Journal: Blood Advances

Article Title: STAT5 is essential for IL-7–mediated viability, growth, and proliferation of T-cell acute lymphoblastic leukemia cells

doi: 10.1182/bloodadvances.2018021063

Figure Lengend Snippet: Cross-analysis of STAT5 ChIP-seq and RNA-seq data on IL-7-stimulated TAIL7 cells. IL-7-deprived TAIL7 cells were cultured with or without IL-7 for 24 hours, and then collected for STAT5 ChIP-seq or RNA-seq enriched for mRNA. (A) Relative STAT5 peak enrichment/depletion in IL-7-stimulated cells. The relative enrichment of each STAT5 peak interval was calculated as described in “Methods.” Value of 0 equals random binding, negative values indicate STAT5 depletion, and positive values indicate STAT5 enrichment. (B) De novo motif discovery and identification on STAT5 ChiP-seq peaks from IL-7-stimulated cells. Enrichment cutoff at 1.5. The “Presence” column denotes the relative presence of the motif on all peaks. “Average motif/peak” denotes the number of times a motif appears on the peak. (C) Graph showing the distance, in base pairs (bp), of RUNX motifs to the STAT motif (horizontal axis) within each peak found in panel B, plotted against the frequency of each occurrence (vertical axis). (D) Venn diagram showing the overlap of genes found in the RNA-seq analysis (purple and yellow sets) and ChIP-seq analysis (green and red sets). Analysis was performed with genes with a STAT5a peak within 20 kb from the transcription start site. The genes with an IL-7-induced STAT5 peak whose expression was up- or downregulated by IL-7 are indicated on the left and right lists, respectively. (E) qPCR validation of ChIP-seq and RNA-seq data. IL-7-deprived TAIL7 cells cultured in medium, IL-7, or IL-7 plus S5i were collected for mRNA extraction and qPCR analysis at 24 hours (HRH2, CISH, OSM, and PIM1) or 48 hours (BCL6 and IL10). Fold change is normalized for the medium condition. Validation results are representative of at least 3 independent experiments. Results represent average of triplicates ± SEM.

Article Snippet: Whole cell lysates were prepared as described elsewhere, 17 resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, and immunoblotted with antibodies against p-JAK1/3 (Y1022-Y1023/Y980) (Sigma), p-Akt (S473), Akt, p-STAT1 (Y701), p-STAT3 (Y705), p-STAT5a/b (Y694/Y699), Bcl-xL, Mcl-1, PIM1 (Cell Signaling Technology), p27kip1 (BD Transduction Labs), STAT5, BCL6, PIM1, ZAP-70, and actin (Santa Cruz Biotechnology).

Techniques: ChIP-sequencing, RNA Sequencing Assay, Cell Culture, Binding Assay, Expressing

Journal: Blood Advances

Article Title: STAT5 is essential for IL-7–mediated viability, growth, and proliferation of T-cell acute lymphoblastic leukemia cells

doi: 10.1182/bloodadvances.2018021063

Figure Lengend Snippet: IL-7 directly modulates PIM1 and BCL6 expression via STAT5. (A) TAIL7 cells were withdrawn or not from IL-7 for 96 hours and collected for immunoblot analysis of BCL6, P-STAT5, and STAT5. (B) Data from ChIP-seq and RNA-seq were uploaded to University of California, Santa Cruz genome browser visualization tool (top 6 tracks). The browser is centered on the human BCL6 gene locus (hg19). Custom tracks are paired as control (Medium) and IL-7. ChIP STAT5 track pair represents peaks found upon STAT5 IP, and the arrow highlights a STAT5 binding peak. Input track represents control input for ChIP. RNA-seq track represents mRNA expression. Peak height is proportional to the expression. The bottom arrow in RNA-seq tracks highlights the decrease in overall BCL6 gene expression and concomitant processing of intron 1 into the mRNA. (C) IL-7-deprived TAIL7 cells were cultured for 72 hours in medium, IL-7, or IL-7 plus S5i and analyzed for P-STAT5 and PIM1. (D) Serum-starved, stably transduced HPB-ALL cells were cultured with or without IL-7 for 24 hours and analyzed for P-STAT5, PIM1, and P-Akt. Results are representative of at least 3 independent experiments.

Article Snippet: Whole cell lysates were prepared as described elsewhere, 17 resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, and immunoblotted with antibodies against p-JAK1/3 (Y1022-Y1023/Y980) (Sigma), p-Akt (S473), Akt, p-STAT1 (Y701), p-STAT3 (Y705), p-STAT5a/b (Y694/Y699), Bcl-xL, Mcl-1, PIM1 (Cell Signaling Technology), p27kip1 (BD Transduction Labs), STAT5, BCL6, PIM1, ZAP-70, and actin (Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, ChIP-sequencing, RNA Sequencing Assay, Binding Assay, Cell Culture, Stable Transfection