EMBO/sd-character-level-ner huggingface dataset:
Figure 5(A) Gene ontology analysis for up-regulated genes based on RNA-seq data in E15 hFOXM1-cKI mice. (B) Gene ontology analysis for down-regulated genes based on RNA-seq data in E15 hFOXM1-cKI mice. (C) Volcano plots shows the gene expression states. The yellow dots are the up-regulated genes, and the blue dots are the down-regulated genes. The Lin28a gene (red dot) is significantly up-regulated. (D) Western blot analysis for Lin28a in cortex of E15 WT and hFOXM1-cKI mice. β-actin was detected as loading control. (E) CHIP analysis for hFOXM1,mFoxm1 and hFOXM1▲exon9 binding to the region of the Lin28a promoter in the N2A cells. (n=3 biological repeats). (F) Staining for Sox2 in E15.5 mice after IUE at E13.5. Arrowheads indicate Sox2+ cell in enlarged SVZ. Scale bar, 50μm. (G) Graph shows the percentage of Sox2+ cells among GFP+ cells in enlarged SVZ (n = 3 each group; two-tailed unpaired t-test, P=0.0154). (H) Graph shows the percentage of Sox2+ cells among GFP+ cells (n = 3 each group; two-tailed unpaired t-test, P=0.0018). (I) LIN28A-shRNA1 rescues the neuronal progenitor cell increase in hFOXM1-cKI mice. Brain slice staining for Sox2 two days after IUE at E13. Scale bar, 50μm. (J) Graph shows the percentage of Sox2+ cells among GFP+ cells (n=3 each group; one-way A
Fig.Figure 5PLDα6-GFP distribution in rice protoplasts with different concentrations of GA3 or IAA. Protoplasts from 12-day old rice (ZH11 background) leaf sheath tissue were collected. Rice protoplasts were transfected with pM999-PLDα6 for 12 hours. After that, GA3 or IAA was added to protoplasts and 2 hours later, confocal images of protoplasts are shown. pM999-GFP refers to the empty vector with GFP only that was transformed as control and Ghd7-RFP was a nucleus marker. Scale bar = 10 μm. Immunoblotting of PLDα6 in subcellular fractions. Total (T), soluble (S) and nuclear (N) proteins were isolated from PLDα6:GFP expressed in rice protoplasts treated with 10 μM GA3 or IAA. Equal amount of each sample was loaded for SDS-PAGE and blotting.
Figure 1Viability of diploid (SC5314) and tetraploid (RBY18) cells grown on PRE-SPO medium at 37°C (n=5, error bars represent S.E.M.). A tetraploid β-tubulin (TUB2)-GFP, histone H2B (HTB)-RFP strain was cultured on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 24 h. Cell images indicate maximum intensity Z projections for GFP and RFP channels, and merged images of the GFP/RFP and GFP/RFP/DIC channels. Assay to monitor for loss of GAL1 in tetraploid strain RBY18. Strain RBY18 is heterozygous for the GAL1 gene and only cells that have lost GAL1 function can grow on 2-DOG medium. Diploid strain RBY1 and tetraploid strain RBY18 were cultured on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 7 days and plated onto 2-DOG medium to monitor for GAL1 loss. 2-DOGR progeny arising from RBY18 on PRE-SPO medium were DNA stained and their ploidy determined via flow cytometry.
Figure 6Mice bearing WEHI 164 sarcomas were treated by systemic (intraperitoneal, i.p.) injections of DACT or PBS as a control (Ctr), and tumor were excised 9 days later for the quantitation of mRNA coding for IFNγ Mice bearing WEHI 164 sarcomas were treated by systemic (intraperitoneal, i.p.) injections of DACT or PBS as a control (Ctr), and tumor were excised 9 days later for the quantitation of mRNA coding for IFNγ Two independent experiments were conducted with a total of 22 mice in the Ctr group and 23 mice in the DACT-treated group, with each data point indicating one tumor. The cycle threshold of the qRT-PCR of IFNγ was normalized to the one of the housekeeping gene peptidylpropyl isomerase A (Ppia) in each mouse and results are shown normalized with respect to controls as a dot plot depicting mean ± SD . The p-value (* p<0.05) was calculated by means of a Student's t test (B). Balb/c mice with palpable WEHI 164 sarcomas (n=7 mice in Ctr and anti-IFNγ groups; n=8 mice in DACT and DACT+anti-IFNγ groups) received two injections of DACT-based chemotherapy (0.5 mg/kg) as well as multiple injections (3 times per week) of neutralizing IFNγ-specific antibody (C). Tumor growth curves are shown for individual mice (D) and as means (E). Statistical difference between DACT-treated tumors and respective controls, which was calculated with a type II ANOVA (* p<0.05), is lost upon IFNγ neutralization (E). Overall survival of mice is also indicated with statistics to respective controls calculated with a Log-Rank test (* p<0.05) (F).
and Ras are required for bronchial tight junction formation.Figure 116HBE cells were stably infected with pSUPER, pSUPER expressing shCdc42, shARHGEF18, shSOS1 hairpins 1-3, All data are representative of 3 independent experiments. (a) Cells were seeded sparsely on glass coverslips, incubated for 3 days, then fixed and stained for ZO-1 (tight junctions) and DNA (nuclei). Scale bar represents 20m. 16HBE cells were stably infected with pSUPER, pSUPER expressing shCdc42, shARHGEF18, shSOS1 hairpins 1-3. All data are representative of 3 independent experiments. (b) Cell lysates were analysed by western blotting for SOS1 and actin; labels as for c. 16HBE cells were stably infected with pSUPER, pSUPER expressing shCdc42, shARHGEF18, shSOS1 hairpins 1-3. All data are representative of 3 independent experiments. (c) Quantification of tight junction phenotype. >500 cells were counted per sample/experiment, across n=3 independent experiments (dots indicate individual data points). Error bars denote mean ± SEM. *, p = 0.0199; **, p = 0.0041; **** p = 0.0001. 16HBE cells were stably infected pQCXIP, or pQCXIP expressing dominant negative myc-HRas N17. All data are representative of 3 independent experiments. (d) Cell lysates were analysed by western blotting for Ras, pERK and total ERK. 16HBE cells were stably infected with pQCXIP, or pQCXIP expressing dominant negative myc-HRas N17. All data are representative of 3 independent experiments. (e) Cells were seeded sparsely on glass coverslips, incubated for 3 days and then fixed and stained for ZO-1 (tight junctions) and DNA. Scale bar, 20μm. (f) Quantification of tight junction phenotype (see c). ***, p = 0.0002.
Figure 7(A) Western blot analyses of ZEB1 and P-ERK in shRNA-control or shRNA-ZEB1-expressing A375 cells +/- 150 nM PLX4032 treatment for 24 h. GAPDH was used as a loading control. (B) Soft agar colony formation assay in A375 cells in the presence or absence of PLX4032 (150 nM). Scale bar = 100 µm. Histograms represent quantitative analyses (mean ± SD, n=3, Student's t-test). (C) Number of weeks of chronic exposure to PLX4032 before emergence of resistance in control or shRNA-ZEB1-expressing cells (n=3, Student's t-test). (D) RPMI7951, A375-R and SKMEL5-R were infected with a retrovirus encoding a constitutive shRNA-ZEB1. Short-term cultures of GOKA and ESP cells, derived from vemurafenib-resistant patients, were infected with a lentivirus encoding an IPTG-inducible shRNA-ZEB1. Western blot analyses showing efficient ZEB1 knock-down in the different models, ±IPTG (200µM), ±PLX4032 (3µM) as indicated. Induction of cell death was assessed by PARP cleavage. GAPDH or actin were used as loading control. (E) Clonogenic assays in the presence of 3 µM PLX4032, and with or without IPTG (200 µM) as indicated. The graphs represent the mean number of colonies in 3 independent experiments (Student's t-test). (F) Western blot analyses of ZEB1, p75 and quantitative PCR analyses of MITF in shRNA-Control or shRNA-ZEB1ESPvemurafenib-resistant patient-derived short-term culture cells. Actin was used as a loading control. mRNA expression levels are represented relatively to control cells (mean ± SD, n=3, Student's t-test). (G) 2,5 x 106 shRNA-Control or shRNA-ZEB1ESPvemurafenib-resistant cells were injected subcutaneously in nude mice. When the tumor reached 5 mm in diameter, ZEB1 expression was silenced by providing mice with IPTG (10 mM) in their drinking water and orally administering vemurafenib (50 mg/kg) daily for 4 weeks. The mean tumor volume for 5 mice is represented (± SEM). (Student's t-test). (H) Upper part: Western blot analyses of ZEB1 in shRNA-Control (1 to 4) or shRNA-ZEB1 (5 to 8) ESPxenografttumors, showing efficient ZEB1 knock-down directly in the tumors, after IPTG ±PLX4032 treatment in vivo. Lower part: Representative pictures of ZEB1 immunostaining in shRNA-Control or shRNA-ZEB1tumors, after IPTG treatment in vivo. Scale bar = 40µm.
Fig.Figure 2A: Control or ELDR plasmid DNA stably transfected NOKs were analyzed for cell proliferation using Trypan blue exclusion at the indicated time points. Live cell numbers are presented. n = 4 biological replicates. Data are represented as the mean ± SD, B: Control or ELDR plasmid DNA overexpressed NOK (3×102) were seeded and allowed to form colonies. After two weeks, colonies were stained with crystal violet and counted. Representative images of colonies in control and ELDR overexpressed cells are presented. The right panel shows quantitation. n = 3 biological replicates. Data are represented as the mean ± SD, C: Cal27, JHU029 and JHU022 cells were transfected with control or ELDR siRNA (50nM), and cell proliferation was analyzed at the indicated time points. n = 3 biological replicates. Data are represented as the mean ± SD, D: Cell lysates from control or ELDR overexpressing NOKs, control or Cal27 and JHU029 cells treated with two different ELDR siRNAs were subjected to Western blot analysis for PCNA expression using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control for normalization. The bottom panel shows a quantitative representation of Western blot band intensities. n = 3 biological replicates. Data are represented as the mean ± SD, E: Cal27 and JHU029 cells were transfected with siELDR, ELDR plasmid DNA or both siELDR and ELDR plasmid DNA. Cell lysates were analyzed by Western blot for PCNA expression using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. The right panel shows quantitative representation of Western blot band intensities. n = 2 biological replicates. Data are represented as the mean ± SD,
Figure 2A) Principal component analysis of the MoMus RNA-seq counts. B) Volcano plot for the F test on differential expression between WT and mdx mice. C) Volcano plot for the F test on differential expression between WT and mdx++ mice. D) Volcano plot for the F test on differential expression between WT and mdx+- mice. E, F) Boxplot comparing the distribution of the Dmd and Prune2 genes in the WT, mdx, mdx++ and mdx+- mice groups. The central band of the boxplot denotes the median; the box denotes the first and third quartile; the whiskers are computed as min(max(x), Q3 + 1.5 IQR) and max(min(x), Q1 - 1.5 * IQR), where min(x) and max(x) denote the minimum and maximum of the distribution, Q1 and Q3 the first and third quartile, and IQR the interquartile range. G) Volcano plot for the F tests on differential expression between mdx++ and mdx+- mice. Scatter plots comparing the estimated logFCs across mice groups. Panel H: mdx vs mdx++. Scatter plots comparing the estimated logFCs across mice groups. ++. Panel I: mdx vs mdx+-. Panel J: mdx++ vs mdx+-. ) Heatmap obtained by Ingenuity Pathway Analysis showing the most affected pathways across mdx, mdx++ and mdx+- mice.
VLCFA-GM3 species synergistically and selectively control human TLR4/MD-2 activation.Figure 5(A) Co-stimulation of human monocytes by GM3 species plus various TLR ligands: LPS (0.13 ng/mL), TLR4/MD-2, Pam3CSK4 (0.5 µg/mL), TLR1/2, Flagellin (50 ng/mL), TLR5, R848 (0.5 µg/mL), TLR7/8, MALP-2 (1.0 ng/mL), TLR2/6. IL-6 production in culture supernatant was quantified by ELISA (shown in a heat map). (B) Co-stimulation of monocytes by GM3 species (16:0, 24:0, 24:1) plus synthetic TLR4 ligands LA506 (15 ng/mL), LA505 (150 ng/mL), or LA504 (150 ng/mL). (C, D) Production of IL-6 (C) and TNF-α (D) in culture supernatant following co-stimulation of monocytes by GM3 species plus human HMGB1. (E) Overexpression of hTLR4, hTLR4/hMD-2, hTLR4 (P714H) /MD-2 in HEK293T cells, and co-stimulation by GM3 22:0 with LPS (5 ng/mL). TLR4 activation was monitored by NF-κB luciferase reporter assay (termed "Relative Luc Activity" on Y-axis). (F, Co-stimulation of hTLR4/hMD-2 by GM3 species plus LPS (5 ng/mL) G) Co-stimulation of hTLR4/hMD-2 by GM3 species plus LPS (5 ng/mL) and further addition of soluble human CD14 (1 µg/mL) (H) Stimulation of Mal-overexpressing HEK293T cells by GM3 species. (I) Co-stimulation of hTLR4/hMD-2 by LPS (5 ng/mL) plus various mixtures of GM3 species. (J) Regulation of hTLR4/hMD-2 by GM3-species balance (schematic).
POMC neuron‐selective loss of autophagy.Figure 1(A) Immunofluorescence microscopy for POMC (green) and Cre (red; n=4), (B) POMC (green) and Atg7 (red; n=4) and (C) POMC (green) and p62 (red; n=6) in MBH sections from Con and Atg7F/F‐POMC‐Cre (KO) mice (D) TUNEL‐positivity (green; n=4) and (E) number of POMC‐positive neurons (red) in MBH sections from Con and KO mice (n=6). Values are mean±s.e.m. P values are as compared with diet‐ and age‐matched controls. *P0.05, ***P0.001. Nuclei are blue (DAPI). Scale inset: 10 μm. White arrows indicate Atg7, Cre, p62 or POMC signal. Yellow arrows indicate merged signal. Con, control; DAPI, 4′‐6‐diamidino‐2‐phenylindole; KO, knockout; MBH, mediobasal hypothalamic; NS, not statistically significant; POMC, proopiomelanocortin; TUNEL, TdT‐mediated dUTP nick end labelling.
Figure 2mRNA expression analysis of epithelial cell differentiation markers Sftpc (AEC II), Hopx (AEC I), Foxj1 (ciliated cells), Muc5ac (secretory cells) and p63 (basal cells) in BALO at days 0, 10, and 21 of culture (n=3 biological replicates with pooled cells from 4 cultures per replicate) Electron microscopy of BALO alveoli showing two cuboidal epithelial cells (AEC II) connected by tight junctions (red square) with numerous mitochondria (M) and lamellar bodies (LB). A LIF with numerous lipid droplets (*) and a MYO with cisterns of rough ER (red arrow) are located at the basal side (left). The alveolar lumen is filled with lamellar surfactant including tubular myelin (white arrow) (lower right). Exocytosis of a lamellar body (*) from an AEC II (upper right). Scale bars indicate 500 nm. Electron microscopy of bronchiolar-like airway depicting columnar ciliated cells (red arrowhead) with basal bodies (black arrowhead) at the left side of the longitudinally sectioned lumen. The boxed area indicates an apical junctional complex between two ciliated cells. 1 = tight junction, 2 = adherens junction. Scale bar indicates 500 nm (in insert: 100 nm). Staining of lamellar bodies in BALO with RFP LysoTracker. Scale bars represent 100 μm. Western blot analysis of the surfactant proteins: pro-SPC, mature SPC, pro-SPB, mature SPB, and SPA in lung homogenate (LH), AEC and day 21 BALO (n=3 biological replicates). F-G. Alveolar diameter (F) and number of alveoli (G) in BALO at days 15, 21, 30 and 40 of culture was measured from n=5 BALO in n=3 biological replicates with tdTomato+ rMC. H-I. Representative scheme and images of day 40 BALO alveoli (H) and airway (I). BALO alveolar-like structures (H) are shown (h') (red arrowheads) in semithin section (0.5 µm) stained with Toluidine blue. Scale bar indicates 100 μm (left). Electron microscopy showing AEC I (h'') ultrastructure within BALO. Scale bars indicate 2500 nm (center) and 1000 nm (right). An airway-like structure (i') is shown with secretory and ciliated cells (red arrowheads) in semithin sections (0.5 µm) longitudinally cut and stained with Toluidine blue. Scale bar indicates 50 μm (far left). Electron microscopy of a bronchiolar-like airway (I) depicting pseudostratified epithelium (i'') with a basal-like cell (BC), not reaching the lumen in which cilia (C) are seen, located between a secretory (SC) and a ciliated cell (Ci). Scale bars indicate 1000 nm (left and right). Mature cilia (i''') in BALO at higher magnification depicting the 9x2+2 structure with central doubled microtubules (insert, red arrowhead), a characteristic for motile cilia. Scale bar indicates 100 nm (far right).
Figure 4A) Scheme of the protocol. ESCs were treated with 10µM KU60019 for 1.5 hours before sequential addition of IdU and CldU for 20 minutes each. DNA was treated and visualised as previously described. B) Distribution of replication rate after treatment with/without ATM inhibitor. Statistical analysis of distributions was carried out using an unpaired Mann Whitney U test. n=4. A minimum of 270 forks was measured for each condition ( **** p< 0.0001). C) Distribution of the average IdU/CldU ratio in Mybl2+/+ and Mybl2Δ/Δ ESCs with or without ATM inhibitor treatment. Percentage of highly unstable forks (ratio above 2) are indicated. Dotted line at 1 indicates positive ratio. Y-axis was cut to change scale and display large values. Statistical analysis was carried out using the Mann Whitney U. At least 260 ratios were calculated per treatment group from 4 separate experiments ( **** p< 0.0001). D) Frequency of new fired origins for Mybl2+/+ and Mybl2Δ/Δ ESCs with or without ATM inhibitor treatment. Error bars represent SEM. Statistical analysis was carried out using unpaired t-test. At least 500 replication structures were counted per treatment group from 4 separate repeats. E) Frequency of anaphases positive for UFB positive cells (based on ERCC/PICH positive immunostaining) for Mybl2+/+, the Mybl2Δ/Δ and the Mybl2+/+ treated for 2 hours with ATM inhibitor (KU60019). Data represents at least 100 anaphases from each group from 2 experimental repeats. Statistical analysis was performed using an unpaired two-tailed t-test. F) Immunoblot showing the levels of phosphorylated ATM (Ser1987) and ATM in the Mybl2+/+ and Mybl2Δ/Δ ESCs with or without CPT treatment (10µM CPT for 2 hours). Bar graph (lower panel) represents average band density of P-ATM in Mybl2Δ/Δ ESCs treated with CPT relative to ATM and relative to wild type CPT treated cells from three independent repeats. Error bars represent SD. Statistical analysis was carried out using a two-tailed unpaired t-test. G) Distribution plot of replication speed in Mybl2+/+and ATM-/- ESC treated or not for 90 minutes with 10µM KU60019 (ATMi), before sequential addition of IdU and CldU for 20 minutes each. Statistical analysis was carried out using the Mann Whitney U test. At least 200 replication forks were counted from 3 separate experiments for wild type and ATM-/- and two separate experiments for ATM-/- treated with ATM inhibitor . ( **** p< 0.0001) H) Quantification of the number of replication factories per cell for the different genotypes and treatments. A minimum of 65 early S-phase nuclei were counted per condition from two independent repeats. Statistical analysis was performed using a two-tailed unpaired unequal variance t-test.( **** p< 0.0001) I) Distribution plot of replication speed in human iPSC and MEFs (primary and immortal). Statistical analysis was carried out using the Mann Whitney U test. At least 400 replication forks for iPSC,200 for primary MEFS and 250 for immortal MEFs were counted from 3 independent experiments. ( **** p< 0.0001)
Figure 4A qPCR of cDNA from L1236 and L428 HL cell RNA obtained 5 days post H2A.B knockdown. Amplification was performed using primers that anneal to the ETS and ITS spliced-out regions to detect nascent rRNA. ETS and ITS, external and internal transcribed regions, respectively. The relative levels of both ETS and ITS were normalised to the average expression levels of the B2M and HPRT1, and presented as mean ± SD for eight biological replicates. The significance of the difference was determined by student's t test and denoted as * p < 0.05; *** p < 0.001. B Cell proliferation assay in the presence (shNTC) or during depletion of H2A.B (shH2A.B). The results are presented as mean ± SD for five biological replicates. The significance of the difference was determined by Student's t test and denoted as ns (not significant); **, p < 0.01; ***, p<0.001.
Figure 1Schematic illustrates the use of different technologies for measuring the absolute or relative abundance of centrosomal proteins in total cell lysates or purified centrosomes. The flow chart refers to the main Figures and also indicates the cell lines and cell cycle stages analyzed in each experiment. Arrows on the right emphasize the use of data from different experiments for comparison and calibration.
Figure 6Silenced TFEB alters the localization of VEGFR2. FACS analysis of surface VEGFR2 expression on human scr-shRNA and sh-TFEB ECs. Bar graph shows the ratio between total and PM VEGFR2 Silenced TFEB alters the localization of VEGFR2. Representative TIRF and epifluorescence images of human scr-shRNA and sh-TFEB ECs after staining with anti-VEGFR2 Ab (scale bars: 10 µm). Bar graphs show the ratio of VEGFR2 analyzed in epifluorescence and TIRF mode analyzed by TIRF Silenced TFEB alters the localization and the phosphorylation state of VEGFR2 and its signal transduction. Representative immunoblot of PM biotinylated portion and total cell lysates of scr-shRNA and sh-TFEB ECs after VEGF-A stimulation (30 ng/ml). Blots of total or PM cell lysates were probed with anti-VEGFR2. Blots of total cell lysates were probed with anti-p-Y1175-VEGFR2, anti-PLCγ, p-PLCγ, anti-ERK-1/2, anti-pERK1/2, anti-p-Src, anti-Src, anti CD31 and α-tubulin Abs. The bar graphs (i,ii) show densitometric analysis of stimulated versus unstimulated scr-shRNA and sh-TFEB ECs expressed as: (i) % of VEGFR2 on PM fraction (n=3, mean±SEM; ANOVA p<0.02; **p<0.001 versus scr-shRNA by Bonferroni post-test), (ii) % of VEGFR2 total Regulaton of autophagy and lysosome pathway by TFEB silencing. qPCR showing the differentially expressed autophagy and lysosome related genes between human scr-shRNA and sh-TFEB ECs. Data are expressed as relative fold-change compared with the expression in control cells after normalization to the housekeeping gene TBP Regulaton of autophagy and lysosome pathway by TFEB silencing. immunoblots showing the differentially expressed autophagy and lysosome related genes between human scr-shRNA and sh-TFEB ECs. Immunoblots of total lysates from human scr-shRNA and sh-TFEB ECs probed with anti-ULK-1, anti-ATG9, anti-LC3-I/II, anti-LAMP-1 and α-tubulin Abs. The bar graph shows the densitometric analysis expressed as the ratio between scr-shRNA and sh-TFEB ECs TFEB silencing inhibits VEGFR2 internalization. Bar graphs of VEGFR2 internalization expressed as the percent of internalized VEGFR2 versus PM VEGFR2 after VEGF-A stimulation
DCV fusion occurs predominantly at axons.Figure 6Schematic representation of NPY-mCherry as optical reporter for DCV fusion with full cargo release (top). Zoom of axonal and dendritic areas of (B), with location of fusion events (middle). Average fluorescence decrease upon NPY-mCherry labeled DCV fusion in axons and dendrites (below). Representative neuron labeled with membrane-targeted EGFP (mEGFP) (black) and localization of axonal (blue dots) or dendritic (purple dots) events upon stimulation. Scale bar: 20 µm. Histogram of axonal (blue) and dendritic (purple) DCV fusion events upon repetitive electrical stimulation (16x50AP at 50 Hz). Cumulative plot of axonal or dendritic fusion events per neuron. Normalized cumulative plot of axonal and dendritic fusion events per neuron. Percentage of axonal fusion events per cell.