Journal: Cells

Article Title: Transcriptomic Response to 1,25-Dihydroxyvitamin D in Human Fibroblasts with or without a Functional Vitamin D Receptor (VDR): Novel Target Genes and Insights into VDR Basal Transcriptional Activity

doi: 10.3390/cells8040318

Figure Lengend Snippet: Top-ranking up- and down- regulated 1,25D-activated VDR target genes in human fibroblasts (CO).

Article Snippet: First-strand cDNA was generated using SuperScript III reverse transcriptase (Thermo Fisher) and quantitative PCR performed in a StepOnePlus Real-Time PCR System (Thermo Fisher) using the following inventoried TaqMan® assays (Thermo Fisher): CYP24A1 Hs00167999 _m1, TGFB2 Hs00234244_m1, RASSF2 Hs00248129_m1, CILP Hs00173647_m1, STC1 Hs00174970_m1, SPP1 Hs00959010_m1, GALNT12 Hs00226436_m1, HBEGF Hs00181813_m1, EFNB2 Hs00187950_m1, and EGR1 Hs00152928_m1.

Techniques:

Journal: Cells

Article Title: Transcriptomic Response to 1,25-Dihydroxyvitamin D in Human Fibroblasts with or without a Functional Vitamin D Receptor (VDR): Novel Target Genes and Insights into VDR Basal Transcriptional Activity

doi: 10.3390/cells8040318

Figure Lengend Snippet: Quantitative RT-PCR validation of differential gene expression findings.

Article Snippet: First-strand cDNA was generated using SuperScript III reverse transcriptase (Thermo Fisher) and quantitative PCR performed in a StepOnePlus Real-Time PCR System (Thermo Fisher) using the following inventoried TaqMan® assays (Thermo Fisher): CYP24A1 Hs00167999 _m1, TGFB2 Hs00234244_m1, RASSF2 Hs00248129_m1, CILP Hs00173647_m1, STC1 Hs00174970_m1, SPP1 Hs00959010_m1, GALNT12 Hs00226436_m1, HBEGF Hs00181813_m1, EFNB2 Hs00187950_m1, and EGR1 Hs00152928_m1.

Techniques: Quantitative RT-PCR, Biomarker Discovery, Gene Expression, Quantitative Proteomics, Microarray

Journal:

Article Title: DNA/DNA hybridization to microarrays reveals gene-specific differences between closely related microbial genomes

doi: 10.1073/pnas.171178898

Figure Lengend Snippet: Strains used in DNA:DNA microarray hybridization analysis

Article Snippet: 184 ATCC 8073 Butter AF387349 * AF387354 * S. woodyi MS32 ATCC 51908, TS Persian Gulf seawater AF003548 AF014944 Environmental isolate W3 18-1 Pacific Ocean marine sediments (630 m, 5–6 cm in core) AF387350 * AF387355 * Environmental isolate PV-4 Pacific Ocean seawater, Naha Vents, Hawaii AF387348 * AF387352 * E. coli K-12 ATCC 10798 Human feces AE000452 AE000447 Open in a separate window Type strains (TS) indicated.

Techniques: Microarray, Hybridization

Journal: PLoS ONE

Article Title: Mesenchymal stromal cell-derived extracellular vesicles reduce lung inflammation and damage in nonclinical acute lung injury: Implications for COVID-19

doi: 10.1371/journal.pone.0259732

Figure Lengend Snippet: (A) Antibody microarray particle analysis of tetraspanin proteins. (B) Electrochemiluminescent multiplexed immunoassay detection of tetraspanin proteins. (C) Western blot analysis of MSC-EV proteins flotillin-1, Annexin-2, Syntenin-1, MHC-I and contaminating proteins MHC-II and calreticulin. (D) Immunogold cryo-transmission electron microscopy imaging for CD63 and phosphatidylserine. Atomic force microscopy for MSC-EV morphology and size. (E) Nanoparticle tracking for MSC-EV size and concentration. (F) MSC-EV particle density correlated to phospholipid content. Error bars are SEM of the mean. *p ≤0.05 vs CD63. Scale bar denotes 100 nm.

Article Snippet: Lysates were volume loaded and separated on a 12% Bolt Bis-Tris Gel and probed using Abcam antibodies against flotillin-1 (ab133497 at 1:10,000 dilution), Annexin-2 (ab41803 at 1:1000 dilution), Syntenin-1 (ab19903 at 1:1000 dilution), MHC-I (ab110645 at 1:1000 dilution), MHC-II (157210 at 1:10000 dilution), and Calreticulin (ab92516 at 1:1000 dilution).

Techniques: Microarray, Western Blot, Transmission Assay, Electron Microscopy, Imaging, Microscopy, Concentration Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Angiogenic patterning by STEEL, an endothelial-enriched long noncoding RNA

doi: 10.1073/pnas.1715182115

Figure Lengend Snippet: Gene regulation in endothelial cells by STEEL RNA. (A) RT-qPCR of STEEL knockdown by two independent siRNA sequences. (B) Volcano plot representing mRNAs regulated by STEEL knockdown in HUVECs and HMVECs from microarray. Red indicates differentially regulated genes. (C–F) RT-qPCR of HOXD1 and HOXD3 mRNA with STEEL knockdown. (D) Western blot of HOXD1 and HOXD3 protein with STEEL knockdown. (E) RT-qPCR of KLF2 and eNOS mRNA with STEEL knockdown. (F) Western blot of KLF2 and eNOS protein with STEEL knockdown. (G) RT-qPCR of KLF2 and eNOS hnRNA (pre-mRNA) with STEEL knockdown. (H) RT-qPCR of RNA Pol II ChIP at KLF2 and eNOS promoters. (I) Promoter luciferase-reporter assays with STEEL knockdown. (J) CHART-qPCR with STEEL knockdown. (K and L) ChIP-qPCR of the core histone, histone H3, and the activating epigenetic mark histone 3 lysine 4 trimethyl (H3K4me3) at the KLF2 and eNOS promoters after STEEL siRNA knockdown in HUVECs. *P < 0.05. Error bars represent SEM.

Article Snippet: Custom lncRNA microarrays on Arraystar Human lncRNA Microarray (Nimblegen 12 × 135 K array platform, 23,155 lncRNAs, 19,484 protein-coding genes) and Human lncRNA Microarray V2.0 (Agilent 8 × 60 K array platform, 33,045 lncRNA probes, 30,215 protein-coding mRNA probes) were used for cell specificity and STEEL knockdown experiments, respectively.

Techniques: Quantitative RT-PCR, Microarray, Western Blot, Luciferase

Journal: Nucleic Acids Research

Article Title: Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells

doi: 10.1093/nar/gkt504

Figure Lengend Snippet: Genome-wide p53 binding after various treatments. ( A ) Representative western blot of p53 levels in U2OS cytoplasmic, nuclear or whole-cell extracts from untreated (NT) cells, or after 24 h treatment with 0.1% DMSO, 0.6 µg/ml DXR or 10 µM Nutlin. Lamin B and actin Hsp90, lamin B and actin are loading controls. ( B ) Number of p53 binding sites identified in U2OS 24 h after treatment with DMSO, DXR or Nutlin. ( C ) UCSC Genome Browser shots summarizing p53 ChIP-Seq and Input control data for known p53 target genes after various treatments. The Y axis corresponds to the number of reads. The same scale is used for all examples presented. ( D ) Genomic distribution of p53 binding sites relative to genic regions after the various treatments: TSS, 5 kb upstream through the first intron; Intragenic, second exon through 3′UTR; and Intergenic, everything else.

Article Snippet: Chromatin was sheared to 100–300 bp using a Bioruptor sonicator (Diagenode, Denville, NJ) for 30 min at high power, 30 s ON, 30 s OFF. p53 (DO-1) antibody (12 μg) or normal mouse immunoglobulin G (IgG) (3 μg) (Santa Cruz) was bound to 200 μl Dynabeads Protein G magnetic beads (Invitrogen) by incubation at 4°C in 5 mg/ml of bovine serum albumin for 6 h. Five percent of the chromatin was saved as input.

Techniques: Genome Wide, Binding Assay, Western Blot, ChIP-sequencing, Control

Journal: Nucleic Acids Research

Article Title: Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells

doi: 10.1093/nar/gkt504

Figure Lengend Snippet: Motif analysis and distribution of p53-binding sites. ( A ) Consensus p53-binding motifs identified from the top 1000 p53-binding sites for each treatment. ( B ) Characterization of p53-binding sites based on the type of sequence bound: zero spacer, consensus motif containing two decamers with no spacer sequence as shown in (A); 1–15 spacer, two decamers with a spacer sequence of length 1–15 nt; half site, one decamer; no motif, no evidence of zero spacer, 1–15 spacer, or a half site. ( C ) Frequency of p53-binding sites containing zero spacer (red), 1–15 spacer (blue), and half sites (green). ( D ) Box-plot depicting distribution of ChIP-over-Input fold enrichment for p53-binding sites containing zero spacer, 1–15 spacer, half site and no motif. Asterisk indicates statistically significant ( P < 10 −7 ).

Article Snippet: Chromatin was sheared to 100–300 bp using a Bioruptor sonicator (Diagenode, Denville, NJ) for 30 min at high power, 30 s ON, 30 s OFF. p53 (DO-1) antibody (12 μg) or normal mouse immunoglobulin G (IgG) (3 μg) (Santa Cruz) was bound to 200 μl Dynabeads Protein G magnetic beads (Invitrogen) by incubation at 4°C in 5 mg/ml of bovine serum albumin for 6 h. Five percent of the chromatin was saved as input.

Techniques: Binding Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells

doi: 10.1093/nar/gkt504

Figure Lengend Snippet: Gene expression analysis following various treatments. ( A ) Number of genes with altered expression following DXR or Nutlin treatment as compared with the control NT or DMSO controls, respectively. ( B ) Heat map showing gene expression changes based on microarray analysis of cells after 24 h of DXR, DMSO or Nutlin treatment. DXR and DMSO changes were assessed in relation to NT, and Nutlin changes were assessed in relation to DMSO. The KEGG/GO terms shown are non-redundant terms manually selected among the most significantly enriched categories for each of the bracketed clusters. The full lists of terms are available in Supplementary Data Set 5 . ( C ) KEGG pathways and GO biological processes enriched among all the differentially expressed genes in (A) after various treatments. ( D ) Breakdown of expression changes of genes with a p53-binding site near the TSS or ( E ) in the intragenic region. ( F ) Number of genes that were bound by p53 near a TSS and were differentially expressed after DXR or Nutlin treatment.

Article Snippet: Chromatin was sheared to 100–300 bp using a Bioruptor sonicator (Diagenode, Denville, NJ) for 30 min at high power, 30 s ON, 30 s OFF. p53 (DO-1) antibody (12 μg) or normal mouse immunoglobulin G (IgG) (3 μg) (Santa Cruz) was bound to 200 μl Dynabeads Protein G magnetic beads (Invitrogen) by incubation at 4°C in 5 mg/ml of bovine serum albumin for 6 h. Five percent of the chromatin was saved as input.

Techniques: Gene Expression, Expressing, Control, Microarray, Binding Assay

Journal: Nucleic Acids Research

Article Title: Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells

doi: 10.1093/nar/gkt504

Figure Lengend Snippet: Correlation between p53 binding, changes in gene expression and histone modifications H3K4me3 and H3K27me3 at 24 h after DXR, Nutlin or DMSO treatments. The heat map represents fold changes in mRNA levels quantified using qRT-PCR, as well as fold changes in p53, H3K4me3 and H3K27me3 occupancy quantified using ChIP-qPCR (see also Supplementary Figures S1D and S2A–C ). Presented are results for 36 genes that were bound by p53 and two genes ( GAPDH and MYOD1 ) that were not bound by p53. Genes are clustered into groups depending on whether the p53 target site contains a consensus p53-binding motif containing two decamers with no spacer (zero spacer), two decamers with a spacer sequence (spacers), or one decamer (half site) or no motif. Fold changes for gene expression were normalized to β-2-microglobulin, whereas p53, H3K4me3 and H3K27me3 ChIP-qPCRs were normalized to the negative controls GAPDH, MYOD1 and GAPDH, respectively. ( A ) DXR/NT, ( B ) Nutlin/DMSO and ( C ) DMSO/NT.

Article Snippet: Chromatin was sheared to 100–300 bp using a Bioruptor sonicator (Diagenode, Denville, NJ) for 30 min at high power, 30 s ON, 30 s OFF. p53 (DO-1) antibody (12 μg) or normal mouse immunoglobulin G (IgG) (3 μg) (Santa Cruz) was bound to 200 μl Dynabeads Protein G magnetic beads (Invitrogen) by incubation at 4°C in 5 mg/ml of bovine serum albumin for 6 h. Five percent of the chromatin was saved as input.

Techniques: Binding Assay, Gene Expression, Quantitative RT-PCR, ChIP-qPCR, Sequencing

Journal: Nucleic Acids Research

Article Title: Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells

doi: 10.1093/nar/gkt504

Figure Lengend Snippet: Validation of new p53 direct target genes. Real-time PCR validation of newly identified putative p53 targets in U2OS cells using shRNA-mediated silencing of p53 expression. Parental U2OS cells treated with ( A ) no shRNA, ( B ) scramble (control) shRNA, ( C ) p53shRNA 3755 or ( D ) p53shRNA 3756. mRNA levels were quantified in U2OS cells 24 h after NT, DMSO, DXR or Nutlin treatment using qRT-PCR and normalized to GAPDH . mRNA levels are shown as fold change after treatment relative to the NT control. Expression of the CDKN1A gene was used as a positive control. Shown are averages of three independent experiments. The dashed line corresponds to a 2-fold change in expression. Error bars represent standard deviation (SD).

Article Snippet: Chromatin was sheared to 100–300 bp using a Bioruptor sonicator (Diagenode, Denville, NJ) for 30 min at high power, 30 s ON, 30 s OFF. p53 (DO-1) antibody (12 μg) or normal mouse immunoglobulin G (IgG) (3 μg) (Santa Cruz) was bound to 200 μl Dynabeads Protein G magnetic beads (Invitrogen) by incubation at 4°C in 5 mg/ml of bovine serum albumin for 6 h. Five percent of the chromatin was saved as input.

Techniques: Biomarker Discovery, Real-time Polymerase Chain Reaction, shRNA, Expressing, Control, Quantitative RT-PCR, Positive Control, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells

doi: 10.1093/nar/gkt504

Figure Lengend Snippet: Proposed models for p53-DNA interaction at half-sites (1 decamer) and full sites (two decamers) with and without a spacer. The minimal p53-binding unit is a half site composed of the consensus decamer RRRCWWGYYY (except for the NT control) based on binding to half sites (decamers) and full sites (two decamers) with a spacer. The binding models assume that p53 exists as a dimer and tetramers are formed following p53 dimer binding to a target sequence. p53-bound half sites as well as full sites containing a spacer are much less frequent across the genome than full sites with zero spacer. ( A ) Efficient tetramer binding at full sites with zero spacer. Strong interactions between dimers and target sequences can occur when the dimers bind to a pair of decamers with no spacer. The dark grey ovals indicates possible changes in p53 (such as configuration) that enable the strong binding. ( B ) Binding to a half site, which is much less efficient than to a full site with no spacer, could be due to weaker interactions of the decamer sequence with just one p53 dimer or with a dimer of dimers, where one dimer makes contact with the decamer sequence, whereas the second dimer interacts non-specifically with nearby DNA. ( C ) Binding to a full site with a spacer is proposed to occur through p53-decamer interactions that are described for the half sites in (B) with the additional possibility of weak interactions between the two bound p53 dimers (lower left panel).

Article Snippet: Chromatin was sheared to 100–300 bp using a Bioruptor sonicator (Diagenode, Denville, NJ) for 30 min at high power, 30 s ON, 30 s OFF. p53 (DO-1) antibody (12 μg) or normal mouse immunoglobulin G (IgG) (3 μg) (Santa Cruz) was bound to 200 μl Dynabeads Protein G magnetic beads (Invitrogen) by incubation at 4°C in 5 mg/ml of bovine serum albumin for 6 h. Five percent of the chromatin was saved as input.

Techniques: Binding Assay, Control, Sequencing