EMBO/sd-character-level-ner huggingface dataset:
Fig.Figure 2A) Number of genes showing significant (adjusted p < 0.05, Wald test) up or downregulation in nuclear or cytoplasmic RNA levels upon transfer of AC16 cells from 37°C to different cold temperatures for 24h. B) Relationship between the log2 fold change in RNA level in the nucleus and that in the cytoplasm for all genes upon transfer of AC16 cells from 37°C to different cold temperatures for 24h. Points are color-coded from low to high density (black < blue < red < yellow). C) Relationship between the log2 fold change in RNA level for all genes upon transfer of AC16 cells from 37°C to 28°C or 18°C (top), 28°C or 8°C (middle) and 18°C or 8°C (bottom) for 24h in both the nucleus and cytoplasm. Points are shaded from low to high density (grey < black). Genes that show significant differential expression at a specific temperature or at both temperatures are highlighted with colored circles or triangles, respectively. D) Venn diagrams showing the overlap between the genes showing significant up or downregulation in nuclear or cytoplasmic RNA levels in AC16 cells exposed to the different cold temperatures.
Classification of MP-SVs in sub-pools at wild-type IHC ribbon synapses(A-E) Representative tomographic virtual sections from resting wild-type.Figure 5Representative tomographic virtual sections from resting wild-type. Five different types of MP-SVs are observed that we classify as sub-pools: non-tethered SVs (A',A''; yellow), single presynaptic density-attached SVs (B',B''; Pd.a, orange, pink arrowhead) or single m-attached SVs (C',C''; Ma., orange, orange arrowhead), or multiple-tethered SVs (D',D''; orange, brown arrowheads) and docked SVs (E',E''; light orange). Ribbon (R), presynaptic density (PD, pink) and AZ-membrane (blue). For clarity, a simplified illustration is provided for each sub-pool. Magnification: 12,000x; scale bar, 40 nm. The graph is showing the percentage of the MP sub-pools shown in A-E of all MP-SVs at inhibitory, resting and stimulatory wild-type synapses.
Full annihilation of PI3Kα activity is required to prevent pancreatic cancer cell migration.Figure 2A, In vitro IC50 of PI3K inhibitors, obtained on recombinant proteins and based on the literature. The Greek letter on the x axis indicates the most potently targeted isoform. B-D B, Murine pancreatic tumour cells R211 were treated for 15min with the vehicle, α, β, β/δ, γ-specific or pan-PI3K inhibitors at 1 or 10 µM in the presence of 10% FBS and the protein levels of pAkt (Ser473 and Thr308), total Akt and β Actin were observed by western blot. P-Akt on C, Ser473 and D, Thr308 were quantified and normalised with β Actin. n = 2 in each group. E, Murine pancreatic tumour cells R211 were treated with the vehicle, α, β, β/δ, γ-specific or pan-PI3K inhibitors at 0.1 or 1 µM and simultaneously subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. Representative image of filter after Crystal violet staining is shown. Scale=200µm. n = 3 in each group. F, Murine pancreatic tumour cells R211 were treated with vehicle, BYL-719 1 µM, AZD4547 2 µM or both treatments and subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. n = 4 in each group. G G, Murine pancreatic tumour cells R211 were treated with the vehicle, α, β, β/δ, γ-specific or pan-PI3K inhibitors and living cells were quantified after 3 days with a MTT colorimetric assay. Metabolically active cells are considered as living cells. n ≥ 3 in each group. H, Murine pancreatic tumour cells R211 were treated with the vehicle, α or pan-PI3K inhibitors and apoptotic (IncuCyte Annexin V) cells were quantified after 2 days. n = 3 in each group. I, Relative p110α, β, γ or δ mRNA expression (compared to scramble siRNA) after inhibition of expression by siRNA targeting p110α or a combination of siRNA targeting each class I PI3K isoform. n ≥ 3 in each group. J, R211 and PDAC8661 cells were treated with siRNA scramble, siRNA targeting p110α or a combination of siRNA targeting each class I PI3K isoform (pools) and subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. n = 3 in each group. K, Relative p110α or β mRNA expression (compared to scramble shRNA stably transduced-cells) in human pancreatic tumour cells PANC-1 stably transduced with shRNA targeting p110α or β. n = 4 in each group. L, PANC-1-transduced cells were subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. n = 5 in each group.
Figure 5A. HeLa cells transfected or not with indicated siRNAs were synchronized in early S phase by double thymidine block, released and pulse-labeled 5,5 h later with 5-azadC for 30 min in the presence or absence of SUMOi. Following 5-azadC removal, cells were subjected to live-cell imaging analysis, and the duration of mitosis (nuclear envelope breakdown (NEBD) to anaphase onset) was quantified (red bars, median; at least 72 cells, pooled from three independent experiments, were analyzed per condition; ****p<0.0001, ns: not significant, Mann-Whitney test). B. Representative time-lapse sequences of mitotic progression in cells in (A). NEBD corresponds to t=0. White arrowheads indicate cells undergoing division. Scale bar, 10 μm. C,D. Quantification of mitotic defects in cells in (A) (mean; n=3 independent experiments; >97 cells quantified per condition; *p<0.05, **p<0.01, paired t-test). E. HeLa cells transfected with RNF4 siRNA were synchronized in early S phase by double thymidine block. Six h after release from the block, cells were pulse-labeled with 5-azadC for 30 min. Mitotic cells were isolated by shake-off 8 h later, washed and incubated or not with MPS1 inhibitor (MPS1i) and collected at the indicated times. Cells were then processed for immunoblotting with indicated antibodies. F. Clonogenic survival of 5-azadC-treated HeLa cells transfected with indicated siRNAs (mean±SEM; n=2 independent experiments). G. Clonogenic survival of 5-azadC-treated WT HAP1 cells and derivative cell lines expressing a truncated form of RNF4 lacking the RING domain (∆RNF4; Fig. EV1H) (mean±SEM; n=2 independent experiments).
Figure 2(A) TIRF image of TMR-labeled DDB molecules (green) bound to either tyrosinated (red) or detyrosinated (blue) MTs. A standard deviation map of the whole time sequence is shown, revealing the preferential association of DDB with tyrosinated MTs versus detyrosinated MTs (pink arrow). Representative kymographs of DDB movement are shown from each type of MT present in the same chamber. Scale bars are 5 μm and 25 sec. See also Movie EV2. (B-C) Quantification of the number of processive (B) or diffusive (C) DDB complexes per μm MT per sec on detyrosinated MTs relative to tyrosinated MTs in the same chamber (n ≥ 20 MTs quantified for each condition from three independent experiments). Error bars represent SD. Data from Δα-CTT MTs (Fig. 1) are re-plotted for comparison. (D) Quantification of the frequency of processive DDB molecules as a function of the amount of tyrosinated tubulin incorporated into the lattice normalized to fully tyrosinated MTs. Data points from two independent experiments are shown. (E) TIRF images of 0.3 nM TMR-labeled p150 (top) or 1 nM TMR-labeled GST-hDyn (bottom) molecules (green) bound to either tyrosinated (red) or detyrosinated (blue) MTs. Right, standard deviation maps of protein binding over an entire time sequence and kymographs from either tyrosinated or detyrosinated MTs are shown. Scale bars are 5 μm and 25 sec. (F) Quantification of mean fluorescence intensity (arbitrary units) per μm MT for TMR-labeled GST-hDyn (2 mM ATP) or p150 bound to the indicated mutant MT relative to WT MTs in the same chamber. The intensity of GST-hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST-hDyn to the MT. (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Error bars represent SD.
Figure 4A K63- and K48-ubiquitination of damaged lysosomes. Untransfected HeLa cells, or cells expressing mCherry-Gal3 (Ch-Gal3) were LLOMe-treated for 1 h. 2 h after washout, cells were stained with antibodies specific for K63 or K48 chains, and LAMP1 or p62 as indicated. Percentage of K63- or K48-ubiquitinated LAMP1 vesicles and percentage of p62-positive K63 or K48 vesicles were quantified from three independent experiments (mean ± SD, Student's unpaired t-test for LAMP1). * = p < 0.05. B Colocalization of K63- and K48-linked ubiquitin conjugates on individual lysosomes. Cells expressing GFP-TAB2-NZF (K63-sensor) were treated as in (A) and stained for K48 chains and LAMP1. Note that the apparent nuclear localization of the sensor is a consequence of the extraction procedure (van Wijk et al., 2012). C Cells expressing GFP-p97-EQ were treated as in (A) and stained with antibodies as indicated.D Quantification of the total number of K63- or K48-ubiquitinated vesicles per cell, and number of p97-EQ positive K63- or K48-ubiquitinated vesicles (mean ± SD).E Percentage of GFP-p97-EQ vesicles positive for K63 or K48 chains was determined (mean ± SD). F ELDR components target K48-conjugates on lysosomes. Detection of GFP-YOD1-CS or UBXD1-GFP alone, or co-transfected with PLAA-HA and GFP-p97-EQ, 2 h after LLOMe washout and co-stained with indicated antibodies. G Scheme of ubiquitination of lysosomes after damage. K48-linked ubiquitination occurs on a subpopulation of lysosomes decorated with K63-linked chains. The ELDR components specifically target K48-linked ubiquitin-conjugates. Scale bars, 10 µm.
The regulation of nutrient starvation‐induced autophagy by BI‐1 depends on IRE1α and TRAF2.Figure 5(A) BI‐1 KO MEFs were stably transduced with lentiviral vectors expressing an shRNA against beclin‐1 (shBeclin‐1) or ire1α (shIRE1α) mRNA or luciferase (shLuc) as control. The levels of LC3, Beclin‐1, IRE1α and Hsp90 were monitored by western blot. (B) BI‐1 KO MEFs were stably transduced with lentiviral vectors as described in A. Cells were exposed to EBSS for 3 h, and then LC3 levels were analysed by western blot. Image was assembled from cropped lanes of the same western blot analysis. (C) Endogenous LC3 distribution was visualized using immunofluorescence and confocal microscopy in BI‐1 KO/shLuc and BI‐1 KO/shBeclin‐1 cells. Quantification represents the visualization of at least 180 cells. Student's t‐test was used to analyse statistical significance. Mean and standard deviation are presented, *P0.001, NS: non‐significant. (D) LC3 was visualized and quantified in BI‐1 KO/shLuc and BI‐1 KO/shIRE1α cells described in (B) by immunofluorescence and confocal microscopy analysis. (E) BI‐1 KO cells were transiently transfected with a TRAF2 dominant‐negative (TRAF2‐DN) construct or empty vector (mock), and after 48 h cells were stimulated with EBSS and the levels of LC3‐II and Hsp90 were monitored by western blot. Right panel: quantification of relative LC3‐II levels normalized with Hsp90 and non‐treated cells. Mean and standard deviation are presented. (F) 293T cells were co‐transfected with expression vectors for HA‐tagged IRE1α (IRE1-HA), TRAF2-FLAG, and MYC‐tagged BI‐1 (BI1-MYC). After 48 h of transfection, HA‐tagged proteins were immunoprecipitated and the possible interaction with TRAF2 was analysed by western blot. Right panel: the percentage of TRAF2 dissociation from IRE1α by the presence or absence of BI‐1 was quantified and normalized with the expression levels observed in the inputs. For comparison, the co‐IP signal observed in the absence of BI‐1 was normalized as 100% co‐IP in each independent experiment (N=3). Mean and standard deviation are presented, *P0.05. Figure source data can be found with the Supplementary Information.
Figure 7A, B, Control and degron ORP2 cells were co-plated, and after 1 day 5 % LPDS, treated with 50 μg/ml LDL and IAA (+IAA; ORP2 depletion) for the indicated times and immunostained for pFAK to quantify its intensity. For rescue experiments, degron ORP2 cells were plated and transfected with GFP-ORP2 or -ORP2-mHHK or -ORP2-∆ELSK for 6 h. Cells expressing GFP-ORP2 constructs at levels similar to endo-GFP-ORP2 were used for quantifying pFAK intensity. Dashed lines indicate cell outlines. See also Fig EV6C. Asterisk indicates cells depleted of endogenous ORP2. Images were acquired by confocal microscopy. Mean ± SD, n = 20-23 cells pooled from 2 independent experiments. Student's t-test. C, Quantification of FAK protein in cytosolic and membrane fractions in degron ORP2 cells with or without IAA. Cells were starved overnight in LPDS and loaded with 50 µg/mL LDL +/- IAA for 2 h. The proportion of FAK signal in the membrane fraction (of total FAK in cytosol+membranes) is presented ± SD. For no IAA n=13 and for IAA n= 11, 2 independent experiments. Student's t-test. D-F, Liposome-co-sedimentation of FAK FERM with increasing concentrations of PI(4,5) P2 and cholesterol. "S" in D indicates the supernatant and "P" pellet containing the FAK FERM-bound to liposomes. The total lipid concentration was kept at 50 μM, and purified FAK FERM concentration at 1 μM. The initial lipid composition was POPC:POPE:POPS:Rhodamine-DHPE:PI(4,5) P2:cholesterol (70:19:10:1:0:0, mol/mol). The increasing PI(4,5) P2 and cholesterol concentrations were compensated by decreasing the amount of POPC. Mean ± SD, n = 5. Student's t-test (*p < 0.05; **p < 0.002). Numbers under the blots indicate fraction of FAK FERM bound to liposomes (pellet) of total FAK FERM (supernatant + pellet). G, H, Binding of Alexa Fluor 488-labeled FAK FERM domain to GUVs containing PI(4,5) P2 and cholesterol. The lipid compositions were POPC:POPE:POPS:Rhodamine-DHPE:PI(4,5) P2:cholesterol (50:19:10:1:20:0) and POPC:POPE:POPS:Rhodamine-DHPE:PI(4,5) P2:cholesterol (20:19:10:1:20:30, mol/mol). Images were acquired by widefield epifluorescence microscopy. Scale bar, 5 μm. Mean ± SD, n = 68-76 pooled from 2 independent experiments. Student's t-test.
Figure 2Immunoblot in lysates and media collected from HEK293DAX cells following 16 h of activation of XBP1s [by 1 g/mL dox] and/or ATF6 [by 10 M TMP], as indicated (Shoulders et al, 2013). Immunoblots of the ATF6 target protein BiP (reactive with the KDEL antibody) and the XBP1s target Sec24D confirm the small molecule activation of these transcription factors.   Representative autoradiogram of [35S]-labeled ERdj3 immunopurified from lysates and media collected from HEK293DAX cells following preactivation of XBP1s [by 1 g/mL dox] and/or ATF6 [by 10 M TMP] (Shoulders et al, 2013). The experimental protocol is shown above. Quantification of relative amounts of [35S]-labeled ERdj3 in media at 8 h collected from cells treated as shown in Figure 2B (n=3). The media [35S]-labeled ERdj3 was measured by densitometry and is normalized to the amount of media [35S]-labeled ERdj3 at 8 h from vehicle-treated cells. Error bars represent standard error from biological replicates (n = 3). * indicates p-value < 0.05; ** indicates p-value < 0.01; *** indicates p-value < 0.001 as compared to the vehicle-treated condition. Quantification of the fraction ERdj3 secreted from autoradiograms as shown in Figure 2B. The fraction of ERdj3 secreted was calculated by normalizing the recovered [35S]-labeled ERdj3 signal in the media at t = 4 or 8 h to the total amount of [35S]-labeled ERdj3 signal in the media and lysates at t = 0 h. Error bars represent standard error from biological replicates (n = 3). * indicates p-value < 0.05; ** indicates p-value < 0.01 as compared to the vehicle-treated condition. Quantification of the fraction ERdj3 remaining from autoradiograms as shown in Figure 2B. The fraction of ERdj3 remaining was calculated by normalizing the sum of the recovered [35S]-labeled ERdj3 signal in the media and in the lysates at t = 4 or 8 h to the total amount of [35S]-labeled ERdj3 signal in the media and lysates at t = 0 h. Error bars represent standard error from biological replicates (n = 3). * indicates p-value < 0.05; ** indicates p-value < 0.01 as compared to the vehicle-treated condition.
Figure 7Electrophoretic mobility-shift assay (EMSA) showing that MYB73/MYB77 binds to the MBSI motif in vitro. Cold probe was added as a competitor. EMSA showing that MYB73/MYB77 binds to the promoter of IAA19. Cold IAA19p or IAA19mp (MBSI motif mutated) was added as a competitor. EMSA results showing that monomeric UVR8 inhibits the DNA-binding activity of MYB73(C) EMSA results showing that monomeric UVR8 inhibits the DNA-binding activity of MYB77(D). ChIP-qPCR analysis showed that UV-B inhibits the DNA-binding activity of MYB73. ChIP-qPCR assays were performed using transgenic seedlings expressing 35S::MYB73-TAP, treated with or without UV-B for 5 h before harvesting samples. Chromatin fragments (~500 bp) were immunoprecipitated by anti-Myc antibody, and the precipitated DNA was analyzed by qPCR using the primer pairs indicated. The IP/Input ratios are shown with SDs (n=3). ChIP-qPCR showing that UV-B inhibits the DNA-binding activity of MYB73 in a UVR8-dependent manner. The IP/Input ratios are shown with SDs (n=3). ChIP-qPCR showing that UV-B inhibits the DNA-binding activity of MYB73 in roots (G) in a UVR8-dependent manner (H). The IP/Input ratios are shown with SDs (n=3). ChIP-qPCR showing that UV-B inhibits MYB77 from binding to the indicated promoters in a UVR8-dependent manner. The IP/Input ratios are shown with SDs (n=3).
Figure 5A-B Differentially expressed genes between shNTC and shH2A.B in L1236 HL cells (A) and L428 HL cells (B). Gene expression fold changes (Log2) are plotted against the gene expression level (Log2 TPM). For both cell lines, RP genes are highlighted in red. The RNA-seq data was analysed employing three biological replicates. C The RNA-seq data were analysed by Exon-Intron Split Analysis. RNA-seq reads were separated into exonic (∆exon) and intronic (∆intron) changes. Exonic changes (∆exon) reflect the combination of changes in transcriptional and post-transcriptional activity changes. Intronic changes (∆intron) reflect changes in transcriptional activity. ∆exon - ∆intron reflects post-transcriptional effects. RP genes in L1236 and L428 HL cells are shown.
Figure 7A) Cells treated with nocodazole are affected in DSB-SPB interaction. Cnm67-CFP and Ddc2-RFP were used as SPB and DSB markers respectively. Diagram depicts the genomic background used. D1: inter-SPB distance; D2: DSB distance to proximal SPB; D3: DSB distance to distal SPB. D1, D2 and D3 distances after inducing a DSB in the presence of nocodazole or mock dimethyl sulfoxide (DMSO) were scored and plotted. Graph represents the mean ± SD of D1, D2 and D3 distances of three independent experiments. At least 100 cells per experiment were scored using the maximum projection of three z-planes. P values were calculated using a 2-tailed unpaired Student t test. A representative picture is shown. Scale bar: 3μm. B) Physical analysis of wild-type cells containing the repair pathway choice assay described in Fig 2B. The diagram with the genomic information, the restriction enzymes used and the location of the probe are shown. Cells were synchronized in G1 by using the α-factor pheromone and released into fresh media for 1 hour. Induction of HO expression was attained by adding galactose for 2 hours. After formation of the DSB, glucose was added to repress the HO in the presence of nocodazole or mock DMSO and samples were taken to analyse the kinetics of the repair. DNA was extracted, digested with EcoRV, separated on agarose gels and blotted. Both probes for the MATa-distal sequence and the HIS3 gene (as loading control) were used. Graphs show the quantification of MATa restoration (NHEJ), mating type switching by gene conversion (HR), and DSB kinetics formation. All data were normalized with the HIS3 gene. Graphs show the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. B) Physical analysis of wild-type cells containing the repair pathway choice assay described in Fig 2B. The diagram with the genomic information, the restriction enzymes used and the location of the probe are shown. Cells were synchronized in G1 by using the α-factor pheromone and released into fresh media for 1 hour. Induction of HO expression was attained by adding galactose for 2 hours. After formation of the DSB, glucose was added to repress the HO in the presence of nocodazole or mock DMSO and samples were taken to analyse the kinetics of the repair. FACS analyses are shown.
a,b) Interaction of Beclin 1 wild-type and monomeric (MutM) mutants with Atg14L or UVRAG in cultured cells.f5(a,b) Interaction of Beclin 1 wild-type and monomeric (MutM) mutants with Atg14L or UVRAG in cultured cells. HEK 293T cells were transfected with GFP-tagged Beclin 1 variant alone (a) or together with FLAG-UVRAG (b). The lysates were immunoprecipitated by GFP antibody, followed by western-blot analysis with anti-Atg14L antibody for detecting binding to endogenous Atg14L (a) or with anti-UVRAG antibody for detecting binding to the FLAG-tagged UVRAG protein (b).
Atg4BC74A-expressing cells (for Atg16L1) or control cells (for PDI, COP II and β-COP) were labelled with specific antibodies against the indicated markers.figf2Atg4BC74A-expressing cells (for Atg16L1) or control cells (for PDI, COP II and β-COP) were labelled with specific antibodies against the indicated markers. The left and right anti-Atg16L1 and anti-PDI images are from two different ER-IM complexes. Arrows, arrowheads and open arrowheads indicate the ER, IMs and VTCs, respectively. Small arrows in the image from the anti-COP II experiment indicate immunogold particles. Scale bars, 200 nm.
Figure 1A Representative siliques from wild-type (WT; Col-0), herk1, anj and herk1 anj plants prior to dehiscence. Siliques were placed on double-sided sticky tape and carpel walls separated from the replum to expose the developing seeds. Scale bar = 5 mm. B Developing seeds per silique in wild-type, herk1, anj and herk1 anj plants. Fully expanded siliques were dissected and photographed under a stereomicroscope. n = 15 (four independent experiments with at least three plants per line and five siliques per plant). Data presented are means ± SEM. *** p<0.001 (Student's t-test). C Percentage of pollen tubes with normal reception at the female gametophyte (black bars; representative image middle centre of figure) and with overgrowth (grey bars; representative image lower centre) as assessed by aniline blue staining. 15 self-pollinated stage 16 flowers from wild-type, herk1, anj and herk1 anj were analysed. Legend scale bars = 50 µm. *** p<0.001 (χ-square tests). D Aniline blue staining of pollen tube reception in reciprocal crosses between wild-type and herk1 anj plants with at least two siliques per cross. Legend as per (C). *** p<0.001 (χ-square tests).