Pmscv flt3 itd y591f y919f

NEAT1_1, ΔNEAT1_1, C/EBPβ, NAP1L1, Trim56 and β‐catenin were cloned into the pCDH lentiviral vector. Plasmid pMSCV‐FLT3‐ITD‐Y591F/Y919F, pMSCV‐FLAG‐MLL‐AF9, and pTY‐EF1a‐Hoxa9‐p2a‐Meisl‐p2a‐GFP were purchased from Addgene (#74499, #71444, #61738) and subcloned into the pMSCV PIG vector to achieve FLT3‐ITD‐GFP, MLL‐AF9‐GFP, and HOXA‐Meisl‐GFP construct, respectively. The shRNAs targeting human NEAT1, NAP1L1, or mouse NEAT1 were cloned into pLKO.1. shRNA sequences are shown in Table S3, Supporting Information.

Human chronic myeloid leukemia (CML) in blast crisis cell line (K562) was procured from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained at 37°C, 5% CO2 in complete medium, containing Iscove’s Modified Dulbecco’s Medium (IMDM), 1X penicillin/streptomycin (Gibco), and supplemented with 10% (v/v) fetal bovine serum (FBS).
To differentiate K562 into Mk, cells were treated with Phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, St. Louis, MO, USA), gilteritinib, quizartinib, dubermatinib (TP-0903), and midostaurin for 48–72 h as previously described (15 (link)). Where indicated, low-serum medium supplemented with 1% FBS and 1 μM imatinib were used.
gilteritinib, quizartinib, dubermatinib (TP-0903), and midostaurin were from Cayman chemical (Ann Arbor, MI, USA). All small molecules were dissolved in DMSO and store in −20°C. pMSCV-Flt3-ITD-Y591F/Y919F was procured from Addgene (Addgene.org). Flt3-ITD-Y591F/Y919F cassette was subcloned into lentiviral vector to produce viral particles using 293T cell.