EMBO/sd-character-level-ner huggingface dataset:
Figure 3A-C, flow cytometric analysis of ROS levels in Du145TXR or MCF-7ADR cells treated with indicated agents for 12 and 36 hours. (A) Representative images and quantification of ROS in (B) Du145TXR or (C) MCF-7ADR cells are shown. One-way ANOVA was used to analyze statistical differences. Mean with ± SD Data information: Results are representative of three independent experiments. D-E, cell viability of (D) Du145TXR or (E) MCF-7ADR treated with indicated agents for 72 hours. One-way ANOVA was used to analyze statistical differences. Mean with ± SD. Data information: Results are representative of three independent experiments. F-G, colony formation assay and quantification of (F) Du145TXR or (G) MCF-7ADR cells treated with indicated agents. One-way ANOVA was used to analyze statistical differences. Data information: Results are representative of three independent experiments. H-I, cellular vitamin E analysis of medium supernatant in MCs and MPCs of (H) Du145TXR or (I) MCF-7ADR cells treated with 50 μM vitamin E (VE) followed by treatment with or without 25 μM ezetimibe for 12 hours. Student's t test was used to analyze statistical differences. Mean with ± SD. Data information: Results are representative of three independent experiments. J-K, cell viability of (J) Du145TXR or (K) MCF-7ADR cells cultured in normal or serum free medium followed by treatment with indicated agents for 72 hours. One-way ANOVA was used to analyze statistical differences. Mean with ± SD . Data information: Results are representative of three independent experiments. L-M, colony formation assay and quantification of (L) Du145TXR or (M) MCF-7ADR cells treated with indicated agents. One-way ANOVA was used to analyze statistical differences. Data information: Results are representative of three independent experiments.
| Pten is required for receptor editing A | Representative analysis of B220 and IgM surface expression in bone marrow (left) and spleen cells (right) of mice from the indicated genotypes.Figure 1| Representative analysis of B220 and IgM surface expression in bone marrow (left) and spleen cells (right) of mice from the indicated genotypes. Representative flow cytometric analysis of splenocytes from mice of the indicated genotypes (pre-gated on B cells: B220+/CD19+) for surface expression of IgM and the 3-83 idiotype (54.1). Shown data are representative 11 - 35 individual mice per genotype. PCR fragments amplified with specific primers for Vκ and Jκ5 from genomic DNA of purified splenic B cells from Ptenf/f x 3-83ki and Ptenf/f x mb1-cre x 3-83ki mice on the respective backgrounds. Genomic tail DNA from a 3-83ki mouse and DNA from purified splenic B cells of a control (ctrl) mouse were used as controls. PCR for the splicing factor Srp20 served as a loading control. Kb and Kd indicate the respective background of the mice, (H2-Kb: +Ag).
REV3L loss compromises heterochromatin-associated DSB repair.Figure 8A. Asynchronous Rev3l+/+ and Rev3l-/- MEFs were UV-irradiated at the indicated doses and harvested 6 h or 24 h later. Cell lysates were analyzed by immunoblotting with indicated antibodies. B. Asynchronous Rev3l-/- MEFs complemented with empty vector (EV) or REV3L were UV-irradiated at the indicated doses and harvested 6 h or 24 h later. Cell lysates were analyzed by immunoblotting with indicated antibodies. C Asynchronous Rev3l+/+ and Rev3l-/- MEFs were pulse labelled for 15 min using 10 μM EdU then UV-irradiated at 4 J/m2. Cells pre-treated with CSK buffer were fixed and 53BP1, RPA and RAD51 were detected by immunofluorescence. Representative images in non-irradiated cells (NI) and quantification of 53BP1 foci in EdU positive cells at different time points are shown Scale bar= 10 μm The number of cells analyzed is indicated in (n) on each graph. ***p < 0.001 (Kruskal-Wallis test). The presented data are representative of 2 repeats). Bars represent the median ± interquartile range Quantification of the intensity of chromatin-bound RPA2 (D) and RAD51 foci (E) 24 hours after UV-irradiation was performed. The number of cells analyzed is indicated in (n) on each graph. ***p < 0.001 (Kruskal-Wallis test). The presented data are representative of 2 repeats). Bars represent the median ± interquartile range F-H. Asynchronous Rev3l-/- MEFs complemented with empty vector (EV) or REV3L were processed as in (C). Quantification of 53BP1 foci in EdU positive cells at different time points is shown (F). Quantification of the intensity of chromatin-bound RPA2 (G) and RAD51 foci (H) 24 hours after UV-irradiation was performed. The number of cells analyzed is indicated in (n) on each graph. ***p < 0.001 (Kruskal-Wallis test). The presented data are representative of 2 repeats. Bars represent the median ± interquartile range. I. Model that summarizes the results obtained in this study.
An UnaG-based hypoxia sensor faithfully marks areas of HIF-1α stabilization in a mouse model of glioblastoma growth.Figure 3a) UnaG-based hypoxia sensing is applicable in vivo as shown here using the human Gli36 glioblastoma model. 500 Gli36 glioblastoma cells, constitutively expressing mCherry and stably transfected with the HRE-dUnaG sensor construct, were stereotactically transplanted into the cortex of a SCIDmouse. Shown is a 30 µm cryosection of a growing tumor 10 days after transplantation. Tumor cells are distinguished from the surrounding cortex by mCherry expression. Blood vessels were contrasted by immunostaining against PECAM-1. Expression of dUnaG was visualized by its green fluorescence and predominates in areas with reduced vascular density (outlined by white line in the composite panel (bottom right).b) Representative area from the tumor shown in a), which is located outside the viewfield in a) positioned more closely to the tumor border. Also at the tumor border UnaG-expressing cells are preferentially observed at a distance to PECAM-1+ vessels. c) Areas of dUnaG expression correlate well with HIF-1α stabilization. Immunostaining for HIF-1α (cyan) revealed the predominantly nuclear localization of stabilized HIF-1α in cells that were also dUnaG-positive (green).d) Analysis of dUnaG expressing cells in c) for mCherry fluorescence and HIF-1α stabilization. dUnaG-positive cells were classified according to their average fluorescence intensity into background level and above background level expression for mCherry and HIF-1α The threshold was set in either case to channel 70 of 256 intensity channels. Using this classification >60% of UnaG positive cells displayed only background level mCherry fluorescence. On the other hand, >98% of the UnaG expressing cells also expressed HIF-1α , which together suggests that here UnaG acts preferentially as a hypoxia sensor in vivo. e) The dUnaG-based senor was efficiently applied in intravital microscopy. 500 Gli36 glioblastoma cells stably expressing mCherry and harboring the dUnaG-sensor construct were positioned at 200µm depth subcortically in a SCIDmouse and observed 4 days later through a cranial window using multiphoton microscopy. We noted two distinct areas of intense UnaG fluorescence (red lines). Shown is a MIP of a 60 µm z stack. The vasculature was contrasted by intravenous injection of non-functionalized Qdots.f) Depth color coding of the MIP shown in e) revealed the spatial relation between the vasculature, (contrasted by Qdots), areas of increased dUnaG expression (bordered by green lines) and tumor cells expressing residual levels of mCherry. The hypoxic areas were located towards the top of the imaged tissue cube and therefore at different levels compared to the large vessel at the lower left corner (red asterisk) and the smaller vessels with a more axial orientation (white arrows). Scale bars = 100µm
Fig.Figure 6(a) Gene expression analysis of macrophages incubated with the indicated stimuli expressed as fold change over RBCL. Dotted red line indicates 2-fold increase in RNA expression. (b) Western Blot analysis of proIL1β. Relative abundance is the average of 3 independent experiments with different donors. (c) FLICA analysis of activated caspase-1. Results are representative of 2 independent experiments with different donors. (d) Two distinct siRNAs against NLRP3 and an irrelevant control siRNA were utilized to knock down RNA levels by lipofectamine transfection in macrophages that were later incubated with iRBCL, or RBCL as control, with or without XO, before quantification of secreted IL1β. Results are representative of 2 independent experiments with different donors. (e) Diagram of macrophage inflammasome activation by XO and iRBC.
circHIPK3 is essential for KSHV replication(A) qPCR analysis of circHIPK3 levels in scr and circHIPK3 KD stably expressing TREx cells.Figure 3(A) qPCR analysis of circHIPK3 levels in scr and circHIPK3 KD stably expressing TREx cells. GAPDH was used as a housekeeper (n=3). (B) qPCR analysis of HIPK3 levels in scr and circHIPK3 KD stably expressing TREx cells. GAPDH was used as a housekeeper (n=3). Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological. (C) Representative western blot of ORF65 levels in scr and circHIPK3 KD cell lines with GAPDH as a loading control. Densitometry analysis of n=3 western blots. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological. (D) qPCR analysis of ORF57 DNA levels for viral load at 72 hours post induction, with scr and circHIPK3 KD TREx cells with uninduced cells as a control and GAPDH as a housekeeper (n=3). Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological. (E) qPCR analysis of ORF57 in HEK-293T cells for reinfection assay (n=3). Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological. (F) qPCR analysis of levels of miR-29b, miR-30c and miR-27a in scr and circHIPK3 KD cells 0 and 24 hours post-induction. SNORD68 was used as a housekeeper (n=3). Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.
Figure 2Aci2 and Pa were both grown in monoculture in the two-resource environment. The same data is shown in both plots. The left plot shows the average of eight technical replicates on a log scale, while the right plot shows each of the replicates on a linear scale. Overlain on the left plot are growth rate fits of gAci2 = 0.88hr-1 and gPa = 0.67hr-1. Data Information: Data in A were collected at 400nm. Aci2 was grown with alanine and glutamate supplied at 25 different ratios from 1:16 to 16:1 with the total supply kept constant at 0.1%w/ Data Information: Data were collected at 600nm. The population size at which Aci2's diauxic shift occurred is linearly correlated to the alanine supply, indicating that Aci2 initially consumes almost entirely alanine. Data Information: Data were collected at 600nm. Instantaneous growth rates were extracted from the Aci2 monoculture data A variation in Aci2's lag time is clearly visible in this representation. Data Information: Data were collected at 600nm. The same monoculture experiments were performed for Pa. The appearance of Pa having a long initial lag is primarily due to the growth that it needs to accomplish before its population size becomes significant on a linear scale as well as a small optical density artifact Data Information: Data were collected at 600nm. Pa's population size at the onset of its diauxic shift correlates to the supply concentration of whichever resource is supplied in a more limiting amount, indicating coutilization. Data Information: Data were collected at 600nm. Instantaneous growth rates extracted from the Pa monoculture data. Data Information: Data were collected at 600nm. Diauxic lag times were fit from the monoculture data and are plotted as circles. Lag times could not be fit for conditions with too little growth on the remaining glutamate. The lag times used in the modeling are shown as lines through the data.
Figure 5L6 myoblasts were transfected with the genetic cytosolic Ca2+ sensor GCaMP6s. Cells were also transfected with non-targeted siRNA (Control) or siRNA directed at endogenous STIM1 (siSTIM1). Some cells with endogenous STIM1 knockdown were also transfected with full-length wild type (WT) or mutant (S257A, S257E or L251S) STIM1-mRuby3. Representative microscopy images of GCaMP6s signal indicating SOCE in L6 myoblasts, L6 myoblasts were transfected with the genetic cytosolic Ca2+ sensor GCaMP6s. Cells were also transfected with non-targeted siRNA (Control) or siRNA directed at endogenous STIM1 (siSTIM1). Some cells with endogenous STIM1 knockdown were also transfected with full-length wild type (WT) or mutant (S257A, S257E or L251S) STIM1-mRuby3. Ca2+ levels over time (fold of 0 mM Ca2+ + EGTA basal) and (C) quantification of SOCE in in L6 myoblasts incubated in buffer containing 1 or 0 mM Ca2+ and treated with 2 µM thapsigargin during the indicated times.
Figure 2Cells were grown in MHB-ca medium without or with 20 mM glucose as indicated. (A) Bacterial growth was measured by optical density at 600 nm. Data are representative of five independent experiments. Cells were grown in MHB-ca medium without or with 20 mM glucose as indicated. (B) PndhC and PndhF YFP reporter constructs were used for following gene expression and data was normalized by growth. Data are expressed as average ± SEM of three independent experiments. *** Denotes p < 0.001 compared to ndhC expression in WT strain grown in MHB-ca medium via two-way ANOVA with Tukey's posttest.
ILK controls proliferation, VEGFR3 signalling, and VEGFR3-β1 integrin interactions in human LECs.Figure 7Images of adult human LECs after 1 hour of BrdU incorporation and previous transfections with control or ILK siRNA. Scale bars: 50 µm LEC proliferation as determined by the number of BrdU-positive cells normalised to the total number of LECs previously transfected with control siRNA or ILK siRNAs in the presence of VEGF-C Cys156Ser (n = 3 independent transfections per siRNA), *P = 0.032 (control versus ILK-1), *P = 0.005 (control versus ILK-2), *P = 0.0003 (control versus ILK-3) VEGFR3 tyrosine phosphorylation as determined by ELISA of lysates from adult human LECs transfected with control siRNA or ILK siRNAs in the presence of VEGF-C Cys156Ser (n = 4 (control siRNA, ILK-1 siRNA, and ILK-3 siRNA), or n = 8 (ILK-2 siRNA) independent transfections per siRNA), *P = 0.0001 (control versus each siRNA) LSM images of VEGFR3/β1 integrin PLA dots in human LECs transfected with control or ILK siRNA. Scale bars: 10 µm Quantification of VEGFR3/β1 integrin PLA dots per human LEC after transfection with control siRNA or ILK siRNAs (n = 5 independent transfections per siRNA), P = 0.234 (control versus ILK-1), *P = 0.024 (control versus ILK-2), *P = 0.001 (control versus ILK-3)
Figure 8Scheme of experiment based on HSC co-culture with MSCs and colony formation from single cells in differentiation media. Frequency of colonies appearing at day 8 and day 12 from HSCs co-cultured with HO-1+/+ or HO-1-/- MSCs. *p< 0.05, ***p<0.001, Fisher exact test. PCA and cluster analysis of the colonies based on initial flow cytometry gating revealed (C) 4 distinct clusters of colonies, that differ by frequency of (D) CD11b+Gr1-, (E) CD11b+Gr1+ PCA and cluster analysis of the colonies based on initial flow cytometry gating revealed (F) cells lacking expression of analyzed lineage markers (Lin-). Analysis of frequency of colonies derived from HO-1+/+ or from HO-1-/- MSC co-cultures among identified clusters. Enrichment in cluster analyzed by Fisher exact test, **p<0.01, two-tailed unpaired t-test. Sdf1α mRNA levels in BM of HO-1+/+ or HO-1-/- mice after administration of Noggin. Data are shown as mean ± SEM. *p< 0.05, two-tailed unpaired t-test, n = 10/group LT-HSC frequency of LT-HSCs after administration of Noggin to HO-1+/+ or HO-1-/- mice. Data are shown as mean ± SEM. *p< 0.05, **p<0.01, two-tailed unpaired t-test, n = 8-10/group cell cycling status of LT-HSCs after administration of Noggin to HO-1+/+ or HO-1-/- mice.
Figure 4Basal R/A-sensitive OCR measured as in Fig. 2A in the absence and in the presence of a cocktail of inhibitors (BPTES + etomoxir + UK5099); End. Sub., OCR measured with endogenous substrates; Exo. Sub., OCR measured with glutamine (Gln), fatty acids (FA) and glucose (Glc) supplemented in the assay medium. The superimposed bars are means ± SEM of n=3 independent biological replicates (3 technical replicates each); Measurement of dependency and flexibility of the OCR to either of Gln, FA, Glc; the assay was carried out according to the MitoFuel protocol (see Material and Methods); the superimposed bars (whose sum corresponds to the capacity) are means ± SEM of n=3 independent biological replicates (3 technical replicates each); LC-MSMS analysis of the cellular content of free aminoacids; the symbols of aminoacids are indicated in x-axis; the amount expressed as μmol/μg protein is indicated in y-axis. The inset shows the content of ornithine, citrulline and arginine. (D)LC-MSMS analysis of the cellular content of acylcarnitines; the length of the acyl moiety is indicated by the number of carbon atoms in abscissa; bars values are means ± SEM of n=3 independent biological replicates; The inset shows the content of carnitine (C0) and acetylcarnitine (C2).
Figure 1Figure 1a. Scheme depicting preparation of AS CDK12 HCT116 cell line. Gate keeper phenylalanine (F) and glycine (G) are indicated in red and adjacent amino acids in CDK12 active site are shown in black letters (left). ATP and ATP analog 3-MB-PP1 are shown as black objects in wild-type (WT) and AS CDK12 (blue ovals), respectively (right). Figure 1b. Genotyping of AS and WT CDK12 clones. Ethidium bromide-stained agarose gel visualizing PCR products from genomic DNA of AS (AS-PCR) and WT (WT-PCR) CDK12 HCT116 cells and their digest with BslI enzyme (indicated as AS- BslI and WT- BslI). Primer positions and BslI restriction sites are depicted at Expanded View Fig. 1a. Numbers on the left and right indicate DNA marker and DNA fragment sizes, respectively. Figure 1c. Detailed insight into sequencing of genomic DNA from WT and AS CDK12 HCT116 cell lines. The genomic region in WT and AS CDK12 subjected to genome editing is shown in red rectangle, gate keeper amino acids F and G are in red. The full ~500 kb sequence surrounding the edited genomic region is in the Appendix Fig. S1a, b. Figure 1d. Effect of CDK12 inhibition on phosphorylation of the CTD of RNAPII. Western blot analyses of protein levels by the indicated antibodies in AS CDK12 HCT116 cells treated with 5 µM 3-MB-PP1 for indicated times. Long and short exp.= long (4-14 min) and short (10-60 s) exposures, respectively. FUS and tubulin are loading controls. A representative image from 3 replicates is shown. Figure 1e, f. Inhibition of CDK12 in AS CDK12 HCT116 cells results in down-regulation of CDK12-dependent HR genes. Graph shows RT-qPCR analysis of relative levels of mRNAs of described genes in AS CDK12 HCT116 (e) and WT CDK12 HCT116 (f) cells treated for indicated times with 3-MB-PP1. mRNA levels were normalized to HPRT1 mRNA expression and the mRNA levels of untreated control (CTRL) cells were set to 1. n=3 replicates, error bars indicate standard error of the mean (SEM).
Parabulin inhibits Tt-MT growth in vitro.Figure 3(A) Plot showing quantification of the T. thermophila MT growth events in TIRF microscopy MT dynamics reconstitution assays in the presence of the indicated drugs. Error bars represent the mean +/- 95% CI. For each concentration, data is obtained from at least 2 independent experiments. For growth rates and growth lengths, n=41(DMSO), n= 4 (0.04uM parabulin), n= 34 (0.04 uM colchicine). Rates were compared using one-way ANOVA with Tukey's multiple comparison test; DMSO vs parabulin P<0001 (****); DMSO vs colchicine p = 0.40 (n.s., not significant). (B) T. thermophila MT growth rates in the presence of the indicated drugs (40 μM). Error bars represent the mean +/- 95% CI (n=4). Rates were compared using one-way ANOVA with Tukey's multiple comparison test; DMSO vs parabulin P = 0.25; DMSO vs colchicine P = 0.20 (n.s., not significant). (C) T. thermophila steady-state MT length in the presence of the indicated drugs (40 μM). Error bars represent the mean +/- 95% CI. For each concentration, data is obtained from at least 2 independent experiments. For growth rates and growth lengths, n=41(DMSO), n= 4 (0.04uM parabulin), n= 34 (0.04 uM colchicine). Rates were compared using one-way ANOVA with Tukey's multiple comparison test; DMSO vs parabulin P = 0.15 (n.s., not significant); DMSO vs colchicine P<0001 (****). (D) Representative kymographs obtained by TIRF microscopy showing the effect of parabulin and colchicine on dynamic Tt-MTs.
Figure 6Mice were injected with control (Ctrl) or STING-KO MC38 cells and treated with PBS or 5-FU. Tumors were harvested 2 weeks after cancer cell injection and intratumoral immune cells were examined by flow cytometry.
Figure 4A TEM image showing fibril morphology of BSP. B-D BSP amyloid aggregation in vitro is pH-dependent in 10 mM EDTA (B), protein concentration-dependent in 10 mM EDTA and at pH 4.5 (C) and strongly inhibited by millimolar Ca2+ at various protein concentrations (D). E-H TEM images showing fibril morphology of BSP12 (E), BSP123 (F), BSP3 (G), BSP45 (H). I-J Amyloid aggregation by BSP subconstructs. BSP12 exhibits more prominent aggregation propensity than BSP3 and BSP45 in 10 mM EDTA and at pH 4.5 (I). The conjunction of module 3 with modules 1 and 2 promotes aggregation (J). K Millimolar Ca2+ inhibits aggregation of BSP and its subconstructs. The inhibition efficiency is defined as the ratio of maximal fluorescence fold change at the end stage of measurement achieved in 10 mM EDTA to that in 10 mM Ca2+. Each experiment was repeated three times, data shown as mean ± SD. L Structural organization of BSP chimeric subconstructs. S (red), signal peptide; module 1 (magenta); module 2 (orange); module 3 (green); module 4 (blue); module 5 (cyan); R-ClfA (yellow), clumping factor R domain; W (purple), cell wall anchor. M Bacterial clumping and biofilm formation of S. aureus V329 and the corresponding S. aureus Δbap strain. For bacterial clumping (bottom), bacteria were cultured overnight with agitation (220 rpm) in LB-glu (pH<5). For biofilm formation (top), bacteria were cultured overnight at 37°C in TSB-glu (pH<5) using sterile 96-well polystyrene microtiter TC-plates under static conditions, assays were repeated for each bacteria. N Biofilm formation of S. aureus Δbap mutants expressing BSP chimeric subconstructs or carrying empty vector (EV) in the absence (cyan) or presence (red) of Ca2+ assayed with crystal violet staining. The EV is a control for S. aureus Δbap bacteria. Each experiment was repeated three times, data shown as mean ± SD.