EMBO/sd-character-level-ner huggingface dataset:
Figure 4(a, b) Delayed vascular outgrowth and hyperdense vascular front in isolectinB4-stained P5 retina from Pald1-/- mouse compared with Pald1+/+ (a). Orange arrow indicates radial expansion of the vascular plexus in the Pald1+/+ retina. Scale bar: 1 mm. Quantification of radial expansion (b) as shown in (a) normalized to wild type litter mates. Mean±SEM, unpaired t-test. n=14 litters, 18 wild type and 23 knock-out pups. Data information: *p < 0.05, **p < 0.01, ***p < 0.001. (c, d) Increased filopodia number in Pald1-/- mouse retina at vascular front, visualized by isolectinB4 staining (c). Scale bar: 50 μm. Quantification of filopodia per 100 µm at the vascular front (d). Mean±SEM, unpaired t-test. n=3 litters, 7 wild type and 5 knock-out pups. Data information: *p < 0.05, **p < 0.01, ***p < 0.001. (e, f) P5 retina vascular front (isolectinB4, green) and endothelial nuclei (Erg, red) (e). Scale bar: 100 μm. Magenta stippled square area quantified in (f). Vascular density was determined in the capillary bed (left) (11 wild type, 12 knock-out) and in area around veins (right) (10 wild type, 9 knock-out, biological replicates). Mean±SEM, unpaired t-test. Data information: *p < 0.05, **p < 0.01, ***p < 0.001. (g, h) pT202/pY204 Erk1/2 immunostaining (black) in Pald1+/+ and Pald1-/- P5 pups. IsolectinB4 (white) visualizes the entire vasculature. Scale bar: 100 μm. Quantification of pT202/pY204 Erk1/2 area (h) as in the black stippled square in (g) (400 µm from the retina rim), normalized to isolectinB4 area. Mean±SEM, unpaired t-test. n=5 litters, 14 wild type and 10 knock-out retinas per genotype. Data information: *p < 0.05, **p < 0.01, ***p < 0.001. (i) Quantitative real-time PCR analysis of P4-P5 retinas from Pald1+/+ and Pald1-/- pups. Ccnd1 transcript levels, normalized to Gapdh. Mean±SEM, unpaired t-test. n=10 Pald1+/+ and 14 Pald1-/- pups. Data information: *p < 0.05, **p < 0.01, ***p < 0.001. (j) Cyclin D1 (black) and isolectinB4 (white) staining of P5 retinas from Pald1+/+ and Pald1-/-pups. Scale bar: 100 µm. (k) Quantification of number of Cyclin D1 positive nuclei normalized for isolectinB4 area in retinas from Pald1+/+ and Pald1-/ P5 pups. Mean±SEM, unpaired t-test. n=7 Pald1+/+ and 6 Pald1-/- pups. Data information: *p < 0.05, **p < 0.01, ***p < 0.001. (l) Quantification of relative radial expansion in vehicle (n=2 litters, 5 Pald1+/+ and 4 Pald1-/- pups) and MEK inhibitor (U0126)-treated pups (n=4 litters, 7 Pald1+/+ and 5 Pald1-/- pups). MEK inhibitor/vehicle was administered twice at 12h interval at P4 and eyes collected at P5. Each dot is one mouse. Mean±SEM, one-way ANOVA, n=4-7. Data information: *p < 0.05, **p < 0.01, ***p < 0.001. (m) Quantification of the tip cell number in vehicle- (n=3 litters, 5 Pald1+/+ and 5 Pald1-/- pups) and MEK inhibitor (U0126)-treated pups (n=3 litters, 5 Pald1+/+ and 4 Pald1-/- pups). MEK inhibitor/vehicle administered twice at P5 at 2-h intervals, and eyes collected 2 h after the second injection. Each dot is one mouse. Mean±SEM, one-way ANOVA. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 7(A) Schematic representation of Rosa26 transgene expression via AAV.Cre recombinase. (B) Time course of optic nerve regeneration experiments using Rosa26 p110αH1047R and Rosa26 p110δ mice. C) Retinal sections from AAV-injected mice, immunolabelled for phospho-S6. Blue colour is DAPI to indicate nuclei. Inset images show individual colours at the same scale as the full image. (D) Percentage of phospho-S6 positive cells in the RGC layer 2 weeks after delivery of AAV-Cre-GFP or AAV-GFP. (E) Retinal whole mounts of AAV-injected mice immunolabelled for the RGC marker Brn3A to indicate cell survival. (F) Analysis of RGC survival 28 days after optic nerve crush (counts of Brn3A positive RGC neurons). G) Representative images of CTB-labelled RGC axons 4 weeks after optic nerve crush in wild type (c57bl/6), Rosa26-p110αH1047R, and Rosa26-p110δ transgenic mice injected with AAV2.Cre.GFP. Dashed line indicates crush site. (H) Regenerating axons at different distances distal to lesion sites.
Involvement of hypothalamic HIF1α and HIF2α in activation of the HPA axis during hypoxia.Figure 5(A) Female C57BL/6J mice were put in normoxia or hypoxia for 2h, hypothalamus was isolated for analyses. N=3-4 per group for a single RNA-seq. Heatmap represents genes upregulated by hypoxia (2h) (LFC > 0.3 and P ≤ 0.05). (B) HOMER motif analysis of hypoxia-induced genes (start offset: −1 kb, end offset: 50 bp downstream of TSS). Enriched motifs with their name and p-value are displayed. (C) Female C57BL/6J mice were put in normoxia or hypoxia for the indicated time points. Hypothalamus was isolated and stress GRE genes were measured via RT-qPCR. N=4-5 per group, one experiment. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, **P<0.01, *P≤0.05. (D) HIF1α and HIF2α protein levels were analysed via western blot using VINCULIN as a loading control. HIF1α and HIF2α protein levels were quantified using FIJI and normalized to VINCULIN levels. P-values were calculated using Mann-Whitney test. N=5 biological replicates per group. N = normoxia, H = hypoxia. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, **P<0.01, *P≤0.05. (E) HIF1α and HIF2α expression was detected in hypothalamus samples via IHC, scale bar 50 µm.
NDST3 is down-regulated in C9orf72-linked ALS patient cells and tissues.Figure 6(A, B) NDST3 protein levels relative to β-tubulin in B lymphocytes derived from healthy controls and C9orf72-linked ALS patients, as examined by immunoblotting (n = 3 biologically independent samples, **P = 0.0026). Data information: Error bars represent ± standard deviation. (C-F) The protein levels of NDST3 relative to GAPDH (D) (**P = 0.0013), Ac-α-tubulin relative to total α-tubulin (E) (*P = 0.0379), and total α-tubulin relative to GAPDH (F) (P = 0.5700), as examined by immunoblotting, in spinal cord tissues from healthy controls and C9orf72-linked ALS patients (n = 4 biologically independent samples in the control group and n = 5 biologically independent samples in the ALS patient group). Data information: Error bars represent ± standard deviation. (G-I) Immunoblotting assays showing down-regulation of NDST3 in C9orf72 shRNA (shC9orf72)-treated RPE1 cells, as compared to control shRNA (shCTRL)-treated cells. Quantification of C9orf72 and NDST3 expression relative to GAPDH is shown in (H) (n = 4 independent experiments, ***P = 0.0004) and (I) (n = 4 independent experiments, ***P = 0.0001), respectively. (J-L) Immunoblotting assays showing up-regulation of NDST3 in RPE1 cells over-expressing Flag-C9orf72 compared to those expressing Flag-EGFP as control. Quantification of C9orf72 and NDST3 expression relative to GAPDH is shown in (K) (n = 3 independent experiments, **P = 0.0011) and (L) (n = 3 independent experiments, **P = 0.0033), respectively. Data information: Error bars represent ± standard deviation. (M, N) Baf A1 sensitivity assay for WT, NDST3 KO, and Noco-treated NDST3 KO RPE1 cells expressing proline-arginine poly-dipeptide (PR82). 24 h after the transfection of PR82, WT and NDST3 KO cells were treated with vehicle or 10 μg/mL Noco overnight. The cell survival measured using calcein AM staining at 48 h post-transfection was quantified as the percentage of the fluorescence intensity for treated cells relative to that of untreated cells (n = 3 independent cultures, *P = 0.0369, ***P = 0.0007). Scale bar, 200 μm. Data information: Error bars represent ± standard deviation. (O, P) Immunoblotting analysis of PR82 levels in WT, NDST3 KO, and Noco-treated NDST3 KO RPE1 cells from (M, N) after the drug treatment. Quantification of the levels of PR82 relative to β-tubulin is shown in (P) (n = 4 independent experiments, *P = 0.0141, **P = 0.0045). Data information: Error bars represent ± standard deviation.
Figure 3(A) Left- HA-tagged ArfGAP1 was transfected in RagA/B knockout (KO) human embryonic kidney 293A (HEK293A) cells. Immunofluorescence images of HA-tagged ArfGAP1 (green) and mTOR (red) were assessed for co-localization (scale bar, 5 μm). Right- Line-scan analysis was performed. Yellow line (left) on image measures intensity along line. Distance across the yellow line is measured in microns. (B) Fluorescence Lifetime Imaging Microscopy (FLIM) and Förster resonance energy transfer (FRET) analysis were performed in wildtype (WT, left) or RagA/B KO (middle) HAP1 cells expressing mCherry-tagged ArfGAP1 and endogenously GFP-tagged Raptor. Cells either had amino acids (+AA) or were starved of amino acids (-AA). At least 20 cells were analyzed in each group. Box plots shows the percentage of pixels with ≥ 30% FRET efficiency of co-transfected cells. RFP1 was expressed and analyzed as a negative control. The whiskers of the boxplot represent the minimum and maximum values, while the box covers the data between 25th and 75th percentiles with a line in the middle plotted at the median. Right- Expression level of RagA/B was confirmed Western blot analysis. Actin was used as a control. (C) Phasor analysis was performed for the FLIM/FRET experiments described in (B) in WT (left) or RagA/B KO (right) HAP1 cells expressing mCherry-tagged ArfGAP1 and endogenously GFP-tagged Raptor. Cells either had amino acids (+AA) or were starved of amino acids (-AA). For each condition, the first panel shows the fluorescence signal of the green channel. The second panel shows the phasor distribution of GFP (black circle), GFP-tagged Raptor (green circle), and GFP-tagged Raptor plus mCherry-tagged ArfGAP1 (red circle). The green line indicates where the GFP phasor would fall when containing autofluorescence. The third panel shows spatial distribution of pixels associated with FRET. Scale bars: 9 μm for WT (left) and 5 μm for RagA/B KO (right) cells, respectively. (D) Raster Imaging Correlation Spectroscopy (RICS) analyses was performed for WT (left) or RagA/B KO (right) HAP1 cells expressing endogenously GFP-tagged Raptor (green curves) with either mCherry-tagged ArfGAP1 (blue curves) or RFP1 as a control (red curves). Auto-correlation (AC) or cross-correlation (CC) curves are shown for samples in conditions with amino acids (+AA, solid curve) or without amino acids (-AA, dashed curve).
Figure 1A, B. Immunoblotting were performed to validate Mettl3 or Mettl14 expression levels in CT26 and B16 cells as indicated. Gapdh served as a loading control. Tumor volume was monitored for control and Mettl3 or Mettl14-depleted tumors with treatment as indicated in CT26 colon cancer Data are mean ± s.e.m of the indicated number of mice in each group. n, the numbers of mice. * P < 0.05; * * * P < 0.001 by Student's t-tests. Tumor volume was monitored for control and Mettl3 or Mettl14-depleted tumors with treatment as indicated in B16 melanoma, respectively. Data are mean ± s.e.m of the indicated number of mice in each group. n, the numbers of mice. * P < 0.05; * * * P < 0.001 by Student's t-tests. E, F. Survival analysis of control tumors and those with depleted genes were recorded as indicated in CT26 colon cancer and B16 melanoma, respectively. Data are mean ± s.e.m. of the indicated number of mice in each group. n, the numbers of mice. * P < 0.05; * * P < 0.01; * * * P < 0.001 by Student's t-tests.
Figure 3 [A] Images of Arabidopsis plants exposed to four individual GMV components, including diacetyl (DA), 2,3-butanediol (BTDL), 2-methyl-1-propanol (MP) and acetoin (ATN). The compounds were applied at dosages that, when the compounds totally evaporate from the agar-containing solid droplets, would yield in volatile concentrations of 9.7 µg (DA), 32.5 µg (BTDL), 7.9 µg (MP) and 28.5 µg (ATN) per mL free space in the petri dish, which resembled the 1 : 3.3 : 0.8 : 2.9 ratio among the four GMV components in natural GMVs22. anthocyanin hyper accumulation (B) were observed in Pi-deficient plants exposed to DA but not to the other three GMV components. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test. PSR gene hyper induction (C) were observed in Pi-deficient plants exposed to DA but not to the other three GMV components. Data information: qPCR results show values of means ± SE of two biological replicates. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test. Compared to the wild type plants, phr1phl1 showed substantially decreased anthocyanin hyper accumulation (D) triggered by DA. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test. Compared to the wild type plants, phr1phl1 showed substantially decreased PSR gene hyper induction (E) triggered by DA. Data information: qPCR results show values of means ± SE of two biological replicates. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.
Figure 6(A-D) Low expression of miR-515-5p is linked to survival in both Lung and Breast cancer. (A) Survival analysis of miR-515-5p from 195 lung adenocarcinoma samples from the TCGA. (B) Survival analysis of miR-515-p from 34 lung squamous cell carcinoma from the TCGA. (C) Survival analysis of miR-515-5p from 60 Breast cancer samples from the Lyng Ditzel Breast Study (GSE37405). (D) Survival analysis of miR-515-5p from 33 Breast cancer from the GSE7405 Breast study. Analysis and plot were obtained with SurvMicro http://bioinformatica.mty.itesm.mx:8080/Biomatec/Survmicro.jsp for (A-C) and with MIRUMIR for (D). (E) Comparison of miR-515-5p expression in 18 primary and matched metastatic lesions from breast cancer patients. miR-515-5p levels were quantified by TaqMan qRT-PCR. Absolute values were normalized to u6 snRNA values and displayed as a dot-plot. Bar; Mean, Whiskers; SEM. (F) MDA-MB-231 cells were inoculated into the mammary fat pads of nude mice and allowed to form metastatic deposits [22]. miR-515-5p and MARK4 levels were then determined in the primary and indicated secondary lesions. Data are average ± SEM of technical triplicates. (G) Luciferase-expressing A549 cells overexpressing miR-515-5p or a non-targeting control (miR-NC) were injected in the tail-vein of SCID mice and the development of tumor burden monitored by whole animal-imaging in response to luciferin administration at week 1 and 4 following injection. (G-left panel) Representative imaging of the same animals for the indicated conditions at week 1 and 4. (G-Right panel) The emitted photons were quantified and the signals normalised to the corresponding readings at week 1. Data represented are representative of three independent experiments. (E-G) P values were calculated by t-test (*, P<0.05; **, P<0.01; ***, P<0.001).
a) Flow cytometry.f5(a) Flow cytometry. Untreated Jurkat T cells, cells treated with etoposide continuously (0.25 μM), or transduced with lentivirus ATG5 or GFP were cultured for 48 h, fixed, permeabilized and exposed to antiphospho-H2AX (Ser139) antibody, then stained with antimouse-APC-conjugated antibody, and analysed using a flow cytometer. (b) Confocal microscopy. Untreated HeLa cells, cells treated with etoposide continuously (0.25 μM), or transduced with lentivirus ATG5 were cultured for 48 h. Additional DAPI nuclear staining was used. Scale bar, 10 μm. Right: phospho-H2AX was quantified by measuring the mean fluorescence intensity (MFI) in five high-power fields of each condition using IMARIS software. Values are means±s.d. (c) Jurkat T cells were treated as in a and analysed by immunoblotting. Besides phospho-H2AX (Ser139), we also detected phospho-ATM (Ser1981) and phospho- ATR (Ser428), two additional markers of DNA damage response. Intensity of the protein bands were quantified with a Licor/Odyssey scanner and software (shown beneath each immunoblot). The fold change in phosphorylation levels compared with no treatment (untreated) are presented. The data in all panels are representative of at least three independent experiments. Full-length immunoblots are provided in Supplementary Fig. S9.
Figure 4HeLa cells were transfected with plasmids to visualize ER (in green) and mitochondria (in red), and then were treated with ApoE3- and ApoE4-ACM for 24 hours. Shown is a box-and-whisker plot comparing co-localization in ApoE3 ACM (n = 15 images analyzed [gray circles, average of co-localization in ~5 cells in each image field]) compared to that in ApoE4 ACM (n = 18 images).
NSMCE2 is a haploinsufficient tumor suppressor A Kaplan–Meyer survival curves of Nsmce2+/+ and Nsmce2+/GT mice.Figure 4A Kaplan-Meyer survival curves of Nsmce2+/+ and Nsmce2+/GT mice. The P‐value was calculated with the Mantel-Cox long‐rank test. ***P 0.001. B Tumor incidence found on Nsmce2+/+ and Nsmce2+/GT mice at the time of death (i.e. moribund mice). C NSMCE2 levels evaluated by Western blot in 2 independent pairs of Nsmce2+/+ and Nsmce2+/GT MEFs. β‐actin was used as a loading control. Numbers shown bellow indicate the relative amounts of NSMCE2 in each sample. D Sister chromatid exchange (SCE) events found per metaphase on Nsmce2+/+ and Nsmce2+/GT MEFs. ***P 0.001. E, F Relative percentages of micronuclei (E) and polynucleated cells (F) on cultures of Nsmce2+/+ and Nsmce2+/GT MEFs. Data are representative of three independent experiments. Error bars indicate SD. ***P 0.001.
TF network uncovers novel functions of Ebf1 and Gata3 in maintaining a multipotent, self-renewing state.Figure 6A, B Bulk RNA-Seq expression levels for Gata3 and Ebf1 in a set of isolated primary cells (data from Immgen (Heng et al, 2008)) compared to Hoxb8-FL cells. Horizontal line indicates mean expression. C Correlation in gene expression changes following Ebf1 and Gata3 loss in Hoxb8-FL cells. Each quadrant corresponds to a set of target genes with a common regulation by Gata3/Ebf1 - I - inhibition/inhibition, II - activation/inhibition, III - activation/activation, IV - inhibition/activation. For each group the sums of scaled expression values in mouse LK/LSK or human BMMC landscapes is plotted to highlight cell types with the highest overall expression. For landscape annotation see Fig 7B andE.. Changes in expression for example genes are provided in Appendix Fig 2G-J. Blue line indicates the linear fit with shaded areas as confidence intervals. D Schematic representation of the experiment performed by (Li et al, 2018). Ebf1 was re-expressed in Ebf1-/- pre-pro B cells thus resuming their differentiation. E Correlation in expression changes after 24h following Ebf1 loss in Hoxb8-FL cells and re-expression of Ebf1 in Ebf1-/- pre-pro B cells (Li et al, 2018). Blue line indicates the linear fit with shaded areas as confidence intervals. F, G Expression of the key marker genes (F) and Ebf1, Pax5, Gata3, Cebpa factors (G) along pseudotime corresponding to the B-cell differentiation trajectory in human foetal liver cells, data from (Popescu et al, 2019).
Sec61 blockade by mycolactone strongly alters the biology and viability of MM cell lines.Figure 1A Effect of mycolactone on MM.1S, JIM3 and KMS-11 cell viability after 48 and 72 h of treatment, as measured by exposure of Annexin V and incorporation of PI. Data are Mean % of live, apoptotic and dead cells from technical duplicates , relative to total cells. B, C Inhibitory effect of mycolactone on cell expression of CD38 (Type II) and CD138, IL-6R, and CD40 (Type I) transmembrane proteins (B). Stimulatory effect of mycolactone on cell expression of BCMA, a Type III transmembrane protein (C). Mean Fluorescence Intensities (MFIs) were measured in live cells 48 h after addition of mycolactone. Data are Mean MFIs ± SD from technical duplicates of 2 independent experiments, relative to vehicle-treated controls (Ctrl). D Inhibitory effect of mycolactone on immunoglobulin (lambda chain) secretion, as quantified by ELISA in culture supernatants after 24 h of treatment. Data are Mean concentrations ± SD of secreted lambda chain (µg.mL-1) from technical duplicates of 2 independent experiment (MM.1S) or Mean of secreted lambda chain concentrations (µg.mL-1) from technical duplicates (JIM3 and KMS-11), relative to vehicle-treated controls. E Correlation between immunoglobulin lambda chain (Ig) secretion after 24 h (D) and MM cell line mortality after 72 h (A), in the 3 cell lines treated with 50 nM mycolactone. Slopes (R) and statistical significance (p values) are indicated.
Figure 4A. Scheme showing the mutations introduced in the anticodon stem loop (ASL) of mt-tRNAMet for analysing tRNA substrate recognition by NSUN3. ASL mutants included G:U basepairs (G:U bp) to affect the stability of basepairing and sequence of the stem, or were generated by swapping G:C basepairs (G/C swap), leading to changes in sequence without affecting basepairing stability. B. In vitro methylation assays using [3H-methyl]-labelled S-adenosyl methionine, the in vitro transcripts of the mt-tRNAMet mutants described in (A) and recombinant His14-MBP-NSUN3. RNA was then separated on a denaturing polyacrylamide gel, stained with ethidium bromide (EtBr), dried and exposed to an X-ray film to detect methylated transcripts (3H-Me). C. In vitro methylation assay using chemically synthesised ASL. The experiment and analysis were performed as described in (B).
Figure 8(A) Nr2f1 is expressed along an anteromedial-low to posterolateral-high gradient (red color code) in the developing mouse cortex, spanning from VZ aRGs to SVZ IPs, bRGs and CP neurons. Upon Nr2f1 removal (KO), the NP pool of the posterior cortex expands leading to occipital macrocephaly. (B) Early to late effects on progenitor and neuron behavior upon Nr2f1 deficiency. Early in development (E10.5-E12-5), Nr2f1 loss causes cell cycle acceleration, increased NP self-renewal and sustained Pax6 expression. Neurogenesis (as well as the expression of neurogenetic factors such as Tbr2 and P21) is delayed, thus allowing amplification of the progenitor pool and lateral expansion of the posterior hemispheres. At later stages (E13.5-E14.5), neurogenesis comes into play and neurons are produced at high rate. Persistent Pax6 expression in the expanded NP population leads to abundant basal RG production at late time points (E16.5-E18.5). High neuronal output results in radial expansion and generation of a thick posterior cortex.