Journal: Cancer cell
Article Title: A transcription factor addiction in leukemia imposed by theMLL promoter sequence
Figure Lengend Snippet: ZFP64 employs a C-terminal transactivation domain to maintain MLL expression and leukemia growth. (A) Experimental strategy used to map the trans-activation domain (TAD) of ZFP64 using GAL4 fusion reporter assays. (B) A series of GAL4-ZFP64 fusion proteins tested in the luciferase assay. FL: full length, ZF: Zinc finger. Number Z1-Z11 describes the number of Zinc finger contained in the construct. Transactivation activity of different ZFP64 mutant by luciferase assay is plotted. Renilla luciferase internal control normalized. (n=3) (C) Western blot confirming detectable protein levels of GAL4-ZFP64 fusion proteins after transfected into 293T cells. (D) RT-qPCR analysis of MLL expression in MOLM-13 cells transduced with various combination of sgRNAs or CRISPR-resistant ZFP64 cDNA. Results were normalized to GAPDH . (n=3) (E) Competition-based proliferation assay in MOLM-13 after transduction with various combination of sgRNAs or CRISPR-resistant ZFP64 cDNA. (n=3) (F) Western blot confirming detectable protein levels of CRISPR resistant ZFP64 cDNA (ZFP64 WT ) and C-terminal deleted ZFP64 mutant (ZFP64 ΔTAD ) in MOLM-13. (G) Domain structure of ZFP64. All bar graphs represent the mean ± SEM. * denotes unpaired T test, p
Article Snippet: For the cDNA overexpression experiments, a full length ZFP64 cDNA (DNASU, clone: HsCD00514448) was cloned into a lentiviral expression vector LentiV_Neo (Addgene: 108101).
Techniques: Expressing, Activation Assay, Luciferase, Construct, Activity Assay, Mutagenesis, Western Blot, Transfection, Quantitative RT-PCR, Transduction, CRISPR, Proliferation Assay