Journal: Cell Reports

Article Title: Persistence of RNA transcription during DNA replication delays duplication of transcription start sites until G2/M

doi: 10.1016/j.celrep.2021.108759

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-NELF-A , Santa Cruz Biotechnoloy , Cat#sc-365004; RRID: AB_10708864.

Techniques: Recombinant, Staining, Transfection, Random Primed, DNA Labeling, Imaging, DNA Library Preparation, Library Quantification, Western Blot, Reverse Transcription, Generated, Software

Journal: Frontiers in Physiology

Article Title: Wolf-Hirschhorn Syndrome-Associated Genes Are Enriched in Motile Neural Crest Cells and Affect Craniofacial Development in Xenopus laevis

doi: 10.3389/fphys.2019.00431

Figure Lengend Snippet: WHS is typically caused by heterozygous microdeletion of numerous genes within 4p16.3. A segment of this region is illustrated here. A microdeletion that spans at least WHSC1, WHSC2 , and LETM1 is currently assumed to be necessary for full WHS diagnostic presentation; children affected by the disorder often possess larger deletions that extend further telomeric and impact additional genes, such as TACC3 .

Article Snippet: Blotting for Whsc2 was carried out using mouse monoclonal antibody to Whsc2 (Abcam, ab75359, dilution 1:3,000).

Techniques: Diagnostic Assay

Journal: Frontiers in Physiology

Article Title: Wolf-Hirschhorn Syndrome-Associated Genes Are Enriched in Motile Neural Crest Cells and Affect Craniofacial Development in Xenopus laevis

doi: 10.3389/fphys.2019.00431

Figure Lengend Snippet: WHS related genes are expressed in the migrating neural crest cells during embryonic development. (A,F) Lateral views of whole mount in situ hybridizations for twist , a CNC-enriched transcription factor. Arrows indicate the pharyngeal arches (PA). (B–E,G–J) In situ hybridizations for whsc1 , whsc2 , letm1 , and tacc3 demonstrate enrichment in CNCs that occupy the PAs ( n = 20 per probe, per timepoint). Scalebar is 250 μm.

Article Snippet: Blotting for Whsc2 was carried out using mouse monoclonal antibody to Whsc2 (Abcam, ab75359, dilution 1:3,000).

Techniques: In Situ

Journal: Frontiers in Physiology

Article Title: Wolf-Hirschhorn Syndrome-Associated Genes Are Enriched in Motile Neural Crest Cells and Affect Craniofacial Development in Xenopus laevis

doi: 10.3389/fphys.2019.00431

Figure Lengend Snippet: WHS related gene depletion affects craniofacial morphology. (A–E) Frontal views of 3dpf embryos (st. 40) following WHS gene single KD. (F–I) Measurements for facial width, height, midface area, and midface angle. A significant 6.54% increase in facial width and 11.43% increase in midface area were observed for Whsc1 KD. Whsc2 KD caused a 12.01% reduction in facial width and a 6.79% reduction in midface area. Letm1 KD caused a 10.33% decrease in facial width and a 8.49% decrease in midface area. Tacc3 KD caused a 21.27% decrease in facial width and a 16.33% decrease in midface area, and an 8.27% decrease in midface angle. Significance determined using a student’s unpaired t -test. (Embryos quantified: Control = 137, Whsc1 KD = 100, Whsc2 KD = 185, Letm1 KD = 115, Tacc3 KD = 79.) ∗∗∗∗ P < 0.0001, ∗∗∗ P < 0.001, ∗∗ P < 0.01, n.s., not significant. Scalebar = 250 μm.

Article Snippet: Blotting for Whsc2 was carried out using mouse monoclonal antibody to Whsc2 (Abcam, ab75359, dilution 1:3,000).

Techniques: Control

Journal: Frontiers in Physiology

Article Title: Wolf-Hirschhorn Syndrome-Associated Genes Are Enriched in Motile Neural Crest Cells and Affect Craniofacial Development in Xenopus laevis

doi: 10.3389/fphys.2019.00431

Figure Lengend Snippet: Knockdown of Whsc2 and Tacc3 impact cartilage morphology. (A–E) Ventral view of 6dpf embryos following single WHS-assoc. gene KD, stained with Alcian Blue to label cartilage elements. (F–I) Measurements of the average area and width of the ceratohyal cartilage, total cartilage area, and width of the brachial arches. Neither Whsc1 nor Letm1 KD caused a significant change in any measured parameter. Whsc2 KD caused a 27.94% decrease in average area of the ceratohyal cartilage, and a 23.87% decrease in area of all craniofacial cartilage. Tacc3 KD caused a 48.5% decrease in the average area of the ceratohyal cartilage, a 24.03% decrease in total cartilage area, and a 28.58% decrease in ceratohyal cartilage width. Significance was determined using a student’s unpaired t -test. (Embryos quantified: Control = 17, Whsc1 KD = 41, Whsc2 KD = 39, Letm1 KD = 34, Tacc3 KD = 11.) ∗∗∗∗ P < 0.0001, ∗∗∗ P < 0.001, ∗ P < 0.05, n.s., not significant. Scalebar is 250μm.

Article Snippet: Blotting for Whsc2 was carried out using mouse monoclonal antibody to Whsc2 (Abcam, ab75359, dilution 1:3,000).

Techniques: Knockdown, Staining, Control

Journal: Frontiers in Physiology

Article Title: Wolf-Hirschhorn Syndrome-Associated Genes Are Enriched in Motile Neural Crest Cells and Affect Craniofacial Development in Xenopus laevis

doi: 10.3389/fphys.2019.00431

Figure Lengend Snippet: Knockdown of Whsc1 and Tacc3 decrease CNC migration in vivo . (A,B,F,G,K,L,P,Q) Anterior lateral views of tailbud stage embryos (depicted at st. 27), following whole mount in situ hybridization against twist . Each column of panels (A,B,F,G,K,L,Q) are lateral views of two sides of the same embryo. (C–E,H–J,M–O,R–T) Measurements were taken for the total area of the three PA (Arch 1-3 extend anterior to posterior), the length of each individual arch, and the migration distance, as measured from the dorsal most tip of each arch to the neural tube. Embryos were stained and quantified at stages 25–30. (K–T) Letm1 or Whsc2 KD did not significantly affect any of the measured parameters. (F–J) Tacc3 KD expression caused an 8.33% decrease in the total PA area, but did not affect length or arch migration. (A–E) Whsc1 KD caused a 23.57% decrease in PA area. Additionally, the length of the second and third pharyngeal arches decreased by 14.72 and 31.70%, respectively. The migration distance of the first, second and third pharyngeal arches decreased by 15.75, 24.04, and 29.29%, respectively. Significance determined using a student’s paired t -test. (Embryos quantified: Whsc1 KD = 13, Tacc3 KD = 18, Whsc2 KD = 12, Letm1 KD = 19.) ∗∗∗∗ P < 0.0001, ∗∗∗ P < 0.001, ∗ P < 0.05, n.s., not significant. Scalebar is 250 μm.

Article Snippet: Blotting for Whsc2 was carried out using mouse monoclonal antibody to Whsc2 (Abcam, ab75359, dilution 1:3,000).

Techniques: Knockdown, Migration, In Vivo, In Situ Hybridization, Staining, Expressing

Journal: Frontiers in Physiology

Article Title: Wolf-Hirschhorn Syndrome-Associated Genes Are Enriched in Motile Neural Crest Cells and Affect Craniofacial Development in Xenopus laevis

doi: 10.3389/fphys.2019.00431

Figure Lengend Snippet: Whsc1 manipulation alters CNC migration speeds in vitro . Dissected CNC explants from control, Whsc1 KD, or Whsc2 KD embryos were plated on fibronectin-coated coverslips, allowed to adhere and begin migration, and imaged for 3 h using 20× phase microscopy. (A) Representative explants at initial timepoint (0 min). (B) Explants after 3 h migration time. (C) Representative tracks generated by FiJi Trackmate plug-in. (D) Mean track speeds of Whsc1 or Whsc2 KD explants compared to their controls. (Explants quantified: 3–4 explants from control and KD embryos were plated for each experiment, explants with neural or epithelial contaminant were excluded from analysis. Three separate experiments were performed for each depletion. Whsc1 controls: 272 cells, 9 explants. Whsc1 KD: 282 cells, 9 explants. Whsc2 controls: 151 cells, 12 explants. Whsc2 KD: 195 cells, 8 explants.) ∗∗∗∗ P < 0.0001, n.s., not significant. Scalebar is 250μm.

Article Snippet: Blotting for Whsc2 was carried out using mouse monoclonal antibody to Whsc2 (Abcam, ab75359, dilution 1:3,000).

Techniques: Migration, In Vitro, Control, Microscopy, Generated

Journal: Frontiers in Physiology

Article Title: Wolf-Hirschhorn Syndrome-Associated Genes Are Enriched in Motile Neural Crest Cells and Affect Craniofacial Development in Xenopus laevis

doi: 10.3389/fphys.2019.00431

Figure Lengend Snippet: Whsc1, whsc2, and tacc3 facilitate normal forebrain development. (A,B,D,E,G,H,J,K) Dorsal view of X. laevis half-embryo gene depletions (6 days post-fertilization), following alpha-tubulin immunolabeling to highlight nervous system. (B,E,H,K) Dorsal view of embryos with superimposed outlines of forebrain and midbrain structures. Internal control is on left (white), depleted side is on right (dashed red). (Alpha-tubulin staining is bilateral; exogenous eGFP on KD side persisted in embryos shown, causing a unilaterally enriched green signal.) (C,F,I,L) Area of forebrain and midbrain. Whsc1 KD reduced forebrain area by 17.65%. Whsc2 KD reduced forebrain area by 17.33% and midbrain area by 4.14%. Letm1 KD caused no significant change in brain size. Tacc3 KD caused a 16.05% decrease in forebrain area. Significance determined using a student’s paired t -test. (Embryos quantified: Whsc1 KD = 14, Whsc2 KD = 18, Letm1 KD = 12, Tacc3 KD = 26.) ∗∗∗∗ P < 0.0001, ∗∗∗ P < 0.001, ∗ P < 0.05, n.s., not significant. Scalebar is 250 μm.

Article Snippet: Blotting for Whsc2 was carried out using mouse monoclonal antibody to Whsc2 (Abcam, ab75359, dilution 1:3,000).

Techniques: Immunolabeling, Control, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-1 transcriptional responses to LPS are regulated by a complex of RNA-binding proteins

doi: 10.4049/jimmunol.1900650

Figure Lengend Snippet: Primary monocytes were mock treated (blue) or treated with 1μg/ml LPS (red) for 30 minutes. A) Promoter contacts from IL1A, IL1B, IL37, IL1RN were analyzed by Capture-C in five separate biological replicates. Interactions were designated based on the geometric mean of the replicates. B) ATAC-Seq signal profile across the IL1A, IL1B, and IL37. This was performed on the same cells. C) H3K27ac, H3K4me1 and H3K4me3 peaks from primary monocytes treated with or without LPS for 1 hour. The ChIP-seq data were extracted from a published study (80). D) The long-range chromatin contacts defined by Capture-C and eRNA locations are displayed as an expanded view for IL1A, IL1B, and IL37.

Article Snippet: Antibodies utilized included those to: CDK9 (a component of P-TEFb), NELF-A, c-JUN, NFκB p65, CBP and p300 antibodies (all from Santa Cruz Biotechnology, Santa Cruz, CA); H3K4me3 antibody (Active Motif, Carlsbad, CA) and H3K27ac antibody (Active Motif) were used in ChIP assays and RNA-immunoprecipitation.

Techniques: Capture-C, ChIP-sequencing

Journal: Biochemistry

Article Title: A Kinase-Independent Activity of Cdk9 Modulates Glucocorticoid Receptor-Mediated Gene Induction

doi: 10.1021/bi5000178

Figure Lengend Snippet: Cdk9 is a decelerator that does not reverse the effects of NELF-A or NELF-B and is not inactivated by chemical inhibitors. (A–G) Cdk9 augments the inhibitory effects of NELF-A and NELF-B. Competition assays of Cdk9 with NELF-A (A–E) or NELF-B (F and G) were performed with the indicated amounts of Cdk9 and NELF plasmids. Graphs of A max vs NELF-A (A) or NELF-B (F) illustrate the additive effects of Cdk9 with each NELF. The other graphs were plotted as in Figure . Similar results were obtained in five additional independent experiments. (H–K) Chemical inhibitors of Cdk9 do not block decelerator activity of Cdk9. Assays were performed as described in the legend of Figure with the indicated amounts of Cdk9 and DRB (H and I) or flavopiridol (J and K) and plotted as A max /EC 50 (H, I, and K) or 1/EC 50 (J). The amount of GREtkLUC reporter was 100 ng in all panels except for F and G, which used 30 ng. Similar results were obtained in two to five additional independent experiments.

Article Snippet: Anti-GR mouse and rabbit monoclonal antibodies [MA1–510 and PA1–516A, respectively (Affinity BioReagents)], anti-NELF-B rabbit polyclonal antibody [ab48336 (Abcam)], anti-NELF-A rabbit polyclonal antibody and anti-Cdk9 mouse monoclonal antibody [sc-23599 and sc-13130, respectively (Santa Cruz)], anti-β-actin mouse monoclonal antibody [A2228 (Sigma)], and anti-Flag mouse monoclonal antibody [F3165 (Sigma)] are commercially available.

Techniques: Blocking Assay, Activity Assay

Journal: Biochemistry

Article Title: A Kinase-Independent Activity of Cdk9 Modulates Glucocorticoid Receptor-Mediated Gene Induction

doi: 10.1021/bi5000178

Figure Lengend Snippet: Dominant negative kinase-dead Cdk9 has activity identical to that of wt Cdk9. (A) dnCdk9 augments the inhibitory effects of NELF-A. Competition assays with the indicated amounts of dnCdk9 and NELF-A plasmids were performed and plotted as in Figure . Similar results were obtained in four additional independent experiments. (B and C) Competition assays were performed as in Figure with 0, 3, 6, and 10 ng each of wt Cdk9 and dnCdk9 plasmids and a constant amount of GR (0.5 ng) and GREtkLUC (100 ng) plasmids. After adjustment for the 3-fold higher level of protein expression of dnCdk9 compared to that of wt Cdk9, plots of A max /EC 50 (A) and EC 50 / A max (B) vs the combined amounts of wt and dnCdk9 were constructed as described in the text. Similar results were obtained in a second independent experiment.

Article Snippet: Anti-GR mouse and rabbit monoclonal antibodies [MA1–510 and PA1–516A, respectively (Affinity BioReagents)], anti-NELF-B rabbit polyclonal antibody [ab48336 (Abcam)], anti-NELF-A rabbit polyclonal antibody and anti-Cdk9 mouse monoclonal antibody [sc-23599 and sc-13130, respectively (Santa Cruz)], anti-β-actin mouse monoclonal antibody [A2228 (Sigma)], and anti-Flag mouse monoclonal antibody [F3165 (Sigma)] are commercially available.

Techniques: Dominant Negative Mutation, Activity Assay, Expressing, Construct

Journal: Biochemistry

Article Title: A Kinase-Independent Activity of Cdk9 Modulates Glucocorticoid Receptor-Mediated Gene Induction

doi: 10.1021/bi5000178

Figure Lengend Snippet: Kinase-defective Cdk9 mutants retain activity of wt Cdk9 with NELF-A and NELF-B in different cells. Competition assays were performed and plotted as in Figure with the indicated amounts of dnCdk9 and NELF-A plasmids in U2OS cells (A) or 293 cells (B and C), with AcMtCdk9 and NELF-A plasmids in U2OS cells (D), with NELF-B in U2OS cells with dnCdk9 (E) or AcMtCdk9 (F), and with NELF-B in 293 cells with wt Cdk9 (G) or dnCdk9 (H). The amount of reporter plasmid was always 100 ng. The amount of GR plasmid used was 0.5 ng in U2OS cells and 5 ng in 293 cells. The range of linear expression of wt Cdk9 (≤40 ng), dnCdk9 (≤20 ng), and NELF-B (≤20 ng) is higher in 293 than in U2OS cells (data not shown). Similar results were obtained in one to four additional independent experiments except for that for NELF-B with AcMtCdk9 in U2OS cells (F), which was performed only once.

Article Snippet: Anti-GR mouse and rabbit monoclonal antibodies [MA1–510 and PA1–516A, respectively (Affinity BioReagents)], anti-NELF-B rabbit polyclonal antibody [ab48336 (Abcam)], anti-NELF-A rabbit polyclonal antibody and anti-Cdk9 mouse monoclonal antibody [sc-23599 and sc-13130, respectively (Santa Cruz)], anti-β-actin mouse monoclonal antibody [A2228 (Sigma)], and anti-Flag mouse monoclonal antibody [F3165 (Sigma)] are commercially available.

Techniques: Activity Assay, Plasmid Preparation, Expressing

Journal: Biochemistry

Article Title: A Kinase-Independent Activity of Cdk9 Modulates Glucocorticoid Receptor-Mediated Gene Induction

doi: 10.1021/bi5000178

Figure Lengend Snippet: Wild-type and mutant NELF-A and -B activities are not altered by mutations that destroy Ckd9 kinase activity. Competition assays were performed with 0.3 or 0.5 ng of GR plasmid and 30 ng of GREtkLUC, and the results are plotted as in Figure with the indicated amounts of wt Cdk9 and 4mtNELF-A (A and B) or wt NELF-A (C) plasmids or dnCdk9 and 4mtNELF-A (D and E) or wt NELF-A (F) plasmids, in U2OS cells. Similar results were obtained from five to eight independent experiments. (G and H) 4mtNELF-B has reduced potency, relative to that of wt NELF-B, in competition assays with dnCdk9. Competition assays were performed with 0.5 or 5 ng of GR plasmid and 100 ng of GREtkLUC, and A max /EC 50 vs NELF-B was plotted as in Figure with the indicated amounts of wt Cdk9 and wt NELF-A (G) or 4mtNELF-A (H) plasmids. The gray bar with the extending line represents the standard error of the mean and average amount of plasmid required for half-maximal reduction of A max /EC 50 from six experiments. To compensate for the 2.09-fold more efficient expression of 4mtNELF-B protein, compared to that of wt NELF-B [as determined by Western blots (not shown)], the x -axis values of panel H should be multiplied by 2.09. Similar results were obtained from five additional independent experiments.

Article Snippet: Anti-GR mouse and rabbit monoclonal antibodies [MA1–510 and PA1–516A, respectively (Affinity BioReagents)], anti-NELF-B rabbit polyclonal antibody [ab48336 (Abcam)], anti-NELF-A rabbit polyclonal antibody and anti-Cdk9 mouse monoclonal antibody [sc-23599 and sc-13130, respectively (Santa Cruz)], anti-β-actin mouse monoclonal antibody [A2228 (Sigma)], and anti-Flag mouse monoclonal antibody [F3165 (Sigma)] are commercially available.

Techniques: Mutagenesis, Activity Assay, Plasmid Preparation, Expressing, Western Blot

Journal: Biochemistry

Article Title: A Kinase-Independent Activity of Cdk9 Modulates Glucocorticoid Receptor-Mediated Gene Induction

doi: 10.1021/bi5000178

Figure Lengend Snippet: Cdk9 is a competitive decelerator acting after the site of action of GR and before that of the reporter gene. Competition assays with the indicated amounts of (A and B) Cdk9 and GREtkLUC reporter, (C) Cdk9 and GR, and (D) dnCdk9 and GR plasmids were performed in U2OS cells and the results plotted as in Figure . Similar results were obtained in five additional (for A and B) and two additional (for C and D) independent experiments.

Article Snippet: Anti-GR mouse and rabbit monoclonal antibodies [MA1–510 and PA1–516A, respectively (Affinity BioReagents)], anti-NELF-B rabbit polyclonal antibody [ab48336 (Abcam)], anti-NELF-A rabbit polyclonal antibody and anti-Cdk9 mouse monoclonal antibody [sc-23599 and sc-13130, respectively (Santa Cruz)], anti-β-actin mouse monoclonal antibody [A2228 (Sigma)], and anti-Flag mouse monoclonal antibody [F3165 (Sigma)] are commercially available.

Techniques:

Journal: Biochemistry

Article Title: A Kinase-Independent Activity of Cdk9 Modulates Glucocorticoid Receptor-Mediated Gene Induction

doi: 10.1021/bi5000178

Figure Lengend Snippet: Competitive decelerator activity of ELL that is augmented by Cdk9 in a manner independent of Cdk9 kinase activity and reversed by accelerators. Competition assays with 0.5 or 1 ng of GR plasmid, 100 ng of GREtkLUC, and the indicated amounts of (A and B) Cdk9 and ELL, (C and D) dnCdk9 and ELL, (E and F) CBP and ELL, and (G and H) TIF2 and ELL plasmids were performed in U2OS cells and the results plotted as in Figure . Wild-type Cdk9, dnCdk9, and ELL are linearly expressed with up to 20 ng of transfected plasmid; CBP is linearly expressed with up to 80 ng of plasmid, so no corrections are needed. TIF2 expression is corrected as described in the text. Similar results were obtained in five (A and B), two (C and D), one (E and F), and three (G and H) additional independent experiments.

Article Snippet: Anti-GR mouse and rabbit monoclonal antibodies [MA1–510 and PA1–516A, respectively (Affinity BioReagents)], anti-NELF-B rabbit polyclonal antibody [ab48336 (Abcam)], anti-NELF-A rabbit polyclonal antibody and anti-Cdk9 mouse monoclonal antibody [sc-23599 and sc-13130, respectively (Santa Cruz)], anti-β-actin mouse monoclonal antibody [A2228 (Sigma)], and anti-Flag mouse monoclonal antibody [F3165 (Sigma)] are commercially available.

Techniques: Activity Assay, Plasmid Preparation, Transfection, Expressing