Journal: Genes & Development
Article Title: JAK2 is dispensable for maintenance of JAK2 mutant B-cell acute lymphoblastic leukemias
Figure Lengend Snippet: Pharmacological inhibition or genetic depletion of JAK2 in MHH-CALL4 B-ALL cells induces an anti-proliferative response and not apoptosis. ( A ) CellTrace violet (CTV)-stained MHH-CALL4 cells were treated with vehicle (dimethylsulfoxide [DMSO]; black) or 1000 nM ruxolitinib (red). Cells were serially passaged for up to 8 d and subjected to assessment of their proliferative capacity by flow cytometry every 2 d. Drugs were replenished to the indicated concentration every 2 d. Data are representative of n = 3 performed in duplicate. ( B ) MHH-CALL4 cells were transduced with constitutive (pLMS-GFP) retroviral vectors harboring shRNAs targeting JAK2 (sh JAK2 .209 or sh JAK2 .2826) or scrambled (sh SCR ). Immunoblotting was performed against the indicated targets using lysates from shRNA + cells (GFP + ) at day 5 after transduction. Tubulin served as loading control. ( C ) Gene set enrichment analysis (GSEA) from 3′RNA sequencing (3′RNA-seq) analysis demonstrates negative enrichment of the indicated JAK/STAT gene set in MHH-CALL4-pLMS-sh JAK2 .209 cells relative to MHH-CALL4-pLMS-sh SCR cells at day 5 after transduction. (NES) Normalized enrichment score; (FDR) false discovery rate. ( D , E ) Transduced and nontransduced populations from B were passaged in vitro for 19 d. ( D ) The percentage of Annexin V + /GFP + cells was assessed by flow cytometry (individual bars represent days 5, 8, 9, 12, 14, 16, and 19 after transduction). ( E ) The percentage of shRNA + cells (GFP + ) was assessed by flow cytometry, and data were normalized to day 5. ( F ) Immunoblotting against the indicated target was performed against MHH–CALL4–Cas9 cells transduced with lentiviral vector (FgH1tUTG-GFP) expressing single guide RNAs (sgRNAs) targeting JAK2 (sh JAK2 .9901 or sh JAK2 .9903) at day 6 after sgRNA induction. β-Actin served as loading control. ( G ) Transduced populations from F were assessed for percentage Annexin V/DAPI positivity by flow cytometry at the indicated time points. ( H ) Transduced and nontransduced populations from F were passaged in vitro for 16 d. The percentage of sgRNA + cells (GFP + ) was assessed by flow cytometry (individual bars represent days 3, 5, 7, 9, 12, 14, and 16 after sgRNA induction). ( I ) MHH-CALL4 cells were transduced with pLMS-sh JAK2 .209 or pLMS-sh SCR . At day 14 after transduction, cell cycle analysis was performed by flow cytometric analysis of BrdU incorporation versus DNA content (7-AAD). Error bars in D , E , G , and I represent SEM ( n = 2); error bars in H represent SEM ( n = 3). (**) P
Article Snippet: sgRNA oligonucleotides (Sigma-Aldrich) targeting JAK2 were annealed and cloned into lentiviral expression vector FgH1tUTG (Addgene, 70183; a gift from M. Herold) for inducible sgRNA expression (see the for sgRNA sequences).
Techniques: Inhibition, Staining, Flow Cytometry, Cytometry, Concentration Assay, Transduction, shRNA, RNA Sequencing Assay, In Vitro, Plasmid Preparation, Expressing, Cell Cycle Assay, BrdU Incorporation Assay