EMBO/sd-character-level-ner huggingface dataset:
Figure 5RNA-seq was conducted on freshly isolated X1 cells from exoc3(RNAi)-treated compared to X1 cells from gfp(RNAi)-treated planarians on day-38 of the injection protocol. The histograms depict the overlap (blue) of DEGs in X1 cells of Smed-exoc3-depleted planarians versus controls (n=3 biological replicates; adjusted P< 0.05) with genes expressed in the indicated sample sets of (A) neoblast stem cells (B-E) The heat maps depict the intersection of DEGs with strong expression signals (log2 fold change > 0.5 or < -0.5) discovered in X1 cells of exoc3-depleted planarians versus controls (n= 3 biological replicates) with the indicated, previously published data sets on gene expression profiles in (B-D) neoblast cells or (E) differentiating progenitor cells of epidermal lineage. The colour scale represents the gene-wise z-score calculated from normalized gene expression levels while purple and green indicate upregulated and downregulated genes in X1 cells of exoc3-depleted planarians versus controls, respectively. RNA-seq was conducted on freshly isolated X1 cells from exoc3(RNAi)-treated compared to X1 cells from gfp(RNAi)-treated planarians on day-38 of the injection protocol. The histograms depict the overlap (blue) of DEGs in X1 cells of Smed-exoc3-depleted planarians versus controls (n=3 biological replicates; adjusted P< 0.05) with genes expressed in the indicated sample sets of (F) differentiating progenitor cells of epidermal lineage from the indicated publications while the sum of overlapping (blue) and set-exclusive (grey) genes represents the total number of genes of a cluster that were analyzed in this study.
a) Proportion of BrdU incorporation into DNA and cell cycle phases, and (b) cell growth in WT and Pink1 KO MEF after 72 h in the absence (denoted as -2DG) or in the presence (+2DG) of the glucose-metabolizing inhibitor, 2-deoxyglucose (2DG, 1 mM).f5(a) Proportion of BrdU incorporation into DNA and cell cycle phases, and (b) cell growth in WT and Pink1 KO MEF after 72 h in the absence (denoted as -2DG) or in the presence (+2DG) of the glucose-metabolizing inhibitor, 2-deoxyglucose (2DG, 1 mM). (c) Proportions of BrdU incorporation into DNA and cell cycle phases, and (d) cell growth in WT and Pink1 KO MEF 72 h after transfection with siHif1α (+siHif1α) or siControl (denoted as -siHif1α). (e) Ki67, Sox2 and DAPI immunostaining in the subventricular zone of WT and Pink1 KO mouse brain, and the corresponding quantification. *P0.05; #P0.05 versus all other conditions (analysis of variance followed by Bonferroni test; n=3-4 independent experiments). All data are expressed as mean±s.e.m. Scale bar, 100 μm.
Functional and biophysical characterization of BakΔTM bound to liposomes.Figure 1All liposome permeabilization assays were performed with C-terminally His-tagged BakΔTM and BclxLΔTM attached to Ni2+-lipid doped MOM-like liposomes. (a-d) Liposome permeabilization assays detecting the release of a fluorescence dye from liposomes. (a) Pore formation of 50 nM BakΔTM (red) is activated by 20 nM cBid (blue). 20 nM cBid alone (orange) shows no liposome permeabilization. Addition of 25 nM BclxLΔTM (green) inhibits pore-formation. (b) Increasing amounts of cBid are titrated to 50 nM BakΔTM in the absence (blue) and presence (green) of 25 nM BclxLΔTM. (c,d) BakΔTM becomes autoactive at increasing concentrations. The concentrations are color coded as indicated in (d). (c) The averaged kinetics for increasing BakΔTM concentrations. Bar diagrams in (b) and (d) represent data from the kinetic experiments averaged between 9000 -10000 s. The standard deviation was calculated from three measurements. ( All CD measurements were performed with C-terminally His-tagged BakΔTM and BclxLΔTM attached to Ni2+-lipid doped MOM-like liposomes. (e) Far-UV-CD spectra of BakΔTM +/- liposomes and the activators cBid or Puma-BH3 peptide as indicated. All CD measurements were performed with C-terminally His-tagged BakΔTM and BclxLΔTM attached to Ni2+-lipid doped MOM-like liposomes. (f) CD-detected thermal melting experiments of BakΔTM (black) with liposomes (red) and Puma-BH3 (blue).
Figure 4A Structural comparison of GGTase-III and other prenyltransferases. The α and β subunits of the prenyltransferases are shown in green and cyan, respectively. The unique N-terminal domain of PTAR1 is shown in yellow. B Overall structure of GGTase-III complexed with Cys195-farnesyl Ykt6ΔAIM and GGPP in two orientations. GGTase-III is colored as in (A). The longin domain of Ykt6 is in pink and SNARE domain is in magenta. C, D Magnified views of site 1 (C) and site 2 (D) depicting the interaction between PTAR1 and Ykt6. Dashed lines indicate hydrogen bonds. E Geranylgeranylation activity of WT GGTase-III and the indicated mutants (100 nM each). Cys195-farnesyl Ykt6ΔAIM (1 μM) and 3H-GGPP (1 μM) were used as substrates (mean ± SEM, n = 3). F Kinetic analysis of WT GGTase-III and the site 1 or site 2 mutant (100 nM each) using increasing concentrations of Cys195-farnesyl Ykt6ΔAIM (mean ± SEM, n = 3 for WT and Y306A, n = 1 for V48A/V50A). Geranylgeranylation of unprenyl WT Ykt6 and the site 1 or site 2 mutants by GGTase-III. WT and mutant Ykt6 proteins (5 μM each) were incubated with GGTase-III (100 nM) and 3H-GGPP (1 μM), and the amount of 3H-geranylgeranyl transferred to Ykt6 was quantified (mean ± SEM, n = 3).
BACE as a new regulator of YAP in metastatic melanoma cells.Figure 7In metastatic melanoma, BACE2 activity assists the secretion of protein aggregates into the extracellular space. The presence of these aggregates might be sensed by Agrin, known to activate the YAP signalling cascade, and is able to induce YAP mediated CTGF transcription. In turn, melanoma cells treated with BACE inhibitors produce fewer protein aggregates and show a lower YAP transcriptional activity.
Both APN and P5 activate AMPK signaling pathway through AdipoR1 in vitro, and P5 induces hair growth factors in DP cells.Figure 2A APN-treated ORS or DP cell lysates were analyzed for p-AMPK and p-ERK1/2. B P5-treated ORS or DP cells lysates were analyzed for p-AMPK and t-AMPK. Densitometric analysis for the ratio of p-AMPK protein to t-AMPK protein (n = 5 (ORS) and 4 (DP) biological replicates / group) C AdipoR1 siRNA-transfected ORS or DP cells were treated with APN or P5. The cell lysates were analyzed for p-AMPK. D-H The relative gene expression levels of growth factors in P5-treated DP cells (n = 4 (VEGF, PDGFA), 5 (IGF1), 6 (HGF, FGF7) biological replicates / group) I-M The relative protein levels of growth factors in P5-treated DP cells (n = 3 biological replicates / group)
Figure 7CRAC/ORAI channel functions down stream of TCR activation, and plays important roles for iNKT cell differentiation and activation. NKT2 cells show higher and prolong Ca2+ signals than NKT1 cells after CRAC/ORAI activation. We propose this is regulated by two mechanisms: Mitochondria in NKT2 cells sequester Ca2+ at the vicinity of the channel to reduce Ca2+ dependent inactivation of CRAC/ORAI and help maintain the channel activity for Ca2+ entry; Lower PMCA expression and slower Ca2+ exportation in NKT2 further contribute to higher and sustained Ca2+ signals in NKT2 cells. Ca2+ in mitochondria is an important co-factor for key metabolic enzymes (PDH: pyruvate dehydrogenase; IDH: isocitrate dehydrogenase; OGDH: 2-oxoglutarate dehydrogenase). Consistently, NKT2 cells show more robust respiration and higher cellular ATP. Note: ER is an important regulator of Ca2+ homeostasis, and is not shown for purpose of simplicity. Figure was generated using BioRender.
Figure 3(A, Effects of the gain- or loss-of-function of Wip1 on TGF-β-induced growth arrest. HEK293T cells were transfected or not with wt Wip1, Wip1(D277A), as indicated and subjected to CCK-8 assays before (0 h) and after stimulation with TGF-β1 (40 ng/ml) (24 h). Data are presented as the mean ± SEM (n=3 biological replicates). *P<0.05, **P<0.01, ***P<0.001 by unpaired Student's t-test. n.s., not significant. B) Effects of the gain- or loss-of-function of Wip1 on TGF-β-induced growth arrest. HEK293T cells were transfected or not with Co siRNA or Wip1 siRNA as indicated and subjected to CCK-8 assays before (0 h) and after stimulation with TGF-β1 (40 ng/ml) (24 h). Data are presented as the mean ± SEM (n=3 biological replicates). *P<0.05, **P<0.01, ***P<0.001 by unpaired Student's t-test. n.s., not significant. Depletion of Wip1 augments TGF-β-induced migration and invasion of MDA-MB231 breast cancer cells. MDA-MB231 cells transfected with Co siRNA or Wip1 siRNA were scratched with a 20-μL pipette tip and then incubated in the absence or presence of TGF-β1 for the indicated times (C, D) Depletion of Wip1 augments TGF-β-induced migration and invasion of MDA-MB231 breast cancer cells. MDA-MB231 cells transfected with Co siRNA or Wip1 siRNA were subjected to transwell assay with or without TGF-β1 for 20 h (E, F).
Figure 1(A) THP1 cells were infected with the shown strains of HSV-1 (MOI 3). Supernatants were harvested from untreated cultures or cells infected for 12 or 18 hours for measurement of type I IFN bioactivity. (B) THP1 cells were treated with 0.1 μg/ml of acyclovir (ACV) and infected with the KOS HSV-1 and F HSV-1 strains (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity. (C) THP1 cells were treated with infectious or UV-inactivated HSV-1 (KOS). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity. (D) MDMs were infected with ICP0-deficient or revertant HSV-1 (KOS), MOI 3. Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity. (E) THP1-derived cells deficient for cGAS or STING were infected with the shown strains of HSV-1 (MOI 3) or stimulated with poly(I:C) (2 µg/ml). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.
The life-extending and autophagy-inducing effects of spermidine in C.Figure 3.The life-extending and autophagy-inducing effects of spermidine in C. elegans are not mediated by Sir2. (A) Fluorescence microscopy of C. elegans transgenic embryos expressing a full-length plgg-1DsRed::LGG-1 fusion protein indicative of autophagic activity. Two representative pictures of wild-type (WT) and sir-2.1 embryos untreated (Co, control) or treated with 0.2-mM spermidine (Spd) supplementation of food are shown. (B) Quantification of autophagic activity through the measurement of DsRed::LGG-1 pixel intensity from images of WT animals shown in A. Data represent means ± SEM (n = 3) with ≥25 images processed for each trial. C) Survival of WT C. elegans during aging with and without (control) supplementation of food (UV-killed E. coli) with 0.2-mM spermidine (n = 110; P < 0.005). (D) Survival of sir-2.1 C. elegans (ok434 phenotype) during aging with and without (control) supplementation of food (UV-killed E. coli) with 0.2-mM spermidine (n = 110; P < 0.01). P-values were calculated using the log-rank test as described in Materials and methods.
Figure 7In the absence of flg22, BIK1 interacts with FLS2, BAK1, MAP4K4 and PP2C38. SIK1 and MAP4K4 stabilizes BIK1 by phosphorylation. PP2C38 maintains BIK1 activity at minimum levels. Upon flg22 treatment, BIK1 is released from FLS2 and BAK1. MAP4K4 phosphorylates PP2C38 at Ser77 thereby enabling BIK1 activation. In parallel or alternatively, SIK1 and MAP4K4 phosphorylate and stabilize BIK1. SIK1 and BIK1 phosphorylate and activate RBOHD producing H2O2.
Figure 4cTAZ repressed the antiviral activity of ectopic STAT1/2. HEK293A were transfected with indicated plasmids, infected with gVSV (MOI = 0.01) for 12 hours, fixed, and subjected to microscopy (Left). Viral infection and replication were marked by green fluorescence. Scale bar, 100 μm. (Right) Quantification of GFP positive cells. About 300 cells (from 3 different fields) were analyzed. Error bars indicate SD. **P<0.01; ***P<0.001; Student's t-test. cTAZ and STAT1/2 expression levels of cells cTAZ effectively repressed antiviral activity of IFN-α. Transfection and infection were performed Top: GFP intensity of cell lysates was measured by a fluorometer. Bottom: viral GFP and ectopic cTAZ protein levels were detected by IB. Depletion of cTAZ repressed viral replication. RKO cell lines (WT and two independent cTAZ -/- clones) were infected with gVSV (MOI = 0.1) for 12 hours. Scale bar, 100 μm. The ratio of GFP positive cells were calculated About 300 cells (from 3 different fields) were analyzed. Error bars indicate SD. ***P<0.001; Student's t-test. Depletion of cTAZ repressed viral replication. Cells were treated GFP intensity of cell lysates was measured. Error bars indicate SD, n = 3. **P<0.01; ***P<0.001; Student's t-test. Depletion of cTAZ repressed viral replication. Cells were treated the viral genomic RNA was quantified by qPCR (normalized to human GAPDH). Error bars indicate SD, n = 3. ***P<0.001; Student's t-test. cTAZ deficiency enhanced ISGs expression. Cells were treated the expression of IFIH1 and DDX58 were assessed by qPCR. Error bars indicate SD, n = 3. *P<0.05; **P<0.01; ***P<0.001; Student's t-test. Overexpression of cTAZ promotes virus infection. DOX-inducible cTAZ-overexpressing RKO cell lines were pre-treated with DOX for 48 hours (1 μg/ml). gVSV infection and GFP Fluorescence intensity quantification were performed Error bars indicate SD, n = 3. **P<0.01; ***P<0.001; Student's t-test. Overexpression of cTAZ repressed ISGs expression. Cells were treated the expression of IFIH1 and DDX58 were assessed by qPCR. Error bars indicate SD, n = 3. *P<0.05; **P<0.01; Student's t-test.
Figure 5A) Confocal image of wing disc expressing the human tetraspanin CD63-GFP expressed in the Hh receiving cells colocalizing with Ptc (Bac.Ptc-cherry) in vesicle-like structures (yellow arrows) along basal cytonemes. B) In vivo confocal frames of abdominal histoblasts showing colocalization of CD63-Cherry and Ptc (Yac.Ptc-GFP) at punctae moving in anterograde direction along cytonemes (white arrowheads show trajectory in time-lapse frames). C) Lateral confocal plane of wing disc showing endogenous Ptc colocalizing with the Drosophila tetraspanin Tsp96F-RFP (orthologous to the mammalian tetraspanin CD9/CD81) (yellow arrows indicate co-localization in punctate structures). D) Basolateral in vivo imaging of abdominal histoblasts expressing Tsp96F-RFP in the Hh receiving cells shows colocalization with Ptc-GFP at mobile punctae (yellow arrows). E) Tsp96F-RFP expressed in wing disc Hh-receiving cells also shows localization in basal cytonemes stabilized by Ihog-CFP expression (yellow arrows indicate Tsp96F-RFP-positive punctate structures). F) Frames showing in vivo localization of Tsp96-RFP punctae to cytonemes stabilized with Ihog-CFP expressed in the abdominal histoblasts Hh-receiving cells, moving in an anterograde fashion towards the P compartment (white arrowheads show trajectory in time-lapse frames). G) Confocal imaging of receiving cells expressing the HA tagged Tsp96F construct also shows co-localization with endogenous Ptc at cytonemes (yellow arrows indicate Tsp96F-HA and Ptc co-localizing punctate structures). H) Western blot against HA-tag of Immuno-Precipitated sample (IP), Depleted Sample (Dep), High Speed Sample (HSS) Whole Sample and Control sample from a co-immunoprecipitation experiment isolating GFP of Tsp96F-HA and Ptc-GFP expressing larvae or GFP and Tsp96F-HA expressing larvae as negative control. Arrowhead indicates the positive band corresponding to Tsp96F-HA expected molecular weight (30-40 kDa), present in all samples including the immuno-precipitated one and not present in the negative control sample.
Figure 3A) Schematic of the single sgRNA CRISPR/Cas9 strategy to remove the duplication of the exons from 18 to 30 in the Dup18-30 mouse model. A single sgRNA targeting intron 21 (i21) was utilized to cut both copies of the duplicated region. Cas9 and the sgRNAs are represented as scissors. B) Two-day-old neonatal Dup18-30 pups were injected with AAV9 carrying Cas9 and i21 gRNA (n=11; 3x1012 viral genomes) via temporal vein and sacrificed 7 weeks later. C) The efficiency of the removal of the duplicated region at the DNA level was assessed via qPCR analysis by normalizing the signal obtained from the duplication junction to that of the total Dmd signal. Dup18-30 untreated, n = 3-4; Dup18-30 treated, n = 5-9. D) The RNA editing efficiency was quantified in the same tissues analyzed in panel B via qPCR utilizing the expression ratio between the duplication junction and the WT Dmd transcript. Dup18-30 untreated, n = 4; Dup18-30 treated, n = 4-5. E) RT-PCR analyzing the removal of the duplication from the heart, TA and triceps muscle of WT, Dup18-30 untreated, and Dup18-30 treated mice. Arrows correspond to primers in exon 17 and exon 33. F, G ) Western blotting detected restoration of dystrophin expression in (F) heart and (G) TA in the Dup18-30 mice. 25% and 50% of the WT proteins compared to Dup18-30 mice have been loaded on the gel. Calnexin was used as a loading control. H) Immunostaining showed restoration of dystrophin expression in the heart and TA. A representative sample is shown. Scale bars, 100 μm. I) Quantification of dystrophin western blot in panels F and G. Dup18-30 untreated, n = 2; Dup18-30 treated, n = 4. J) Percentage of dystrophin positive fibers from the immunostaining in the heart and TA of and Dup18-30 untreated and Dup18-30 treated mice. Dup18-30 untreated, n = 5; Dup18-30 treated, n = 10-11.
Figure 4Mice were subjected to ONC and i.v.-injected with either an anti-VCAM-1 blocking antibody or an isotype control antibody. Eyes were collected after 2 or 3 days for flow cytometry. (A) Flow cytometry quantification of myeloid cells in retinas from mice that were treated with anti-VCAM-1 antibody, as compared to isotype-treated controls. n = 3 per group. Asterisks above bars indicate significant differences compared with noninjured tissue; significant differences between different antibody treatments are indicated by asterisks between bars. Mice were subjected to ONC and i.v.-injected with either an anti-VCAM-1 blocking antibody or an isotype control antibody. Eyes were collected after 2 or 3 days for Real-Time PCR analysis, respectively. (B) Expression ratios of Tnf, Il12a and Il1b, compared to Tgfb2, showing that blocking VCAM-1 results in skewing the retina towards a pro-inflammatory milieu after ONC. n = 11-13 per group. Data shown are representative of two independent experiments. Bar graphs throughout the figure show mean ± SE of each group. *, P < 0.05; **, P < 0.01; *** P < 0.001. noninj - noninjured control; ONC - optic nerve crush.
Figure 4(A) Schemes of the Far1(1-150)-GFP constructs used in 'B'. The constructs contain either wild-type phosphorylation sites or have all TP sites mutated to SP sites (SP mutant). The predicted Cks1 docking connections are shown with the arrows below the scheme. (B) The degradation of Far1(1-150)-GFP with either wild-type or all serine-based phosphorylation sites and the indicated docking motifs was measured in time-lapse microscopy. Plot shows mean±SEM of GFP fluorescence intensities from Start. (C) Phos-tag SDS-PAGE autoradiographs showing the Cks1-dependency of Clb5-Cdk1-mediated multisite phosphorylation of Far1(1-150) with either NLxxxL (WT) motif or RxL motif. The experiment was performed twice, a representative example is shown. (D) Plot showing the kinetics of Clb5-Cdk1-mediated phosphorylation of Far1(85-150) with either wild-type (NLxxxL) or RxL docking motif. The data is from two replicates. (E) Scheme of Far1(85-150)-GFP with either NLxxxL, RxL or mutated cyclin docking motif and with or without T3 primer site introduced upstream of the S87 S91 degron. (F) Mean±SEM GFP fluorescence intensities of the indicated Far1(1-85) constructs, synchronized at the time of Start. (G) Plot showing the timing of 50% degradation of the indicated Far1(85-150) constructs in single cells. The numbers above the plot show median values. The bars show median ±95% CI. **** and ns indicate p-values <0.0001 and >0.05 by Mann-Whitney test for pair-wise comparisons with WT.
NSCLC cells to apoptosis induction by inhibition of the Raf-ERK-pathway.Figure 7A 1205Lu melanoma cells carrying dox-inducible GFP, GFP-Usp27x or GFP-Usp27xC87A were treated with doxycycline (dox) as indicated. After 48 hUO126 (10µM) was added for another 48 h. Apoptosis was measured by staining for active caspase-3, followed by flow cytometric analysis. Data (means/SEM) are from n=3 (GFP) or from n≥6 separate experiments (GFP-Usp27x and GFP-Usp27xC87A). P-values (t-test) for statistically significant differences are shown. N.s., p>0.05. The addition of QVD inhibited cells from active Caspase-3 positive staining (not shown). A stain for active Bax is shown in Appendix Fig. S7B. B HCC827 NSCLC cells carrying dox-inducible GFP-Usp27xC87A, GFP-Usp27x, or two separate lines established from these GFP-Usp27x-cells where the Bim-locus had been targeted for deletion using CRISPR/Cas9 (Bim-KO), were treated with combinations of dox and the EGFR-inhibitor gefitinib as indicated. Apoptosis was measured after 72h of treatment by staining for active caspase-3, followed by flow cytometric analysis. Data (means/SEM) are from n=≥15 (maternal GFP-Usp27x line) or from n≥5 (for Bim1KO) or from n≥6 (for Bim2KO), or n=4 (for GFP-Usp27xC87A) separate experiments. P-values (t-test) for statistically significant differences are shown. Again, the addition of QVD inhibited cells from active Caspase-3 positive staining (not shown). The inset shows Bim-knock-out-efficiency (n=2).
Single gene encoding Esyt expressed in Drosophila melanogaster.Figure 1(A) Schematic showing the phylogeny of extended synaptotagmin in different organisms: Drosophila melanogaster (Dm), Saccharomyces cerevesiae (Tricalbin), Homo sapiens (Hs), Mus Musculus (Mm), Rattus norvegicus (Rn), Caenorhabditis elegans (Ce), Danio rerio (Dr) and Xenopus tropicalis (Xt). Bold lines - Sub nodes. Numbers on the nodes - Bootstrap value. Colours - Esyt1, Esyt2 and Esyt3 clusters. (B) Schematic of dEsyt-PA protein domain structure aligned with mammalian Esyts and yeast tricalbins (Image generated using IBS, Illustrator for Biological sciences Version 1.0 software http://ibs.biocuckoo.org/)(Liu et al., 2015). Amino acid numbers in each protein are indicated. SMP-SMP domain. C2A, B, C, D, E are the various C2 domains. (C) Quantitative real time PCR analysis showing head enrichment of dEsyt; the X-axis indicates the wild type tissues from which RNA was isolated and Y-axis indicates the dEsyt transcript level expression normalized to the loading control (RP49- Ribosomal protein 49), n=3 replicates (biological). (D) Quantitative real time PCR analysis showing dEsyt transcript level expression in head RNA samples from wild type and sOD. Y-axis indicates the dEsyt transcript level expression normalized to the loading control (RP49- Ribosomal protein 49), n=3 replicates (biological).