBP1 to total 4EBP1 ratio from three independent experiments, one-way ANOVA analysis versus starvation treated wild-type cells, ***p < 0.001). (H) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO (Control) or Torin-1 (250 nM) treatments for 1 h (mean ± SD of three independent experiments, one-way ANOVA analysis versus DMSO treated wild-type cells, ***p < 0.001; n>400 cells per condition).
Altered expression of respiratory components and ROS scavengers by atg mutant cells.Figure 4(A) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) medium for the indicated time. Lysates were prepared using the alkaline-trichloroacetic acid method and subjected to immunoprecipitation with anti-Cox2, anti-Cox4, anti-Tim17 and anti-Pgk1. Pgk1 was used as loading control. (B) WT and atg1Δ cells expressing Cta1-3×FLAG or Ctt1-3×FLAG were nitrogen-starved as in (A) for the indicated time. Cell lysates were prepared as (A) and subjected to immune precipitation with anti-FLAG and anti-Pgk1. Pgk1 was used as loading control.
Retinoic acid signaling is involved in HSC activation.Figure 1Phenotypic changes of HSCs by citral. Phase-contrast images (left), oil red O-stained images (middle), and autofluorescence images (right) are shown for HSCs after passage 1 with or without the treatment of citral (100 μM) for 24 h. Scale bar = 10 μm. Western blot analysis of cell lysates from the citral-treated HSCs in (A). α-tubulin was used as a loading control. The quantitative densitometric data are expressed as the percentage of untreated control and represent the means (SD) for three independent experiments. **P-value = 0.002, paired t-test (compared to control). Protein expression of ALDH1A1 and ALDH1A2 during the culture activation of HSCs. Cell lysates were prepared from HSCs at 3 days after plating (HSCs-d3) and from HSCs after passage 1 (P1), 2 (P2), and 3 (P3), and analyzed by Western blotting. Real-time PCR analysis for the expression levels of ALDH1A1 and ALDH1A2 during the culture activation of HSCs. The PCR data are expressed as the percentage of HSCs-d3. **P-value, paired t-test (n = 3) (compared to HSCs-d3), ALDH1A1/HSCs-P3: 0.003, ALDH1A2/HSCs-P1: 0.0018, ALDH1A2/HSCs-P3: 0.001. Effect of ALDH1A suppression on HSC activation. HSCs-P1 were transfected with non-targeting siRNA or siRNA specific to ALDH1A1 and/or ALDH1A2, and after 48 h, whole-cell lysates were subjected to Western blotting. **P = 0.0023, paired t-test (n = 3) (siRNA-ALDH1A1/2 compared to control). Reverse-phase HPLC analysis of whole-cell lysates from the siRNA-ALDH1A-treated HSCs in (E). Typical chromatograms of all-trans retinoic acid and 13-cis retinoic acid. The retention time is 14 min for 13-cis-RA and 23 min for all-trans RA. Phenotypic changes of HSCs by RAR antagonist. HSCs-d3 were incubated with AGN193109 (1 μM) ± all-trans RA (10 μM) for 3 days and subjected to Oil red O staining. Scale bar = 10 μm. Western blot analysis of cell lysates from AGN193109 ± RA-treated HSCs in (G). **P = 0.003, paired t-test (n = 3) (AGN193109 compared to control).
Figure 1(A) rBCV biogenesis in BMMs infected with either wild type (2308), VirB-deficient (ΔvirB11), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria, measured as the percentage of LAMP1-positive BCVs over time. Data are means ± SD of n=3 independent experiments. Asterisks indicate statistically significant differences (P<0.05, two-way ANOVA followed by Dunnett's multiple comparisons test) compared to control (2308). (B) Brucella replication in BMMs infected with either wild type (2308), VirB-deficient (ΔvirB11), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria, measured as number of bacteria per cell at 24 h pi. Data are means ± SD of n=3 independent experiments. Grey dots represent values from individual cells analyzed (n>300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, one-way ANOVA followed by Dunnett's multiple comparisons test) compared to control (2308). (C) Brucella replication in BMMs expressing either GFP or GFP-BspF and infected with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria, measured as number of bacteria per cell at 24 h pi. Data are means ± SD of n=3 independent experiments. Grey dots represent values from individual cells analyzed (n>300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, two-way ANOVA followed by Dunnett's multiple comparisons test) compared to controls.
Figure 2A. ShRNA targeting Nrg1 type III were validated in rat purified neurons. Western blot analysis for Nrg1 shows reduction of its expression (pro-protein at 150 KDa) using two different harpins, representative of two independent experiments. Clx is calnexin. B. ShRNA#1 and 2 downregulate Nrg1 type III expression also in myelin-forming Schwann cell/DRG neuron co-cultures analyzed after 7 days of ascorbic acid treatment, as shown by western blot analysis, representative of two independent experiments. Clx is calnexin. C, D. Titration of shRNA #1 (C) and of shRNA #2 (D) LVs (lentiviral vectors) on WT Schwann cell/DRG neuron co-cultures analyzed after 7 days of ascorbic acid treatment, with quantification of the mean number of Mbp positive segments and Schwann cell number. In (C) n= 20 (SCR, Scramble), n= 11 (LV #sh1, 12.5%), n= 16 (LV #sh1, 25%), n= 9 (LV #sh1, 50%) DRG/coverslips from two independent experiments. Results are mean ± SEM, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test. In (D) n= 14 (SCR), n= 10 (LV #sh2, 12.5%), n= 12 (LV #sh2, 25%), n= 79 (LV #sh2, 50%) DRG/ coverslips, representative of two independent experiments. Results are mean ± SEM, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test. E. Representative confocal images of Mtmr2-/- co-cultures transduced using LV expressing Nrg1 type III shRNA #1, with quantification of the percentage of myelin outfoldings, n= number of DRG/coverslips, n= 9, 382 fibers (SCR), n=9, 368 fibers (LV #sh1, 12.5%), n= 8, 327 fibers (LV #sh1, 25%). Asterisks indicate fibers with myelin outfoldings. Results are mean ± SEM, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test, representative of two independent experiments. E. Western blot analysis of Nrg1 and Akt phosphorylation (S473) shows that activation of Akt decreases following Nrg1 type III downregulation (p-Akt/Akt/Actin, ratio between Mtmr2-/- Scramble-treated and Mtmr2-/- sh#1-treated using 12.5% LV is 1 : 0.85). Each lane is a pool of at least 10 different DRG/coverslips, representative of two independent experiments. Bar in (C, D) is 100 μm; in (E) is 20 μm.
Figure 5Residue-scale validation: Receiver operating characteristic (ROC) curves for CS predictors derived by leave-1-out validation. The dashed black line at y=x corresponds to performance expected by chance. Top left = final predictor trained by stacking structure- and sequence-based models. Top middle = predictor trained only on structure-based features. Top right = predictor trained only on sequence-based features. Bottom left = theoretical maximum predictive power for a probability estimator (AUC = 0.98). Bottom middle = same algorithm as used for final predictor but with all features shuffled beforehand. Bottom right = CSPD model developed using metal-catalyzed oxidation (MCO) site data from E. coli. See also Fig EV3 and EV4. Protein-scale validation: Comparison between predicted CS enrichment from leave-1-out validation to CS enrichment computed from all carbonylated peptides measured for E. coli (left) and D. radiodurans (right). Each point represents a different protein species. Predicted probability-weighted CS enrichment = (sum of carbonylation probabilities across training set sites)/(number of residues in corresponding peptides from experiments). Experimentally measured probability-weighted CS enrichment = (sum of empirical oxidation probabilities across training set sites)/(number of residues in corresponding peptides from experiments). The solid line is the fitted regression line, and dashed lines indicate the boundaries of the 95% confidence interval.
Figure 1A. The expression of EP subtypes in bone marrow myeloid cells and colon tumor myeloid cells. The short black lines indicate individual expressions, the blue and yellow lines indicate the average expression level in each condition. The grey dotted lines represent the overall average between Ptger1 and Ptger3 or Ptger2 and Ptger4. B. The expression of EP subtypes in CD11b+ cells isolated from tumor tissues and spleen of CT26 tumor-bearing mice by magnetic bead separation (n=3). C. Schematic diagram of the granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin (IL)-4-induced mouse monocyte DCs/macrophages differentiation assay. Cells were treated with specific antagonists of distinct EP receptors: ONO-8711, EP1; PF-04418948, EP2; L-798106, EP3; and E7046, EP4. D. Representative flow cytometry plots revealing the effects of prostaglandin E2 (PGE2) and EP1-4 antagonists on mouse bone marrow monocyte dendritic cell (DC)/macrophage differentiation (n = 3). E. The frequencies of F4/80+CD11C− macrophages and F4/80−CD11C+ DCs under varying treatments as analyzed by flow cytometry analysis (n = 3). F. Schematic representation of the GM-CSF/IL-6-induced mouse bone marrow monocyte myeloid-derived suppressor cell (MDSC) differentiation assay (n = 3). G. Representative flow cytometry plots revealing the effects of PGE2 and EP1-4 antagonists on mouse bone marrow derived-MDSC differentiation. Upper subpanel: vehicle group and PGE2 group. Lower subpanel: ONO-8711 (EP1 antagonist) group, PF-04418948 (EP2 antagonist) group, L-798106 (EP3 antagonist) group, and E7046 (EP4 antagonist) group. (n = 3). H. The frequencies of Ly6C+Ly6G− mMDSC and Ly6CmidLy6G+ PMN-MDSCs under varied treatments as analyzed by flow cytometry analysis (n = 3).
Figure 3(A-B) Representative dSTORM images of GlyRs and GABAARs at synapses in side view (A) or en face view (B). The top row of images shows the pointillist representation of the detections, the second row the rendered images and the bottom row the corresponding segmented SSDs. GlyRs were labelled with Alexa 647 and GABAARs with Cy3B in these experiments. Scale bar: 200 nm. (C) Image correlation analysis (ICA) of GlyR and GABAAR clusters at the same PSDs. The ICQ values between GlyR and GABAAR (blue trace, 0.36 ± 0.06, n = 395 synapses from three independent experiments) are significantly lower than between GlyR clusters labelled with two secondary antibodies conjugated with different fluorophores (yellow trace, 0.38 ± 0.04, n = 218 synapses; MW test, p < 0.0001). (D) The mean diameter of GlyR SSDs is 65 ± 29 nm (mean ± SD, n = 218 synapses from two independent experiments), that of GABAAR SSDs is 69 ± 30 nm (mean ± SD, n = 395 synapses from three independent experiments). (E) Partial overlap between the SSDs of GlyRs and GABAARs. The fraction of overlapping SSDs was calculated based on the binarized images for each synapse (without selection for synapse orientation), dividing the number of overlapping SSDs by the total number of SSDs for each receptor. Data are shown as median, 25% and 75% quartiles of the population (only synaptic clusters with ≥ 2 SSDs in the respective channel; n = 183 for GABAAR-A647, n = 277 for GlyR-Cy3B, from three independent experiments). GlyRs labelled with both Alexa 647- and Cy3B-conjugated secondary antibodies for the same primary antibody were used as positive control (n = 158 GlyR-A647 and n = 171 GlyR-Cy3B clusters from two independent experiments). The GABAAR-GlyR overlap is significantly lower than the GlyR-GlyR match (KW test, p < 0.0001). (F) Distance measurement between GlyR SSDs and GABAAR SSDs (blue bar; mean ± SD, n = 395 synapses from three independent experiments) and between double labelled GlyR SSDs (yellow, mean ± SD, n = 218 synapses from two independent experiments).
Figure 2Total carbonyl-bearing proteins detected by shotgun redox proteomic measurement in three biological replicates each of E. coli and D. radiodurans with and without irradiation. The left axis is the number of sequence-unique proteins detected as carbonylated. The right axis is the number of sites in total detected as carbonylated (red) or not oxidized (black) in peptides bearing at least one carbonyl. Stripes indicate carbonylated proteins and carbonylatable sites detected only in irradiated samples. Volcano plots for relative protein abundance changes measured by mass spectrometry in E. coli (left) and D. radiodurans (right) after irradiation using the same biological replicates as in Fig 2A. Black-circled points are those proteins with significant changes (paired, 2-sided t-test p-value < 0.05) of >2-fold or <0.5-fold. Red points are proteins with at least one carbonylated peptide detected. Fold change and p-value cutoffs considered for significance are indicated by dashed lines.
TAK1 mediates TRAIL‐induced AMPK activation and autophagy.Figure 4(A) The lysates of MCF10A cells treated with 500 ng/ml TRAIL for the indicated times were analysed by immunoblotting. The control lane (0 min TRAIL) originates from the same blot and has been treated and exposed identically. Similar results were obtained in three independent experiments. (B, C) The lysates of MCF10A-eGFP-LC3 cells transfected with indicated siRNAs for 48 h and left untreated (−) or treated with 500 ng/ml TRAIL for 2 h were analysed by immunoblotting. (C, lower panel) MCF10A-eGFP-LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs by RT-PCR. Similar results were obtained in two independent experiments. (D, E) MCF10A-eGFP-LC3 cells were transfected with indicated siRNAs for 48 h, treated as in Figure 3C and analysed for LC3 translocation (left) and sub‐G1 DNA content (right). The values represent mean±s.d. of a minimum of three independent experiments except for the right panel in E (two experiments). *P‐value 0.05 and **P‐value 0.01 as compared with cells treated in a same way, but transfected without siRNA.