Journal: JCI Insight

Article Title: Endothelial cell–glucocorticoid receptor interactions and regulation of Wnt signaling

doi: 10.1172/jci.insight.131384

Figure Lengend Snippet: Mouse lung endothelial cells (MLECs) were serum starved in 0.5% FBS for 4 hours and then treated with 10% Wnt3a conditioned media for 6 hours. Dexamethasone (DEX) 100 nM was added for 1 hour at the completion of the media incubation period. (A and B) qPCR for Sox17 (A) and Axin2 (B) was performed. (C) Control siRNA or GR siRNA MLECs were treated with Wnt3a 200 ng/mL for 4 hours in the presence or absence of DEX 100 nM for 1 hour, and qPCR for Sox17 was performed. (D) MLECs were stably transfected with a TCF/LEF luciferase construct and subjected to either control siRNA or GR siRNA treatment, with and without Wnt3a 200 mg/mL for 4 hours. (E) MLECs were treated with either control siRNA or GR siRNA, and lysates were subjected to Western blot for GR and β-catenin expression. (F) Quantification of GR and β-catenin expression by densitometry. Data represent 3 independent experiments. One-way ANOVA with Tukey’s post hoc test was used to analyze data in A, B, and D. Student’s t test was used to analyze data in C and F. *P < 0.05; **P < 0.01.

Article Snippet: Primary antibodies used include the following: GR (Thermo Fisher Scientific, MA1-510), β-catenin (BD Biosciences, 610153), and Hsp90 (Affinity Bioreagents, MA3-011).

Techniques: Incubation, Stable Transfection, Transfection, Luciferase, Construct, Western Blot, Expressing

Journal: Science Advances

Article Title: The glucocorticoid receptor associates with the cohesin loader NIPBL to promote long-range gene regulation

doi: 10.1126/sciadv.abj8360

Figure Lengend Snippet: ( A ) Schematic representation of ChIP-seq experiments after double treatment of dexamethasone and auxin of the HCT116 RAD21mAID cells. ( B ) Genome browser screenshot of representative GR-bound chromatin location in HCT116 RAD21mAID cells. Two biological replicates are combined for each sample. ( C ) Heatmap representing GR ChIP-seq intensity at GR chromatin–bound locations ( n = 371) identified in HCT116 RAD21mAID cells. Data combine two independent biological replicates. ( D ) Nascent RNA quantification before and after dexamethasone treatment before and after 6 hours of auxin treatment in RAD21mAID cells. Log 10 fold inductions (dexamethasone/EtOH) are normalized on GAPDH mRNA. Five biological replicates are shown for each column. Columns depict mean with error bars representing SD between experiments. P values derived from unpaired Mann-Whitney-Wilcoxon test. ( E to G ) YY1, H3K27ac ChIP-seq, and ATAC-seq in RAD21mAID cells, before and after auxin treatment. No substantial changes are found in YY1 chromatin binding and chromatin accessibility after acute depletion of cohesin subunit RAD21. Data combine two independent biological replicates each. All heatmaps and genomic data are normalized to a total of 10 million reads and further to local tag density. Homer and bedtools algorithms were used to identify unique and common sites between each experimental condition.

Article Snippet: The following are the antibodies used in this study: NIPBL (Bethyl Laboratories, A301-779A; Thermo Fisher Scientific, MA1-72534), YY1 (Active Motif, 61779), H3K27ac (Active Motif, 39133), GR (Santa Cruz Biotechnology, sc-393232; Thermo Fisher Scientific, MA1-510), SMC1a (Bethyl Laboratories, A300-055A; Abcam, ab133643), SMC3 (Abcam, ab9263), RAD21 (Abcam, ab992), H2B (Abcam, ab61250), and tubulin (Abcam, ab6160).

Techniques: ChIP-sequencing, Derivative Assay, MANN-WHITNEY, Binding Assay