EMBO/sd-character-level-ner huggingface dataset:
Figure 4HUVECs transfected with CCP5 and/or CEP41 siRNAs were scratched and incubated to evaluate wound closure. The extent of wound closure in co-transfected cells with CCP5 and CEP41 siRNAs was compared with that in control or CEP41 siRNA-transfected cells at the indicated time points (A). Scale bars, 600 μm. Quantification of the extent of wound closure in (A) is presented graphically by measuring the distance between the dotted lines at each time point in (B). Data are shown as mean ± SD of three independent experiments (n ≥ 3 scratches per experimental condition). The indicated siRNA-transfected HUVECs were subjected to in vitro angiogenesis assays. Tubulogenesis of HUVECs depleted for CEP41 and CCP5 was compared to that of control, CEP41 single-knockdown, or CCP5 single-knockdown cells (C). Scale bars, 600 μm. Quantification of tube node number in (D) and tube length in (E) from data examined within equivalent fields of view at the indicated time points using the ImageJ angiogenesis analyzer. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition. The blood vessels in the trunks of cep41-mutant/morphant zebrafish were compared to those of zebrafish co-injected with ccp5 MOs (2 ng) at 40 hpf in (F). Asterisks and arrowheads indicate impaired ISVs and DLAVs, respectively. A, anterior; P, posterior. Scale bars, 100 μm. Quantification of the number of defective ISVs and DLAVs from data observed in equivalent fields of view (within eight somites). Data are median of three independent experiments with ≥ 20 embryos per condition. HUVECs co-transfected with CEP41 and CCP5 siRNAs were immunostained with ARL13b- and GT335-specific antibodies. The staining results were compared to those of control, single CEP41, or CCP5 siRNA-transfected cells. Representative images indicated with rectangles appear in magnified images in the lower panels. Scale bars, 20 μm. Quantification of relative intensity of GT335/ARL13b signals in the cilia ciliary length (J) in images (H) are the result of three independent experiments with ≥ 200 cells per condition (median). **
Formation of double-membrane vesicles in P.Fig 9A) Scanning electron microscopy (SEM) of MoDCs of the early interaction of MoDCs with Pg381 and Mfa1+Pg (upper panel 10000x and lower panel 25000x). B) SEM for MoDCs interacting with Pg381 (green stains for bacteria are computer generated). C) Transmission electron microscopy (TEM) of autophagosome like structures within MoDCs infected with Pg381, Mfa1+Pg and FimA+Pg for 12 hours (upper, middle and lower panels, respectively). The right and left sections show the different magnifications of randomly selected sections. Pg381 and FimA+Pg strains are mostly enclosed in the characteristics double-membrane intracellular vesicles (Orange arrows). Contrary, Mfa1+Pg escaped these autophagic (double-membrane) vesicles and enclosed within single membrane structures or freely occupy the cytoplasm (Green arrows). D) The ratio of bacteria included in the double membrane were compared to total number of bacteria within MoDCs and plotted as percentage. Counting of the bacteria included in single or doubled membrane vesicles after 12 hours of infection. Each strain was counted in three randomly selected grids for each sample. The analysis of the bacterial counts used Kruskal-Wallis test of different groups and Dunn's test for multiple comparisons (*p<0.01).
Figure 3A. Sub-clustering of eight neuronal populations using unsupervised graph-based clustering within the transcriptomes of CT7.5 (top) and CT15.5 (bottom) SCN cells. The heatmaps show the respective up-regulation of three representative marker genes in each sub-population. When compared, three subpopulations showed strong transcriptional similarity between the time-points of sequencing (Groups A, B and C), two showed partial similarity (Groups D and E) and three were transcriptionally distinct to either CT7.5 or CT15.5 sequenced cells, giving rise to the identification of eleven SCN neuronal sub-populations. B. Violin plots depicting the distribution of the quantified average cellular expression of Prok2, ProkR2, Vip, Vipr2, Avp, Avpr1a, Grp and Grpr in each sub-cluster in CT7.5 (left) and CT15.5 (right) datasets. C. Visualisation of the location within the pooled t-SNE plot of cells positively expressing >90% of the overall expression range of Prok2, ProkR2, Vip, Vipr2, Avp, Avpr1a, Grp and Grpr. This shows a contrasting expression pattern of the respective genes in cells collected at CT7.5 versus CT15.5. D. Venn diagram summarising the degree of non-/overlapping signalling axes: Prok2 (purple) - ProkR2 (brown), Vip (green) - Vipr2 (red), Avp (blue) - Avpr1a (orange) and Grp (pink) - Grpr (grey). The number SCN neurons collected at CT7.5 positively expressing each gene, which is associated to the respective signalling axis, is further annotated within the population circles.
Anti-autophagic Activity of the vBcl-2 Mutant Proteins.Figure 3(A) NIH3T3 cells stably expressing the WT and mutant forms of vBcl-2 were transfected with GFP-LC3. At 18 h posttransfection, cells were incubated under normal or starvation conditions for 4 h. Autophagy was quantified as mean±SEM of the combined results from three independent experiments. **, P<0.0001. (B) NIH3T3 cells expressing the WT or mutant forms of vBcl-2 as indicated were transfected with GFP-LC3 and treated with 2 µM rapamycin for 6 h. GFP-LC3 was detected using an inverted fluorescence microscope (top). Autophagy was quantified as mean±SEM of the combined results from three independent experiments. Scale bar, 5 µm; **, P<0.01. (C) NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2, as indicated, were treated with 2 µM rapamycin. LC3-I and LC3-II levels were then determined by immunoblotting with an antibody against LC3 (top). Densitometric quantification of the LC3-II/LC3-I ratios under normal and rapamycin treatment conditions is shown at the bottom. Similar results were obtained from three independent experiments.
Figure 7A-B. Immuno-electron micrographs showing the buds and the neck region of two β'-COPVN•Dsl3pVCcells. Both parts contain an overview image (left) and two zoomed details (right). Slides were decorated with antibody against COPI and visualized with protein A-conjugated 10 nm gold particles. Gold particles mark electron-dense areas in apposition to membranes and/or vesicular structures, which are localized at the side of the bud and the bud neck of the mother cell. Scale bar 100 nm. C. Fluorescence micrograph of a budding cell carrying β'-COPVN•Dsl3pVC (green) and RFPHDEL (red). Arrowheads mark BiFC signals which may correspond to the localizations found in (A) and (B). Scale bar 2 µm. D. COPI-positive, electron-dense structures in close apposition to perinuclear ER. 10 nm gold particles mark COPI. Scale bar 100 nm. E. Accumulation of COPI-positive vesicles. 10 nm gold particles mark COPI. PM: plasma membrane, nuc: nucleus. Scale bar 100 nm.
Figure 2(A-D) THP-1 cells were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. Flow cytometry results for annexin V single positive (A and C) and annexin V and zombie dye double positive (B and D) cells. Percentage of positive cells are depicted at 12 hpi or 24hpi. (E and F) THP-1 cells were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. LDH release measured from supernatants of mock treated or infected cells at 12 hpi (E) or 24hpi (F). G THP-1 Caspase 1 -/- cells were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. LDH release measured from supernatants of mock treated or infected cells at 12 hpi or 24hpi. n=3 for 12hpi, n=5 for 24hpi. Medians are indicated, statistical analysis was performed by one-way ANOVA.
Protein degradation rates govern dynamics of p53 targets induced under oscillatory dynamics (a) p53 protein levels from mass-spectometry under oscillatory dynamics were used to fit the production and degradation rates (kpmRNA and kdmRNA, respectively) for MDM2 mRNA.Figure 3(a) p53 protein levels from mass-spectometry under oscillatory dynamics were used to fit the production and degradation rates (kpmRNA and kdmRNA, respectively) for MDM2 mRNA. The MDM2 mRNA levels (from RNA-seq data in Fig 1) were used to fit the production and degradation rates (kpprot and kdprot, respectively) for MDM2 protein. The experimentally observed and fitted measurements are shown, along with the R2 value of the correlation between the two. (b-c) The method described in (a) was used to fit parameters for all (b) mRNAs and (c) proteins induced under oscillatory dynamics. The distribution of R2 for the fits is shown. (d) mRNA and protein degradation rates were calculated for each gene in Fig 2a-b, as shown in (a). The distribution of mRNA and protein degradation rates for each cluster (Fig 2a-b) under oscillatory p53 dynamics is shown. White lines indicate the median, and box edges and whiskers extend to the 25-75% and 5-95% quantiles, respectively. Individual dots represent outliers. (e) The protein expression trajectories of two representative p53 target genes showing the mRNA and protein dynamics indicated at the left were modeled for the indicated values of kdprot. Pink box shows the value of kdprot that best fits the experimentally measured protein dynamics. Cyan box shows protein expression at high kpprot. (f) The production and degradation rates in (a) were used to predict the MDM2 mRNA and protein expression trajectories under rising p53 dynamics. R2 values compare the observed to expected expression patterns. (g) The kpprot and kdprot parameters calculated for each gene in (c) under oscillatory p53 dynamics were used to predict the protein expression trajectories of the corresponding genes under rising p53 expression. The distribution of R2 values comparing the predicted to observed expression patterns is shown.
Fig.Figure 3(A) Schematic illustration showing the genomic loci of the circLMP2A (black arrow). The sequence of circLMP2A was validated by Sanger sequencing. The red arrow indicates the "head-to-tail" splicing sites of circLMP2A. (B) Real-time PCR analysis of the expression of circLMP2A in SNU-4th, SNU719 and YCCEL1 cells. (C) The existence of circLMP2A was validated by RT-PCR (up) and real-time PCR (down) in SNU-4th, SNU719 and YCCEL1 cells treated with or without RNase R digestion. GAPDH was used as a negative control. (D) Real-time PCR analysis of circLMP2A and LMP2A expression after treatment with actinomycin D at the indicated time points in SNU-4th cells. (E) Real-time PCR analysis of circLMP2A and LMP2A expression in either the cytoplasm or nucleus in SNU-4th cells. GAPDH and U6 were used as controls in cytoplasm and nucleus, respectively. (F) RNA ISH for circLMP2A. U6 and 18S are mainly localized in the nucleus and cytoplasm, respectively, and were used as controls. (G) Kaplan-Meier survival curve analysis showing the correlation between circLMP2A expression and OS. n= 69, P < 0.001, log-rank test. (H) Kaplan-Meier survival curve analysis showing the correlation between circLMP2A expression and progression-free survival (PFS). n = 69, P < 0.001, log-rank test.
Figure 5(A,B) HP1-HP2 distance distributions for (A) EAAT1 (measured between residues 383 and 443) and (B) GltPh (measured between residues 276 and 355) obtained from unguided simulations as a function of K+ occupancy. Inset, snapshots of the HP2 gate in open (gray) and closed (blue) states. The boundary between open and closed states was defined as the first local minimum of the K1-bound histogram. (C) K+-induced conformational changes of the EAAT1 transport domain. Overlay of TM7 before (yellow cartoon) and after (blue) K1 occupation.
TLR3 and TLR4 promote mTORC1 signaling in a TBK1-dependent manner in RAW264.Figure 5A. Pharmacologic TBK1 inhibition reduces mTORC1 signaling upon activation of TLR3 and TLR4 in cultured macrophages: RAW264.7 cells cultured in full serum were pre-treated with amlexanox ([50 μM or 100 μM]) (2 hr.), rapamycin [20 ng/mL] (30 min), or Ku-0063794 [1 μM] (30 min) and stimulated -/+poly (I:C) [30 μg/ml] or LPS [100 ng/ml] (60 min). Whole cell lysate (WCL) was immunoblotted as indicated. B. Knockdown of TBK1 with shRNA reduces TLR3- and TLR4-stimulated mTORC1 signaling: RAW264.7 macrophages were co-infected with shRNA-containing lentiviruses targeting TBK1 and IKKε shRNA or infected with scrambled control (Scr), selected in puromycin, and treated as in A. C. Pharmacologic TBK1 inhibition reduces mTORC1 signaling upon activation of TLR3 and TLR4 in primary macrophages: Bone marrow derived macrophages-monocytes (BMDMs) were pre-treated with amlexanox [100 μM] (2 hr.) or Ku-0063794 [1 μM] (30 min) and stimulated -/+ poly(I:C) or LPS as in A.  D. Pharmacologic TBK1 inhibition reduces mTORC1 signaling upon activation of TLR3 in HEK293-TLR3 cells. Cells were serum starved (20 hr.), pre-treated with amlexanox or Ku-0063794 as in C above, and stimulated -/+ poly(I:C) [50 ug/mL] (60 min). E. TLR4/LPS-stimulated mTORC1 signaling requires TBK1: TBK1+/+ and TBK1-/- MEFs were serum starved (20 hr.), pre-treated with amlexanox [50 μM] (2 hr.) or Torin1 [100 nM] (30 min), and stimulated -/+ LPS as in A.
Figure 4A Cytoplasmic extracts from MEG-01 cell lines stably expressing AS-RBM15, Exon 1, or shRNA-02 against AS-RBM15 were fractionated through sucrose gradients (15%-60%) (n=4). Global polyribosome profiles of these cell lines were generated by UV absorbance at 254 nm (A254). The arrow indicates increasing polyribosome weights. Low molecular weight, LMW; high molecular weight, HMW. B The distributions of RBM15 mRNA (top), GAPDH mRNA (middle), and AS-RBM15 (bottom) were measured by real-time PCR and represented as percentages of total RNA in the fractions (n=3). C MS2 pull-down assays: AS-RBM15 full-length (FL) and exon 1 were fused to MS2RNA as baits. Plasmids expressing MS2 binding protein ( HA-tagged) and fusion baits were co-transfected into 293T cells. HA antibody was used to immunoprecipitateHA-MS2 binding protein as shown by a WB on the bottom panel. RBM15 mRNA and MS2 fusions were assessed by real-time PCR (n=3, mean ± SD). Fold enrichment was calculated as 2 to the power of [(CI.R-Cb.R)-(CI.MS2-Cb.MS2)]. I: input i.e. the RNA in solution before immunoprecipitation, R: RBM15 mRNA, b: bead-bound mRNA, MS2: RNA levels of MS2 fusions, C: number of cycles in real-time PCR. One-way ANONA for p value: p<0.05. D WB analysis of the endogenous RBM15 protein levels in 293T cells overexpressing two different doses of AS-RBM15-MS2 and MS2 (n=3). E RBM15 mRNA interacts with AS-RBM15 via RNA-RNA interaction. AS-RBM15-MS2-associated RNAs were purified by phenol extraction and measured by reverse transcription and PCR for detection of RBM15, AS-RBM15 and MS2 under denaturing (heat) and non-denaturing conditions (n =3). F RNase A protection assays for detecting endogenous association of AS-RBM15 and RBM15 mRNA (n=4). RNA was extracted from MEG-01 cells with phenol and digested with RNase A. cDNA was synthesized after RNase A treatment and detected using AS-RBM15 exon-specific primers as shown in the schematic diagram on top. RT: reverse transcriptase. G MEG-01 cells expressing AS-RBM15 or vector control were treated with DMSO or rapamycin for 45 minutes. Cells were harvested for WB using antibodies against GAPDH and RBM15 (n=2). H The RBM15 5'UTR reporter remains sensitive to rapamycin treatment with or without overexpression of antisense AS-RBM15 (n=4, mean ± SD). P values were calculated by one-way ANOVA test (**p<0.01 and ***p<0.001).
Figure 6Pre-existing immune activation exacerbates SARS-CoV-2-dependent inflammation. (A) Schematic of experimental design. (B-H) MDM were primed with 100ng/ml LPS for 2h before exposure to SARS-CoV-2 (MOI 0.02 TCID50VERO/cell). (B) Expression of genomic and subgenomic viral E RNA at 48 h post exposure in indicated conditions. (C-G) Host gene expression of (C) CCL5, (D) ISG56, (E) IFIT2, (F) IL-6 or (G) IL-1b was measured 48hpi. (H) IL-1b secretion was detected in culture supernatants at 48 hpi by ELISA. (I) Schematic of experimental design. MDM were exposed to SARS-CoV-2 (MOI 0.02 TCID50VERO/cell) for 48 h and subsequently stimulated with 100ng/ml LPS for 24 h. (J-P) (J) Expression of genomic and subgenomic viral E RNA 72 h post-exposure in indicated conditions. (K-O) Host gene expression of (K) CCL5, (L) ISG56, (M) IFIT2, (N) IL-6 and (O) IL-1b. (P) IL-1b secretion was detected in culture supernatants at 48 hpi by ELISA. Data Information: (A-P) Gene expression is shown as fold induction over untreated controls. Data from 8-13 independent donors is shown. Groups were compared as indicated by Wilcoxon matched-pairs signed rank test, *, p<0.05, ** (p<0.01), *** (p<0.001). Calu-3 cells were infected with SARS-CoV-2 (MOI 0.04 TCID50VERO/cell) in the presence or absence of 10ng/ml IL-1b. (Q-T) Gene expression of (Q) IFNβ (R), CXCL10, (S) IL-6 and (T) IFIT1 was measured after 24h. Fold induction over untreated mock infection is shown, n=3. Calu-3 cells were infected with SARS-CoV-2 (MOI 0.04 TCID50VERO/cell) in the presence or absence of 10ng/ml IL-1b. (U) Non-adherent cells were collected at 48h post infection and live/dead stained. Cells were acquired by flowcytometry and cell counts determined by time-gating. For statistical comparison, total cell numbers were compared. (V) Viral release into culture supernatants at 24 h was measured by TCID50 on VeroE6 cells. (Q-V) Mock and SARS-CoV-2 infected conditions were compared with or without IL1b-treatment, respectively, by unpaired T test (n=3). *, p<0.05; n.s., non-significant. Mean +/- SEM shown.
Figure 5A. Top: Schematic experimental set up of the clonogenic assay. Bottom: Bright-field microscopy images of WT, TP53KO and onco-RNF43/TP53KO human colon organoid lines at day 2 and 14. Scale bars represent 1000 μm. B. Relative outgrowth of WT, TP53KO and onco-RNF43/TP53KO organoid lines treated with the PORCN inhibitor C59 (1 μM) for 7 days. Graph shows the number of organoids at 5 days after splitting (day 14) relative to the number of organoids at day 2. Error bars represent ±s.d. of the mean of n = 3 experiments. C. Model for mode of action RNF43 LOF and onco-RNF43 variants. (Left) RNF43 performs a bifunctional tumor suppressor role by (I) targeting Wnt receptors for endocytosis and lysosomal degradation, and (II) by transiently interacting with the destruction complex to reconstitute its activity in the cytosol and re-establish Wnt pathway inhibition. This second suppressor role involves CK1-mediated phosphorylation and an unknown molecular activity of the flexible cytosolic tail of RNF43. (Middle) Classical loss-of-function (LOF) mutations prevent RNF43 function at the plasma membrane, leading to Wnt receptor overexpression and, consequently, hypersensitivity of cancer cells to Wnt. (Right) Onco-RNF43 truncated variants are stably expressed at the plasma membrane and retain E3 ligase-dependent Wnt receptor downregulating activity. However, due to loss of the cytosolic tail, these mutants selectively trap CK1 and Axin1 at the plasma membrane, which prevents destruction complex assembly and drives uncontrolled β-catenin-mediated transcription of target genes.
Figure 3A Schematic representation of the luciferase-reporter constructs used to assay miR-1236 activity against a CDS target site in FOXL2 mRNA. The 231-bp human FOXL2 segments harboring either the C402 (WT) or the G402 (mutant) nucleotide were inserted in-frame into the CDS of the luciferase gene in the pGL3 control vector. B Luciferase activity of the reporter constructs shown in (A) were measured in KGN cells after transfection with an miR-1236 mimic for 48 h. The black arrow indicates the position of 402C>G mutation site. C A schematic diagram of the luciferase-reporter constructs generated by inserting the predicted miR-1236-target sequences of WT and 402C>G FOXL2 mRNAs in the 3′-UTR of luciferase. D Luciferase activities were measured in KGN cells, using the reporter constructs shown in (C), after transfection with a control miRNA or an miR-1236 mimic for 48 h. E, F miR-1236 mutants, in which the C that pairs with G402 of the FOXL2 mutant was substituted with either G (miR-1236-G) (E) or U (miR-1236-U) (F), were cotransfected into KGN cells with one of the reporter constructs described above. Luciferase activities were subsequently determined. Arrows indicate the mismatched sites. G In vitro annealing kinetics of miR-1236 with 230 nt-long transcripts of WT or variant FOXL2. 32P-labeled miR-1236 (0.5 nM) was incubated with increasing concentrations of synthetic FOXL2 transcripts (0, 2.5, 12.5, 25, or 50 nM). FOXL2 mRNA-miR-1236 complexes were resolved on a 6% native gel and detected by autoradiography (left). The predicted Kds for the WT and 402C>G FOXL2 transcripts are presented in the right graph.
Figure 6A. Representative H&amp;amp;E staining of lung tumors from EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not during four days with erlotinib (12.5 mg/kg/day), with the AKT inhibitor G594 (25 mg/kg/day), or in combination with both drugs. Scale bar: 500 µm. B. Quantification of the tumor/lung ratio of mice treated or not with the individual drugs or with a combination of both. C. Representative Ki67 immunostaining of lung tumors from EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not with erlotinib alone (12.5 mg/kg/day) or in combination with the AKT inhibitor G594 (25 mg/kg/day) for four days (scale bar: 50 µm), and the corresponding quantification (D).
Figure 4A. Quantification of mRNA expression by RT-qPCR of candidate miR-34/449 targets that might regulate spindle orientation in E14 cortex samples from miR-34/449 KO mice compared with littermate controls (Het). Expression was normlaized (norm.) to Phosphoglycerate kinase (PGK) mRNA levels. Significance was tested by Welch's t test: p = 0.009756 (JAM-A (Het) vs. JAM-A (KO)), all the rest Het vs. KO comparisons p=n.s. (n = 4 cortices per genotype group, 2 independent litters). All subfigures were statistically tested as in A. * indicates p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤ 0.001. Red bar indicates median. B. RT-qPCR quantification of JAM-A mRNA expression in HeLa cells 48 h after transfection of miR-449a mimic compared to cells transfected with non-targeting negative control siRNA. JAM-A mRNA Expression was normalized (norm.) to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels. p = 0.0001218 (Neg. ctrol. vs. miR-449a mimic) (n = 9 measurements from 3 independent replicates). C, D. mouse J110 cells were transfected with either miR-449a mimic or a negative control mimic. Whole cell extracts were subjected to immunoblot analysis to detect endogenous JAM-A and ACTIN. ACTIN was used to normalize JAM-A expression (norm.) as loading control. p = 0.04454 (Neg. ctrol. vs. miR-449a mimic) (n = 4 independent wells, 2 independent transfections). E, F. HeLa cells were cotransfected with either miR-449a mimic or a negative control mimic, together with JAM-A-GFP fusion protein and GFP. Whole cell extracts were subjected to immunoblot analysis to detect GFP. GFP was used to normalize (norm.) by transfection efficiency as loading control. p = 0.001176 (JAM-A WT Neg. ctrol. vs. miR-449a mimic) (n = 9 independent wells, 4 independent transfections). G, H. Confocal images of coronal sections from E14 brains of wild type mice stained with anti-JAM-A antibody (green), phalloidin to label actin (gray) and DAPI to label cell nuclei (blue). Scale bars: 10 μm (G), 5 μm (H).I. Quantification of JAM-A protein expression in the ventricular zone of E14 brain coronal sections from miR-34/449 KO mice compared with littermate controls (Het) by immunofluorescence. ACTIN (phalloidin signal) was used to normalize (norm.) the expresion levels of JAM-A. p = 0.04842 (n = 5 (KO) or 7 (het) cortices per genotype group, 3 independent litters).