EMBO/sd-character-level-ner huggingface dataset:
mATG9 Is Internalized by Clathrin-Mediated Endocytosis.Figure 1(A′) HeLa cells transfected with pEGFP-RAB11 were fixed in basal conditions and stained for mATG9. Pearson's coefficient: 0.452 ± 0.034 (SEM).(A″) HeLa cells were transfected with ATG9L1-pEGFP, fixed in basal condition, and labeled for RAB11. Pearson's coefficient: 0.413 ± 0.020 (SEM). (B) HeLa cells were incubated for 1 hr at 37°C or 18°C to reach the correct temperature, loaded with transferrin Alexa-555 for 1 hr, fixed, and labeled with anti-ATG9 and anti-TGN46 (marker for Trans Golgi Network). Histogram shows Pearson's coefficient between ATG9 and transferrin (Tf) or TGN46. Error bar, SEM. ∗∗∗p < 0.001. Inserts show merge at higher magnification. (C) HeLa cells were incubated in serum-free medium containing Dynasore 100 μM or DMSO for 2 hr and loaded for 30 min with transferrin Alexa-555. Cells were then fixed and labeled for mATG9 and TGN46. Pearson's coefficient between mATG9 and transferrin or TGN46 was quantified. Error bar, SEM. ∗∗∗p < 0.001. Inserts show the merge at higher magnification. (D) Cells treated or not treated with Dynasore or transfected with Dynamin K44A mutant or AP2 siRNA were fixed and then analyzed by TIRF microscopy. Pictures are at the same magnification and brightness. Cells transfected with Dynamin mutant were identified by abnormal transferrin accumulation at the plasma membrane. (E) HeLa cells were incubated for 1 hr at 4°C with ice-cold NHS-LC-Biotin (Pierce) solution, rinsed, and lysed in RIPA buffer. Biotinylated proteins were precipitated using streptavidin-agarose beads (Pierce) and blotted for mATG9 to assess protein localization at the plasma membrane. (F) HeLa cells treated as in (C) were processed for immunogold labeling on cryosections and stained with anti-mATG9 antibody. Arrows show clathrin-coated structures. Scale bar in a and c, 150 nm; scale bar in b, 100 nm. (G) HeLa cells transfected with GFP-ATG16L1 were fixed, labeled for mATG9, and then analyzed by TIRF microscopy. Control cells were cotransfected with GFP-ATG16L1 and Straw-ATG16L1. Pictures are at the same magnification and brightness. See Figure S1J for quantification. (H) HeLa cells treated with Dynasore were processed for immunogold labeling on cryosections and double labeled with ATG16L1 (10 nm) and mATG9 (15 nm). Scale bar, 100 nm. (I) HeLa cells treated as in (H) were processed for triple labeling on cryosections (clathrin, 5 nm; ATG16L1, 10 nm; and mATG9, 15 nm). Scale bar, 150 nm.(J) Table shows quantification of numbers of clathrin-coated structures carrying mATG9 and ATG16L1 alone or together or neither protein. A minimum of 50 clathrin-coated structures were examined from three different experiments. We constructed a 2 × 2 table with 0 or 1-3 gold particles for mATG9 or ATG16L1 for a X2 test to avoid statistical cells with 0 counts. X2 = 10.4, df = 1, and p = 0.001.
Heterogeneity of the OM of the smooth B.Figure 1Localization of R-LPS (green) and S-LPS (red) in exponential phase bacteria. Scale bars: 2 μm. Distribution of R-LPS and S-LPS in exponential phase culture analyzed by flow cytometry (one representative example among 3 biological replicates, n = 20,000 events). Numbers in each corner corresponds to % of relative frequencies.
Figure 1(A) Analytical scheme of multi-layer systems omics. Multiple association studies among 4 layers between genotype and wing size phenotypes are performed and, based on the information, a prediction model is constructed. (B) Wing size variation in absolute centroid size (CS) and relative CS (adjusted for body size). (C) Reproducibility of RNA-seq experiment. Pairwise Spearman correlation coefficients between RNA levels showed higher correlations among biological replicates than among non-replicates. (D) Comparable variability of RNA and protein levels for genes quantified at both levels. (E) RNAs of the genes quantified at both RNA and protein levels have the largest means and smallest variabilities among the whole RNAs quantified. (F) Weak correlation between RNA and protein levels. The median Spearman correlation coefficient was 0.1 and only 18 % of genes compared showed a positive correlation with statistical significance (p < 0.05).
Figure 3(A) Immunostaining of CDC50A on Cdc50a knockdown neurons and controls. Accell Cdc50a siRNA pool was used to knock down Cdc50a in neurons for 7 days. CDC50A expression alongside neuronal processed was reduced after Cdc50a siRNA treatment. Scale bar, 10 µm. (B) Quantification of relative CDC50A fluorescence intensity in Cdc50a-RNAi and controls. N = 4 for biological replicates, mean ± SD, **** p < 0.0001 by Student's t-test. (C) Live neurons were imaged with or without Cdc50a siRNA treatment under a confocal microscope. Propidium iodide (PI) was used to label dead cells. In controls, live neurons exhibit a few weak pSIVA signals. However, in Cdc50a-RNAi neurons, more and stronger pSIVA signals were observed alongside neuronal processes. Scale bar, 20 µm. (D) IHC of Cdc50a-RNAi neurons exhibited pSIVA colocalized with synapsin I, a general synapse marker. The dotted square was zoomed-in shown on the right side. Arrows indicated where pSIVA colocalized with synapsin I. Scale bar, 5 µm.
Figure 1(A) Growth curves of S. pombe cultures in minimal medium without (EMM) or with antimycin A (EMM + antA). Cultures were supplemented with either the complete amino-acid mix (all AA mix, black), without any amino acids (no AA, red), with arginine only (R, blue), or with individual amino acids other than arginine (individual AAs, grey). (B) Growth curves as in (A) for cultures supplemented with complete amino-acid mix (black), without any amino acids (red), or amino-acid mix without arginine (green). (C) Scatter plot of normalised colony sizes of cells grown on EMM against normalised colony sizes of cells grown on EMM supplemented with arginine (EMM+R) (Dataset file 1). Mutants that passed our filtering (Methods) and whose colony-size ratios were 30% bigger when supplemented with arginine are shown in red; mutants encoding known genes of the arginine biosynthesis pathway are shown with blue circles. (D) Scheme of arginine biosynthesis pathway, with protein localisation based on PomBase annotations (Lock et al, 2019). (E) Box plot of mean colony size ratios of mutants grown with vs without arginine for all mutants (grey) and respiratory deficient mutants (orange, based on previous screens (Malecki & Bähler, 2016) (Welch's t-test pval** < 0.002). The same analysis was performed for mutants grown on EMM (left; 4 biological repeats, Dataset file 1) or on EMM supplemented with antimycin A (right; 2 biological repeats, Dataset file 2). Boxplots were created using the R boxplot command with default settings. The central band indicates the median, the upper and lower limits of the box indicate first and third quartiles, and the whiskers show the most extreme data, limited to 1.5-time interquartile range.
Figure 5Ribosome fractionation of mESCs treated with control or INK128 (200 nM) for two hours in 2iL. Fraction 1, free ribosome subunits and monoribosomes. Fraction 2, polyribosomes. Abs 254 nm, absorbance of light at 254 nm. 10%-50%, sucrose gradient, also hereafter in (E). Relative enrichment changes of translation as in (A). Ratio (fraction 1 to fraction 2, or F1/F2) of relative expression of each mRNA (normalized by Actb) was calculated. mRNAs undergoing translation suppression (more in fraction 1, z-score < 1.5, 3083 mRNAs) or activation (more in fraction 2, z-score > 1.5, 177 mRNAs) were indicated. GO analysis of mRNAs undergoing translation suppression (more in fraction 1). Relative enrichment heatmaps of mitochondrial ribosome subunit and ETC complex component mRNAs as in (A). Ribosome fractionation of mESCs transduced with Rptor shRNA (two targets mixed) in 2iL. RT-qPCR for pluripotency and mitochondrial ribosome subunit genes as in (E) (mean values ± SD). n = 3 biological replicates.
Figure 6Diagrams and averaged fluorescence traces of the thermal stability assays of PABPN1L in the presence and absence of poly(A) variants (n = 6). The RNA substrate comprised 20 non-poly(A) nucleotides followed by a 10/20/30/60-adenosine poly(A) tail. PABPN1L-bound substrates were prepared with 2/1/0.5/0.2/0.1 RNA molecules per PABPN1L. The midpoint of the unfolding curve was determined as the melting temperature (Tm). Diagrams and averaged fluorescence traces of the thermal stability assays of PABPN1L in the presence and absence of poly(A) variants (n = 6). The RNA substrate comprised 20 non-poly(A) nucleotides followed by a 10/20/30/60-adenosine poly(A) tail. PABPN1L-bound substrates were prepared with 2/1/0.5/0.2/0.1 RNA molecules per PABPN1L. The midpoint of the unfolding curve was determined as the melting temperature (Tm). D RNA immunoprecipitation results showing the interaction of PABPN1L with indicated transcripts. Fully grown GV oocytes of WT mice were microinjected with mRNAs encoding PABPN1L (WT, RRM-deleted, or R171A mutant), and were released from meiotic arrest at 12 h after microinjection. Total RNAs were extracted from 350 MI stage oocytes in each sample. RNAs recovered from the immunoprecipitants were subjected to RT-qPCR of the indicated transcripts. Fold change values of both Input and IP samples were normalized by RIP results of the FLAG-PABPN1L microinjection group. Error bars, S.E.M. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed Student's t-test comparing with RNA levels pulled-down by PABPN1L WT in the second column.
Figure 5 A Exponentially growing cell cultures were serially diluted (1:10) before being spotted out onto YEPD plates with or without CPT at the indicated concentrations  (B-E) G1-arrested cell cultures (αf) were released in YEPD supplemented with CPT (50 μM). Sample collected at the indicated time points were subjected to western blot analysis with anti-Rad53 antibodie  (B-E) G1-arrested cell cultures (αf) were released in YEPD supplemented with CPT (50 μM). Sample collected at the indicated time points wer stained with propidium iodide to analyze the kinetics of nuclear division (C, E)  (B-E) G1-arrested cell cultures (αf) were released in YEPD supplemented with CPT (50 μM). Sample collected at the indicated time points were subjected to western blot analysis with anti-Rad53 antibodies (B, D  (B-E) G1-arrested cell cultures (αf) were released in YEPD supplemented with CPT (50 μM). Sample collected at the indicated time points wer stained with propidium iodide to analyze the kinetics of nuclear division (C, E)  F Exponentially growing cell cultures were serially diluted (1:10) before being spotted out onto YEPD plates with or without CPT  G Colony-forming units on YEPD plates with or without CPT at different concentrations. Plates were incubated 3 days at 25°C. Plotted values are the mean values with error bars denoting SD (n = 3)
Figure 1A, Schematic illustration of the expression cassette in TAPa vector. The vector allows translational fusion of TSN or GFP at their C-termini to the TAPa tag. The expression is driven by two copies of the cauliflower mosaic virus 35S promoter (2x35S) and a tobacco mosaic virus (TMV) U1 X translational enhancer. The TAPa tag consists of two copies of the protein A IgG-binding domain (IgG-BD), an eight amino acid sequence corresponding to the 3C protease cleavage site (3C), a 6-histidine stretch (His), and nine repeats of the Myc epitope (myc). A Nos terminator (Nos ter) sequence is located downstream of each expression cassette. B, Immunolocalization of TSN2-TAPa and GFP-TAPa fusion proteins in root cells of 5-d-old seedlings. The seedlings were grown under no stress (NS) conditions (23ºC) or incubated for 40 min at 39ºC (HS) and then immunostained with α-Myc. Scale bars = 10 μm. C, Schematic representation of three classes of TSN-interacting proteins, i.e. stress-dependent, stress-sensitive and stress-independent interactors, identified upon the comparison of TSN2_NS and TSN2_HS protein pools. D, Venn diagram showing the comparison of TSN2_NS interactome with human and yeast SG proteomes Jain et al., 2016(). E, Venn diagram showing the comparison between TSN2_NS and TSN2_HS protein pools. TSN2-interacting proteins are divided into three classes: HS-sensitive, HS-independent and HS-dependent. Within each class, the proteins are further classified into two groups. Group 1 contains known human or yeast SG proteins (Jain et al., 2016), including SG remodelers (marked in grey color), whereas group 2 represents components of recently isolated Arabidopsis RBP47-SG proteome (Kosmacz et al., 2019). The full lists of TSN2-interacting proteins, including previously uncharacterized and potentially novel SG components not belonging to either group 1 or group 2, are provided in Dataset EV1. F, Frequency of RBPs and proteins with prion-like domains or ATPase activity found in TSN2_NS and TSN2_HS protein pools in comparison with yeast and human SG proteomes Jain et al., 2016().
Figure 5(A). Primary mouse chromaffin cells were transduced with a lentivirus containing the gene for wild-type PTP-MEG2 or different mutants with a GFP tag at the C-terminus. Positive transfected cells were confirmed with green fluorescence and selected for electrochemical analysis. Typical amperometric current traces evoked by AngII (100 nM for 10 seconds) in the control (transduced with control vector) (top left panel), WT (top right panel), G334R (bottom left panel), and D335A (bottom right panel) are shown. (B). The schematic diagram shows key residues of PTP-MEG2 defining the substrate specificity adjacent to the pY83 site of NSF. (D). Statistical diagram of the quantal size in Fig. 5A and Appendix Figure S6A-D. The secretion amount of each group was standardized with respect to the control group. Data information: (B Green color represents the amino acids which are found to modulate foot probability based on structure analysis, and red color means amino acids which does not regulate foot probability. , * indicates PTP-MEG2 mutants overexpression group compared with the WT overexpression group. # indicates the overexpression group compared with the control group. **, P<0.01; *** P<0.001 and #, P<0.05; ##, P<0.01; ### P<0.001. N.S. means no significant difference. Data were from 6 independent experiments. All the data were analyzed using one-way ANOVA and showed as the mean ± s.e.m. (C). The distribution of the quantal size of chromaffin cells transduced with lentivirus containing the genes encoding different PTP-MEG2 mutants. (D). Statistical diagram of the quantal size in Fig. 5A and Appendix Figure S6A-D. The secretion amount of each group was standardized with respect to the control group. Data information: Green color represents the amino acids which are found to modulate foot probability based on structure analysis, and red color means amino acids which does not regulate foot probability. , * indicates PTP-MEG2 mutants overexpression group compared with the WT overexpression group. # indicates the overexpression group compared with the control group. **, P<0.01; *** P<0.001 and #, P<0.05; ##, P<0.01; ### P<0.001. N.S. means no significant difference. Data were from 6 independent experiments. All the data were analyzed using one-way ANOVA and showed as the mean ± s.e.m. (E-G). Calculated parameters of secretory dynamics, including the spike amplitude (E), PSF frequency (F), and SAF frequency (G). Data information: Green color represents the amino acids which are found to modulate foot probability based on structure analysis, and red color means amino acids which does not regulate foot probability. , * indicates PTP-MEG2 mutants overexpression group compared with the WT overexpression group. # indicates the overexpression group compared with the control group. **, P<0.01; *** P<0.001 and #, P<0.05; ##, P<0.01; ### P<0.001. N.S. means no significant difference. Data were from 6 independent experiments. All the data were analyzed using one-way ANOVA and showed as the mean ± s.e.m.
Figure 7A, B Three independent C9 patient iPSC-MN lines (Becker S6, M211R2, C90202) were treated with GELD, SPL, FSK, H89 or DMSO vehicle control and levels of poly-GA (A) and poly-GP (B) measured after 7 days. Values are displayed as percentages of mean DMSO signal. No significant changes in either DPR were observed (linear mixed effects model, all not significant, P>0.05; P = 0.1 DMSO vs FSK). Treatments were performed in four technical replicates. C, D Three independent C9 patient iPSC-MN lines were treated with 10 µM H89 and levels of poly-GA (C) and poly-GP (D) were measured after 14 days. Values are displayed as percentages of mean DMSO signal. H89 significantly reduced poly-GA *P = 0.039 and poly-GP levels *P = 0.018 (linear mixed effects model). Treatments were performed in four technical replicates. E No increased LDH release was observed after 14-day 10 µM H89 treatment compared to vehicle treatment in two independent C9 patient iPSC-MN lines (unpaired t-tests, Becker S6: significant decrease in LDH, **P = 0.0064; M211R2: not significant, P>0.05). In Becker S6, mean LDH release was 22% lower after H89 treatment compared to vehicle. Treatments were performed in eight technical replicates. Data information: Error bars reported as ± SD. Not significant = P>0.05, *P<0.05, **P<0.01. F Relative fold changes of C9orf72 variant RNA levels normalised to RPL13A, after 14-day 10 µM H89 treatment vs vehicle treatment (N=1 C9 patient line, four technical replicates). No significant changes were observed between H89 and vehicle for any of the isoforms (2-way ANOVA with Šídák's multiple comparisons test, all not significant, P>0.05). Data information: Error bars reported as ± SD. Not significant = P>0.05, *P<0.05, **P<0.01. T
Figure 4(A) Representative images of IL-33 immunostaining on WT skin. Inset highlights the IL-33-negative nuclei in the epidermis (scale bar: 100 μm). Skin tumor outcomes in WT mice treated with DMBA/TPA carcinogenesis protocol in combination with SB431542 (test, n=9) or acetone (carrier control, n=5). (B) Tumor latency (log-rank test) Skin tumor outcomes in WT mice treated with DMBA/TPA carcinogenesis protocol in combination with SB431542 (test, n=9) or acetone (carrier control, n=5). (C) skin tumor counts per mouse over time Skin tumor outcomes in WT mice treated with DMBA/TPA carcinogenesis protocol in combination with SB431542 (test, n=9) or acetone (carrier control, n=5). (D) skin tumor volume at the completion of the DMBA/TPA carcinogenesis protocol. (E) Representative images of IL-33 immunostaining on K14-IL33tg,ST2KO skin. Inset highlights the IL-33-positive nuclei in the epidermis (scale bar: 100 μm). Skin tumor outcomes in K14-IL33tg,ST2KO mice treated with DMBA/TPA carcinogenesis protocol in combination with SB431542 (test, n=6) or acetone (control, n=5). (F) Tumor latency (log-rank test) Skin tumor outcomes in K14-IL33tg,ST2KO mice treated with DMBA/TPA carcinogenesis protocol in combination with SB431542 (test, n=6) or acetone (control, n=5). (G) skin tumor counts per mouse over time, Skin tumor outcomes in K14-IL33tg,ST2KO mice treated with DMBA/TPA carcinogenesis protocol in combination with SB431542 (test, n=6) or acetone (control, n=5). (H) skin tumor volume at the completion of the DMBA/TPA carcinogenesis protocol. (I) SMAD signaling proteins levels in WT and K14-IL33tg,ST2KO skin treated with acetone (control) at the completion of the DMBA/TPA carcinogenesis protocol. Tissue lysates prepared from the whole back skin of the animals were subjected to immunoblot with p-SMAD2/3, p-SMAD1/5, SMAD6, SMAD2/3 and SMAD1 antibodies. GAPDH is used as the control housekeeping protein (n=5 in each group). (J) Representative images of IL-33 and p-SMAD2/3 immunostaining on the adjacent sections of human squamous cell carcinoma (SCC) and normal skin (scale bar: 100 μm). Data information: Graphs show mean + SD, NS: not significant, unpaired t-test.
Higher VEGF-A levels in the lens cause ERK-pathway hyperactivation and age-dependent progressive nuclear cataracts.Figure 3A. Increased ERK1/2 phosphorylation is observed in lenses from VEGF-Ahyper mice (KI) with increased VEGF-A levels already prior to cataract formation (11-months old mice with no cataracts (0 cataract)), which is maintained in lenses with mature cataracts (lens of a 26-months old VEGF-Ahyper mouse with +3 cataract). Quantification of ERK1/2 phosphorylation in 11-months old lenses (n=3/group). Graph shows mean ± SEM. ***P-value: 0.0005. B.-C. Cataracts with opacification of the lens (B., black arrow) and extruded nuclei (C., white arrow) are observed with progressive age in VEGF-Ahyper mice (here a representative 30-months old lens with a cataract is shown). D.-E. Histology of lens from b.-c. shows extrusion of sclerotic nucleus (white arrows in C. and D.) and massive degenerative changes of the lens cortex with vacuolization (black arrows in D. and E.). Scale bar 500 μm in (D.) and 100 μm in (E.). F.-G. Representative histological images of 21-months old eyes from WT and VEGF-Ahyper mice. The sclerotic nucleus (red arrow in G.) was extruded and the lenscortex and capsule are separated from the nucleus (white arrow in G.). The retina in aged VEGF-Ahyper mice is atrophic and shows degenerative changes (yellow arrow in G.), while the retina (*) and lens (**) appears normal in age-matched WT littermate mice. Scale bars 200 μm. DAPI (blue, nuclei); phalloidin (green).
Quantification of the Ape1, Ams1, Atg19 protein abundances in living cells.Figure 4 Figure 4: Quantification of the Ape1, Ams1, Atg19 protein abundances in living cells. (A) Epifluorescence microscopy images of the prApe1-GFP/Atg19-mCherry/ypt7Δ, Ams1-GFP/Atg19-mCherry/ypt7Δ, Atg19-GFP/Ape1-mCherry/ypt7Δ S. cerevisiae cells that were used to quantify the fluorescence intensity of GFP spots. Only co-localizing GFP and RFP spots were considered. BF represents bright-field images. (B) Bar plot showing the number of molecules of prApe1, Ams1 and Atg19 found in Cvt vesicles in living cells. Data information: data are representative of two independent experiments in which a total of 217 prApe1-GFP, 181 Ams1-GFP and 233 Atg19-GFP spots were analyzed. Error bars indicate standard deviation (SD). ***P < 0.001; *P = 0.0176 (Z test).
Figure 4A, B Recombinant cGAS (10 μM) and the indicated amounts of Cy5-labeled dsDNA were incubated, with or without G3BP1 (2 μM). The DNA-induced cGAS-DNA liquid droplets were analyzed with time-lapse imaging. Data information: Representative images are shown Scale bars, 10 μm C Quantitative analysis of (A). The partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background. n = 3 biological replicates. Data information: Error bars, mean with s.d. , **P < 0.01, two-tailed t-test. D cGAMP production assays. Recombinant cGAS (10 μM) was incubated with the indicated amounts of dsDNA, with or without G3BP1 (10 μM). The produced cGAMP was quantitatively analyzed by LC-MS/MRM. n = 3 technical replicates. Data information: Error bars, mean with s.d. , **P < 0.01, two-tailed t-test. E, Immunofluorescent staining of cGAS in WT and G3BP1-/- U937 cells were transfected with ISD (2 μg ml-1) for 3 h (E). Hoechst (blue), nuclear staining. Data information: Representative images are shown Scale bars 20 μm ISD, interferon stimulatory DNA. , F The percentage of cells with cGAS foci was quantified, n = 3 biological replicates (F). Data information: Error bars, mean with s.d. G qPCR analysis of IFNB mRNA levels in WT and G3BP1-/- U937 cells transfected with dsDNA as indicated. n = 3 technical replicates. Data information: Error bars, mean with s.d. , **P < 0.01, two-tailed t-test.