Journal: Heliyon

Article Title: Neutrophil extracellular traps (NETs) reduce the diffusion of doxorubicin which may attenuate its ability to induce apoptosis of ovarian cancer cells

doi: 10.1016/j.heliyon.2022.e09730

Figure Lengend Snippet: The effects of NETs on the diffusion of doxorubicin (DOX). Neutrophils (1 × 10 7 /4 ml) stimulated with PMA (A) or LPS (B) were placed in the bottom chamber as described in Figure 2 legend and DOX added at a final concentration of 10 μM and incubated for another 30 min. In some wells, DNAse I was added at a final concentration of 1000 u/ml at 30 min before the addition of DOX. Then, culture inserts containing 2 mL of HBSS were placed in the bottom chambers and auto fluorescence intensities of DOX in the upper chamber measured at the indicated time points. In each set of experiments, the relative ratios of fluorescein intensities were calculated against the value of the samples measured at 1 h after incubation in control wells which did not contain NETs and DNAse I. Data are shown as mean ± standard deviation in 3 (PMA) and 3 (LPS) different experiments. ∗: p < 0.05, ∗∗: p < 0.01.

Article Snippet: SYTOX green nucleic acid stain (≥99%) and DNAse I (≥90%) were purchased from Thermo Fisher Scientific (Waltham, MA) and Worthington Biochemical Co. (Lakewood, NJ), respectively.

Techniques: Diffusion-based Assay, Concentration Assay, Incubation, Fluorescence, Standard Deviation

Journal: Heliyon

Article Title: Neutrophil extracellular traps (NETs) reduce the diffusion of doxorubicin which may attenuate its ability to induce apoptosis of ovarian cancer cells

doi: 10.1016/j.heliyon.2022.e09730

Figure Lengend Snippet: Peritoneal tumors of SKOV-3 were induced as described in Materials and Methods, and similar sized tumors (approximately 3∼5 mm in diameters) were soaked in 50 mM DOX diluted in 4 ml of HBSS buffer with unstimulated (A) or PMA-stimulated (B) neutrophils in 15 ml tube. In (C), DNase I (1000 u/ml) was added with PMA-stimulated neutrophils at the start of the experiment. After 3 h, the tumors were taken out, fixed with dry-iced acetone and 10-μM cryostat sections of post-fixed frozen samples were created. After the counterstaining the nuclei with DAPI, the infiltration of DOX from the tumor surface was evaluated with the detection of autofluorescence under fluorescence microscopy (BZ8000; Keyence, Osaka, Japan). Figures show the merged images for DOX (red) and DAPI (Blue).

Article Snippet: SYTOX green nucleic acid stain (≥99%) and DNAse I (≥90%) were purchased from Thermo Fisher Scientific (Waltham, MA) and Worthington Biochemical Co. (Lakewood, NJ), respectively.

Techniques: Fluorescence, Microscopy

Journal: Heliyon

Article Title: Neutrophil extracellular traps (NETs) reduce the diffusion of doxorubicin which may attenuate its ability to induce apoptosis of ovarian cancer cells

doi: 10.1016/j.heliyon.2022.e09730

Figure Lengend Snippet: KOC-2S or SKOV3 cells were embedded in collagen gel droplets as described in Materials and Methods and cultured in 2 ml media containing 15 μM doxorubicin (DOX) with neutrophils (1 × 10 7 ) and/or DNAse I (1000U/ml). NETs (-) and (+) show the data in the presence of unstimulated and LPS-stimulated neutrophils, respectively. After 12 h, apoptotic cells were examined with FACSCalibur. (A) Representative FACS Profiles of KOC-2S (B) Data are shown as mean ± standard deviation in triplicate from one of 4 (KOC-2S) and 3 (SCOV-3) different experiments. ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.001.

Article Snippet: SYTOX green nucleic acid stain (≥99%) and DNAse I (≥90%) were purchased from Thermo Fisher Scientific (Waltham, MA) and Worthington Biochemical Co. (Lakewood, NJ), respectively.

Techniques: Cell Culture, Standard Deviation

Journal: PLoS ONE

Article Title: Nucleolus association of chromosomal domains is largely maintained in cellular senescence despite massive nuclear reorganisation

doi: 10.1371/journal.pone.0178821

Figure Lengend Snippet: ( A ) Distribution of NADs along human autosomes. NADs are indicated by red rectangles over the ideograms of the chromosomes. Note that the p-arms of the five acrocentric chromosomes (13, 14, 15, 21 and 22), centromeres and some pericentromeric regions were not analysed because they are not present in the current human genome assembly. ( B ) Histogram of NAD sizes. Median = 361kb, a total of 1,646 NADs were identified. ( C ) 3D immuno-FISH analysis of NAD and inter-NAD regions (iNADs) in IMR90 cells. Nucleolus association of a chromosomal domain is illustrated by showing the Z-projection of an IMR90 nucleus on the left and the corresponding single light optical sections with the associated and non-associated allele on the right. BAC hybridization signals are shown in green, nucleolar staining in red and DAPI counterstain in blue (scale bar: 1.6 μm). ( D ) Hybridization signals (percentage of nucleolus-associated alleles) are plotted against the according microarray signals (average log2-fold difference of the nucleolar signal over the background). Red and grey circles indicate genomic regions that reside in NADs and iNADs, respectively (see for further details). The positions of the BAC clones used in 3D immuno-FISH experiments to monitor NADs and iNADs are shown also in ( A ) by red and grey circles, respectively.

Article Snippet: Human IMR90 embryonic fibroblasts were obtained from Coriell Repositories (Cat. No. I90-79) and cultivated in DMEM (Gibco Cat. No. 21885–025 supplemented with 10% v/v Foetal Calf Serum, 100 U/mL Penicillin, 100 μg/mL Streptomycin) at 37°C in humidified, 5% CO 2 atmosphere and regularly tested for mycoplasma contamination.

Techniques: Hybridization, Staining, Microarray, Clone Assay

Journal: PLoS ONE

Article Title: Nucleolus association of chromosomal domains is largely maintained in cellular senescence despite massive nuclear reorganisation

doi: 10.1371/journal.pone.0178821

Figure Lengend Snippet: ( A ) Venn diagrams and Jaccard coefficients show the extent of overlap between NADs and LADs. LAD1: LADs of Tig3 cells , LAD2 and LAD3: LADs of IMR90 cells [ , ]. ( B ) Bar graphs show Gencode v19 and UniProt gene frequencies in NADs (red), iNADs (grey), LADs (black), and iLADs (white) based on UCSC Table Browser data. ( C ) RefSeq gene (ZNF, OR and DEF indicate zinc finger, olfactory receptor and defensin gene families, respectively) frequencies in NADs, iNADs, LADs, and iLADs. ( D ) Non-coding RNA gene (‘RNA genes’) and ( E ) repeat frequencies in NADs, iNADs, LADs, and iLADs. The SINE repeat bars are divided with a horizontal line into MIR (bottom) and Alu (top) sub-groups.

Article Snippet: Human IMR90 embryonic fibroblasts were obtained from Coriell Repositories (Cat. No. I90-79) and cultivated in DMEM (Gibco Cat. No. 21885–025 supplemented with 10% v/v Foetal Calf Serum, 100 U/mL Penicillin, 100 μg/mL Streptomycin) at 37°C in humidified, 5% CO 2 atmosphere and regularly tested for mycoplasma contamination.

Techniques:

Journal: PLoS ONE

Article Title: Nucleolus association of chromosomal domains is largely maintained in cellular senescence despite massive nuclear reorganisation

doi: 10.1371/journal.pone.0178821

Figure Lengend Snippet: ( A ) Bar graph of nucleolus number in young, proliferating (‘Y’) and senescent (‘S’) IMR90 cells. Proliferating cells have 3.0±1.2 and senescent cells 1.7±1.1 nucleoli per nucleus. Z projections of mid-sections of representative confocal microscopy images are shown on the top. Nucleolar staining is shown in red and DAPI counterstain in blue (scale bars: 1.6 μm). ( B ) Maps of NADs on chromosome 5 from young and senescent cells. Genomic regions associated with nucleoli only in young (¬S) or senescent (¬Y) cells are shown also as individual tracks. ( C ) Y-only and S-only NADs are enriched in protein-coding genes compared to all NADs and the genome. RefSeq gene data were obtained from the UCSC Table Browser. ( D ) Boxplots show positive correlation of senescence-related loss of nucleolus association and gene activation. Global gene expression changes (log2 fold change in senescent versus young cells) in constitutive (Y∧S), Y-only and S-only NAD genes are shown. The notches are defined as +/-1.58*IQR/sqrt(n) and represent the 95% confidence interval for each median. Group means are significantly different for all comparisons (p-value < 0.05, Tukey HSD). ( E ) Stacked columns show that the association frequency of five selected genomic regions is similar in young and senescent cells. Nucleolus-association data were collected from 50 cells for each category. BAC clones 1 to 5: RP11-44B13, RP11-173M10, RP11-828F4, RP11-125O21, RP11-81M8.

Article Snippet: Human IMR90 embryonic fibroblasts were obtained from Coriell Repositories (Cat. No. I90-79) and cultivated in DMEM (Gibco Cat. No. 21885–025 supplemented with 10% v/v Foetal Calf Serum, 100 U/mL Penicillin, 100 μg/mL Streptomycin) at 37°C in humidified, 5% CO 2 atmosphere and regularly tested for mycoplasma contamination.

Techniques: Confocal Microscopy, Staining, Activation Assay, Expressing, Clone Assay

Journal: PLoS ONE

Article Title: Nucleolus association of chromosomal domains is largely maintained in cellular senescence despite massive nuclear reorganisation

doi: 10.1371/journal.pone.0178821

Figure Lengend Snippet: ( A ) Semi-quantitative immunoblots show severely decreased H3K9me3 and Lamin B1 levels, less strongly decreased H3 levels and no detectable alterations in Lamin A/C, tubulin and GAPDH levels in senescence. The same amounts of whole cell extracts of young and senescent cells were loaded as serial two-fold dilutions on SDS-PA gels and analysed on immunoblots (see the entire dataset from three independent experiments in ). ( B ) H3K9me3 is accumulated at spatially compact satellite repeat clusters. 3D immuno-FISH shows strong co-localization of H3K9me3 and HSATII staining. Mid-section of a representative confocal microscopy image is shown. HSATII FISH signals are in red, DAPI counterstain in blue, and H3K9me3 immunofluorescence signals are in green (scale bar: 1.6 μm). ( C ) Quantitative immunofluorescence analysis of H3K9me3 distribution. The areas of interest are illustrated on a light optical section of a representative confocal microscopy image. The lamina- and nucleolus-associated areas label 240 nm distances from the edges of the DAPI and nucleolus staining, respectively. ( D ) Bee swarm plots of relative fluorescence intensities show senescence-dependent small decrease in H3K9me3 levels at the nuclear periphery and strong reduction at the perinucleolar space. Proliferating and senescent IMR90 cells were stained for H3K9me3 and the relative immunofluorescence intensities were measured at the nuclear periphery (lamina) and at the perinucleolar space (No). Values measured in proliferating cells (‘Y’) are shown in red, values measured in senescent cells (‘S’) are shown in blue. Results from individual cells are illustrated as single data points (n Y = 88, n S = 88). A solid line indicates the median, and thin lines the upper and lower quartile. Median: Y.lamina = 0.095, S.lamina = 0.084; Y.No = 0.052, S.No = 0.022. ( E ) Bee swarm plots indicate more heterogeneous H3K9me3 staining in the nucleus and at the nuclear periphery of senescent cells, but no change in the perinucleolar space. The heterogeneity of staining was calculated as coefficient of variation (C.V. = standard deviation/mean of fluorescence intensity) for the total nucleus (Nu), the nuclear periphery (lamina) and the perinucleolar space (No). Plot labels are as in ( D ). Median: Y.Nu = 0.573, S.Nu = 0.677; Y.lamina = 0.632, S.lamina = 0.709; Y.No = 0.576, S.No = 0.555, n Y = 88, n S = 88. ( F ) Bee swarm plots illustrate robust rearrangement of the most heterochromatic regions in the perinucleolar space. The distribution of the 10% brightest pixels was quantified at the nuclear periphery (lamina) and the perinucleolar space (No). Ratios were calculated compared to the whole nucleus. Plot labels are as in ( D ). Median: Y.lamina = 0.157, S.lamina = 0.137; Y.No = 0.056, S.No = 0.013. n Y = 88, n S = 88.

Article Snippet: Human IMR90 embryonic fibroblasts were obtained from Coriell Repositories (Cat. No. I90-79) and cultivated in DMEM (Gibco Cat. No. 21885–025 supplemented with 10% v/v Foetal Calf Serum, 100 U/mL Penicillin, 100 μg/mL Streptomycin) at 37°C in humidified, 5% CO 2 atmosphere and regularly tested for mycoplasma contamination.

Techniques: Western Blot, Staining, Confocal Microscopy, Immunofluorescence, Fluorescence, Standard Deviation