EMBO/sd-character-level-ner huggingface dataset:
Figure 5(A) HT1080 (WT) and HT1080PARN KO (KO) cells stably transfected with GFP plasmid (empty) were transfected with siRNAs against p53 for 72h before RNA extraction and qRT-PCR. siRNAs against luciferase (siLuci) were used as control. p53 expression levels were normalized first to ACTB and then to WT/empty/siLuci cells. Experiment was performed in triplicate. Error bars indicate s.e.m. (B) Cells were treated as in (A) and transcript levels of the indicated genes were measured by qRT-PCR. cDNA expression levels were normalized first to ACTB and then to WT/empty/siLuci cells. Experiment was performed in triplicate. Error bars indicate s.e.m. (C) qRT-PCR analyses of the indicated genes were performed on RNA extracted from P1's primary or SV40T-transformed fibroblasts. cDNA expression levels were normalized to primary fibroblasts.
Figure 4(A) Schematic representation of the constructs used to monitor insertion efficiency in the ER membrane. All constructs contain a HA-tagged SQS cytosolic domain fused to a varying TMD sequence, which is C-terminally fused to an opsin tag (sequence indicated). Glycosylation of the C-terminal opsin tag acts as an insertion readout. (B) Sequences of the TMD regions used for cloning and the immediate flanking residues are represented. The regions defined as TMDs were determined using TOPCONS (Tsirigos et al, 2015) and are in bold/underlined. Red residues indicate mutations made in the Xport-A TMD to increase its hydrophobicity. To the right of each sequence is the "transmembrane tendency" score of Zhao & London (C-G) (C-D, F) Western-Blot of WT and EMC6KO (6KO) U2OS cell lines transiently transfected with the opsin-tagged constructs probed for HA. When indicated, denatured samples were treated with PNGase to confirm glycosylation. Glycosylated proteins shift upwards in the gel and are indicated by arrows (). (E, G) Western-Blot of WT and EMC6KO (6KO) U2OS cell lines transiently transfected with the opsin-tagged constructs probed for Actin and EMC6.
Fig.Figure 2A-C WISH of fst in FLT3/WT- (A), FLT3/ITD- plasmid DNA injected zebrafish embryos without (B) or with (C) quizartinib treatment (2.5 μM) from 6 to 36 hpf. fst expression was expanded by FLT3/ITD DNA in 86% of embryos (B, arrow, 32/37) which could be effectively blocked by treating with FLT3 inhibitor quizartinib in 83% of embryos (C, 29/35). Data information: Scale bar = 500 μm. Generation and characterization of FLT3/ITD-transgenic zebrafish. Diagrammatic representation (D and E) of the generation of Runx1-FLT3/ITD transgenic zebrafish Generation and characterization of FLT3/ITD-transgenic zebrafish. GFP expression was detected by florescent microscopy (F-H) and in blood circulation and thymus by WISH (I and J, blue arrow) in WT sibling and Runx1-FLT3/ITD transgenic zebrafish (F1) embryos at 4 dpf. FLT3/ITD-positive zebrafish (F1) were confirmed by PCR genotyping of GFP and FLT3/ITD using genomic DNA from fin clip of WT siblings and Runx1-FLT3/ITD transgenic zebrafish (F1) at two months old. Fish 4, 5 and 6 showed germline transmission of FLT3/ITD transgene (K). Data information: Scale bar = 500 μm. Kidney marrow (KM) was collected from Runx1-FLT3/ITD transgenic zebrafish (F1) at 18 months old. The morphology and hematopoietic composition of KM from WT siblings (n = 6) and Runx1-FLT3/ITD transgenic (n = 6) zebrafish were examined by Giemsa staining (L) and flow cytometry (abbreviation for panel M: M, Myeloid cells; P, Progenitor cells; L, Lymphoid cells; E, Erythroid cells). Data are presented in box plot. **P<0.01 (Student's t-test). Kidney marrow (KM) was collected from Runx1-FLT3/ITD transgenic zebrafish (F1) at 18 months old. The morphology and hematopoietic composition of KM from WT siblings (n = 6) and Runx1-FLT3/ITD transgenic (n = 6) zebrafish were examined by flow cytometry Data are presented in box plot. The whiskers, boxes, and central lines in panel N represented the minimum-to-maximum values, 25th-to-75th percentile, and the 50th percentile (median), respectively. **P<0.01 (Student's t-test). O Expression of fst was detected by RT-qPCR in KM from WT sibling and Runx1-FLT3/ITD transgenic zebrafish at 18 months old. The RT-qPCR experiments were performed in triplicates and data were presented as mean ± SEM. **P<0.01. P Detection of FST expression, p-ERK1/2, and p-CREB in mononuclear cells from normal peripheral blood stem cell (PBSC) and FLT3/ITD AML patients (diagnostic samples with leukemia blasts > 80%) by Western blotting. ^: non-specific staining of p-ATF1 protein due to the conserved motif.
Figure 3A qRT-PCR analysis of MdCAX3 in the roots of MdCAX3-suppressed Mx. B Fluorescence ratio (488/440 nm excitations) of BCECF fluorescence in the roots of MdCAX3-suppressed Mx. C Ratio (488/440) image of BCECF fluorescence in the roots of MdCAX3-suppressed Mx. Scale bars: 100 μm. D Rhizosphere acidification in MdCAX3-suppressed Mx Bromocresol purple was used as a pH indicator for visualization. Scale bars: 1 cm. E qRT-PCR analysis of MdCAX3 in the leaves of MdCAX3-suppressed in leaves Md/Mx. F TaqMAN real-time PCR analysis of MdCAX3G expression in the roots of MdCAX3-suppressed in leaves Md/Mx under Fe deficient and Fe replete conditions for 3 days. G Fluorescence ratio (488/440 nm excitations) of BCECF fluorescence in the roots of MdCAX3-suppressed in leaves Md/Mx. H Ratio (488/440) image of BCECF fluorescence in the roots of MdCAX3-suppressed in leaves Md/Mx. Scale bars: 100 μm. I Rhizosphere acidification of MdCAX3-suppressed in leaves Md/Mx. Bromocresol purple was used as a pH indicator for visualization. Scale bars: 1 cm.
Figure 2The number of sim-expressing nuclei as counted by automated spot detection over time (A) Continuous opto-Notch activation initially induces sim expression across a large number of nuclei. Over time, the majority of nuclei adapt to the input and terminate expression. The number of sim-expressing nuclei as counted by automated spot detection over time (B) Inactivation is not due to developmental timing, as nuclei remain responsive when activation is delayed to a later time point. The number of sim-expressing nuclei as counted by automated spot detection over time for (C) Sustained nuclear localization of NICD is required for adaptation to take place, as pulsatile activation can overcome adaptation. sim expression could not be measured during dark phases of pulsatile photo-activation as its visualization requires 488 λ nm excitation which causes photo-activation; dashed lines are a visual aid connecting data from the same embryo. (D-E) Quantification of nuclear localisation of mCherry::NICD during opto-Notch photo-activation. Raw data was bleaching-corrected with an exponential fit and shown is the nuclear/total ratio, which further normalises bleaching and embryo-to-embryo variability. NICD displays persistent nuclear enrichment during continuous activation (D) and shuttles back and forth between nucleus and cytoplasm under the pulsatile protocol (E).
Figure 2A. Expression profiling of differentially expressed circRNAs between the tumor and normal groups. Rows represent circRNAs, and columns represent samples. Rows were ordered by fold change, and columns were ordered by their group. The sample of N8 was not included due to low sequencing library size. B. Expression profiling of differentially expressed circRNAs between the recurrence and nonrecurrence groups. Both the row and column were unsupervised and clustered with the hierarchical clustering method. C. The 4 of 22 differentially expressed circRNAs were confirmed by qRT-PCR, which were retained after marker selection procedure. **P < 0.01, Student's t-test, mean ± SD. D. Bar plot shows the resample model inclusion proportion (RMIP) of qualified circRNAs calculated in the training dataset. The red line presents the threshold used to obtain the final markers. E. Time-dependent AUC analysis of individual circle RNA and cirScore for predicting recurrence in the training dataset. P-values are shown for the indicated comparison of AUC between each marker and cirScore. Student's t-test, AUC=area under the curve.
The loss of YME1L in the nervous system does not trigger brain atrophy.Figure 3Brain weights were monitored at 31-32 weeks of age (WT, n = 15; NYKO, n = 14). Unpaired t-test, ns = not significant. Data are means ± SEM. B Nissl stainings of sagittal sections across brain regions from 31-32 week old WT and NYKO mice. 4x and 40x enlargements are shown in the left and right panels, respectively. Scale bars 4x = 500 µm, 40x = 50 µm. C Morphometric analysis of different brain regions from 31-32 week old WT (n = 6) and NYKO (n = 6) mice. GCL = Granule cell layer, ML = Molecular layer. Unpaired t-test, ns = not significant. Data are means ± SEM. Immunoblot analysis of tissue lysates (FB = forebrain; CB = cerebellum) from 31-32 week old WT and NYKO mice, using GFAP-specific and, as a loading control, GAPDH-specific antibodies. Unpaired t-test, ns = not significant. Data are means ± SEM.
Figure 3A, B. Effect of tamoxifen on replication in the spleen (A) and intestine (B) of Ssrp1fl/fl; CreERT2+/+ mice. EdU was given to mice three days after the end of tamoxifen treatment (1 hour before tissue collection). Organ sections were stained for total DNA (Hoechst), EdU, and SSRP1. Scale bars - 100 μM. C. Quantitation of EdU staining. Mean number of EdU positive cells per field of view +/- SD. n > 5 fields of view per mouse. 3 mice were analyzed. D. Quantitation of cleaved caspase-3 staining. Mean number of positive cells per fields of view +/- SD, n > 5 fields of view per mouse Two male and female mice were analyzed. Data information: P-values for C and D were calculated using the unpaired t-test.
a) NRK cells stably expressing CFP–LC3;LAMP1–YFP were transfected with nonspecific (NS)- or PIP5K1A-RNAi and starved for 10 h.figf4(a) NRK cells stably expressing CFP-LC3;LAMP1-YFP were transfected with nonspecific (NS)- or PIP5K1A-RNAi and starved for 10 h. The insets represent enlarged regions of interest. Scale bar, 5 μm. (b) Cells from a were assessed for the presence of elongated reformation tubules in a blind fashion after starvation and quantified. n = 100 cells from three independent experiments. Error bars indicate the s.d. (c) Cells from a were assessed for the length of reformation tubules in a blind fashion after starvation and quantified. 50 cells were counted. The average tubule length is indicated above each column. (d) Diagram of reformation tubules. R, radius of main body; L, length. (e) Representative image of reformation tubules in nonspecific- or PIP5K1A-RNAi-transfected cells. The top and bottom panels show two randomly selected images of the respective samples. (f) Cells from a were assessed for the L/R ratio of reformation tubules in a blind fashion after starvation and quantified. 25 cells were counted. The average L/R is indicated above each column. (g) Purified reformation tubules were monitored by TEM. Scale bar, 250 nm. (h) Purified reformation tubules stained with antibodies against PI(4) P, PtdIns(4,5) P2 and PIP5K1A. Scale bar, 250 nm. (i) Fractions from Fig. 2f were analysed by western blotting with an antibody against PIP5K1A. The outline indicates the LAMP2-positive fraction. (j) NRK cells stably expressing CFP-LC3;LAMP1-YFP were starved for 8 h, and the budding of proto-lysosomes was monitored by spinning-disc microscopy. Scale bar, 100 nm. (k) NRK cell lines stably expressing LAMP1-Cherry;clathrin-GFP were starved for 8 h and observed by confocal microscopy. The right panels show enlarged regions of interest. Scale bar, 5 μm. (l) NRK cell lines stably expressing LAMP1-Cherry;clathrin-GFP were transfected with nonspecific- or PIP5K1A-RNAi and starved for 8 h. Scale bar, 5 μm. The middle panels show enlarged regions of interest from the upper panels. The distance between clathrin dots on each reformation tubule in the upper panels is shown in diagrammatic form in the lower panels. Black line: reformation tubules; red dot: clathrin. (m) The distance between two clathrin dots on reformation tubules (RTs) in cells from l was accessed in a blind fashion. Twenty-five cells were counted. The average distance between two clathrin dots is indicated above each column. Uncropped images of blots are shown in Supplementary Fig. S6.
Figure 2(A) HeLa cells were treated with control or indicated siRNA for 72 hours and SL biosynthesis measured by [3H] - sphingosine pulse-chase assay. GSL levels are expressed as fold changes with respect to control. CERT and FAPP2 knockdowns (blue bars) were used as controls. (B, C) Effect of GRASP55 depletion on SL biosynthesis monitored by [3H] - sphingosine pulse-chase assay in GRASP55 KO cells or cells treated with GRASP55 siRNA or following expression of GRASP55-GFP in GRASP55 depleted cells. GSL levels are expressed as fold changes respect to control. (C) The levels of GM and Gb were quantified and represented as GM/Gb ratio. (D) Control and GRASP55KO cells were processed for Cy3-conjugated Shiga Toxin (ShTxB) and Alexa488-conjugated Cholera Toxin (ChTxB) staining followed by flow cytometry analysis. Mean fluorescence intensity was measured and represented. SL levels as assessed by LC/MS in control and GRASP55 KO (#2) cells. F) SL levels as assessed by MALDI-MS (Gb3) in control and GRASP55 KO (#2) cells.
Figure 2A IL-8 secretion in HEK cells stably expressing hTLR4 but not MD-2 (HEK-isoTLR4), transfected with empty vector (EV) or MD-2 and left untreated (nil) or treated with 1 ng/mL LPS, 200 μM nickel chloride, 2 μg/ml HMGB1, 100 μM platinum(II) chloride, 100 μM platinum(IV) chloride or 25 μM cisplatin (n=3 or 4 independent biological replicates). C IL-8 secretion in HeLa cells transfected with non-targeting (siNT) or TLR4-targeting (siTLR4) siRNA and left untreated (nil), or treated with 30 μM cisplatin (n=3 independent biological replicates). Mock cells were not subject to siRNA treatment prior. Data Information: Actual individual data are plotted as box (25th and 75th percentile borders; median central band) with Tukey whiskers (A, C) Statistical analyses were determined in comparison to nil treatments using 2-way (A, C) ns, not significant; *, P<.05; ****, P<.0001 (Dunnett's test). B Fold IL-8 secreted (relative to nil treatment) in MD-2 deficient HeLa cells treated with 10 or 100 ng/mL LPS or 25 μM cisplatin (n=4 independent biological replicates). Data Information: Actual individual data are plotted as box (25th and 75th percentile borders; mean and standard deviation (B). Statistical analyses were determined in comparison to nil treatments using one-way (B) ANOVA. ns, not significant; *, P<.05; ****, P<.0001 (Dunnett's test).
3D architecture of membrane rearrangements induced 16 h after HCV infection.Figure 6(A) Huh7.5 cells were infected with 100 TCID50/cell of Jc1, fixed 16 h later and after HPF-FS processed for ET as described in materials and methods. Left: slice of a dual axis tomogram showing the various membrane alterations. Right: 3D reconstruction of the complete tomogram. Note the high number of DMVs. Panels B, C and D are part of the tomogram displayed in panel A and their position in panel A is highlighted by either a yellow dashed square (B and C) or by a star (D). In the 3D models shown on the right, the ER is depicted in dark brown, the inner membrane of DMVs and DMTs in yellowish brown and their outer membrane in semi-transparent light brown. Single membrane vesicles are colored in pink, intermediate filaments in dark blue and the Golgi apparatus in green. (B) Left: serial single slices through the same tomogram shown in panel (A) displaying a connection between the outer membrane of a DMV and the ER membrane (black arrows). Right: 3D surface model showing the membrane connection. (C) Left: serial single slices through the same tomogram illustrating a lasso-like structure of a DMV that after rendering reveals a pore-like opening that connects the interior of the DMV with the cytosol. The position of this opening in the 2D slice is marked with a black arrow. Right: 3D view of this DMV showing the 'pore'. (D) Left: serial single slices through the same tomogram showing a DMV with a large inter-membrane space between its inner and outer membranes. Right: 3D view of this DMV. Scale bars represent 100 nm. This tomogram is shown in movie S1.
Figure 1(A) Schematic of pulses of p53 expression in response to DNA double strand breaks. p53 dynamics are differentially decoded to induce target genes with diverse downstream functions, including mediating cell cycle arrest and p53 regulation. (B) Schematic of modulation of p53 expression by Nutlin-3, a small molecule inhibitor that prevents p53 degradation by MDM2. Cellular exposure to media with and without Nutlin-3 potentially generates six drug dosing regimens that modulate p53 pulse amplitude, frequency, and duration. (C) Clonal cell lines expressing p53-Venus were engineered to monitor p53 expression dynamics ("input") and two canonically regulated p53 target promoters expressing mCherry ("output") in response to Nutlin-3 treatment. (D) Representative phase contrast and yellow fluorescent (indicating p53-Venus levels) images at the indicated time points for a cell exposed to the "natural dynamics" Nutlin-3 dosing regimen. (E-F) p53-Venus expression in response to the "natural dynamics" Nutlin-3 dosing regimen. Single cell traces (gray; E) and the mean (red; E) are shown for p53-Venus expression in response to each Nutlin-3 regimen. Heat map (F) shows an alternative representation of all traces as shown in (E). N = 51 cells.
PI3K-dependent recruitment of COPII to ERGIC after starvation.Figure 3.(A) Atg5 KO MEFs were either untreated or starved with or without 20 nM wortmannin for 30 min. Cells were harvested and ERGIC membranes were isolated by pooling fractions 3 and 4 (F2 in Figure 2C,D) of the OptiPrep gradient in the three-step fractionation approach followed by immunoblot to examine the amount of indicated markers on ERGIC membranes. (B) Atg5 KO MEFs were transfected with a plasmid encoding an HA-tagged SEC31A. After transfection (24 hr), the cells were treated as shown in (A). Immunofluorescence was performed with anti-ERGIC53 and anti-HA antibodies and the cells were examined by confocal microscopy. Bar: 10 μM (C) Quantification of SEC31A overlapped with ERGIC53 shown in (B) using the Pearson's correlation coefficient (PCC). Error bars represent standard deviations from ∼20 cells in three independent experiments. p values were calculated by T-test. (D) A proposed model. Starvation activates autophagic PI3K to induce COPII recruitment on ERGIC. COPII vesicles generated from ERGIC serve as templates for efficient LC3 lipidation. These membrane precursors may collaborate with other membranes such as the endoplasmic reticulum (ER), plasma membrane (PM), ATG9 compartment and mitochondria, to form a mature autophagosome.
Reconstitution of Atg5–Atg12/Atg16 and Atg8 on GUVs.Figure 4(A) Confocal microscopy images showing eGFP-Atg8 recruitment to GUVs is dependent on the Atg5-Atg12/Atg16 complex and PE. The membrane was labelled by incorporation of rhodamine‐PE. (B) Single confocal section showing colocalization of Atg5-Atg12/Atg16-eGFP and mCherry-Atg8 on GUVs. (C) Quantification of GUV‐associated eGFP-Atg8 in the presence of the Atg5-Atg12/Atg16 complex and the Atg5-Atg12 conjugate. The first graph shows the percentage of GUVs associated with eGFP-Atg8 fluorescence. The second graph shows the intensity of eGFP-Atg8 on the GUVs. The quantification is based on four independent experiments. Inset: anti‐Atg12 western blot using the rabbit anti‐Atg12 antiserum showing the amounts of Atg5-Atg12/Atg16 complex and Atg5-Atg12 conjugate in one of the assays used for the quantification shown in (C). (D) Quantification of membrane recruitment of Atg3-mCherry by the Atg5-Atg12/Atg16 complex, the Atg5-Atg12 conjugate and a buffer control. The graph is based on two independent experiments. (E) Widefield images showing individual frames of a time series of GUVs incubated with Atg3, Atg7, Atg8 and the Atg5-Atg12/Atg16 complex (see Materials and methods for details). The membrane was labelled by incorporation of rhodamine‐PE. The time is indicated in minutes. (F) Widefield images of GUVs incubated with Atg3, Atg7, eGFP-Atg8 and the Atg5-Atg12/Atg16 complex for 35 min (left) or 70 min (right). The membrane was labelled by incorporation of rhodamine‐PE. Note the massive accumulation of eGFP-Atg8 at the interface between individual GUVs. The arrows point to some of these interfaces. For the quantifications, the averages and standard deviations are shown. P‐values were calculated using Student's t‐test. Scale bars: 10 μm. Figure source data can be found with the Supplementary data.
Figure 2DNA content analysis of various G1 arrests. RPE1-hTERT cells were treated for 7 days with doxorubicin (Doxo; 100 ng/ml), palbociclib (Palbo; 5 μM), or nutlin3 (Nutlin; 10 μM). Total number of cells analyzed is indicated by n in each condition. Results were comparable between 2 biological replicates. Schematics of EdU incorporation assay. Drugs were applied to RPE1-hTERT cells 12h after initial cell plating. 6 days later (144h), cells were switched to drug medium containing 5-ethynyl-2'-deoxyuridine (EdU; 10 μM) for 24 hours before fixation and analysis. The percentage of EdU positive cells after doxorubicin (Doxo), palbociclib (Palbo), or nutlin3 (Nutlin) treatment. EdU incorporation was performed as described in (B). At least 100 cells were analyzed per condition per replicate. Individual data points and mean are shown; n=2 biological replicates. Cell proliferation (in the absence of NK cells) after 7 days of doxorubicin (Doxo), palbociclib (Palbo), or nutlin3 (Nutlin) treatment. The drugs were washed out after 7 days and the cells were re-plated for live cell imaging. Cell proliferation was measured as described in Figure 1D. The dashed line indicates the starting cell number (ninitial = 50). Dot plot of individual data points and mean are shown; n=2 biological replicates. NK cell-mediated killing for doxorubicin (Doxo), palbociclib (Palbo), and nutlin3 (Nutlin) treated samples (NK cell: target cell =2.5:1). n=3 biological replicates; mean± SEM. Ctrl vs. Doxo, p< 0.0001; Ctrl vs. Palbo, p< 0.0001; Ctrl vs. Nutlin, p< 0.0001; KS test. The percentage of EdU positive cells after 7 days of torin1 treatment was determined as described in (B) and (C). Individual data points and mean are shown; n=2 biological replicates. NK cell-mediated cytotoxicity towards torin1-treated cells. Torin1 treated (Torin) cells were generated as described in (F) and the NK cell killing assay was performed as described in Figure 1. n=3 biological replicates; mean± SEM. Ctrl vs. Torin, p= 0.79, not significant (n.s.); KS test.