Journal: Cell stem cell

Article Title: Reversal of malignant ADAR1 splice isoform switching with Rebecsinib.

doi: 10.1016/j.stem.2023.01.008

Figure Lengend Snippet: Figure 2. Development of a lentiviral ADAR1 A-to-I RNA editing reporter (A) Schematic diagram demonstrating the synthetic RNA sequence containing an ADAR1-sensitive stop codon that, upon A-to-I editing, reads through to produce nanoluciferase and GFP proteins separated by a T2A cleavage site. (B) ADAR protein expression levels in 293T cells co-transfected with the ADAR1 nanoluciferase-GFP (nanoluc-GFP) reporter and increasing amounts of FLAG- tagged wild-type (WT) ADAR1, catalytically inactive mutant ADAR1 (E912A), or wild-type ADAR2. b-actin was used as a loading control. (C) Relative luciferase signals in 293T cells prepared as in (B). Data are represented as mean ± SEM. (D) Live-cell fluorescent imaging of GFP expression in human myeloid leukemia TF-1a cells transduced with the ADAR1 nanoluc-GFP reporter vector (lower panels) compared with untransduced controls (upper panels). (E) Detection of nanoluciferase expression via in vivo bioluminescence (IVIS) imaging of no transplant control (far left), K562-nanoluc-GFP and pCDH vector transduced, and K562-nanoluc-GFP and ADAR1 wild-type or E912A mutant transduced human leukemia cells (K562) transplanted into Rag2/gc/ mice. (F) Luminescence-based quantification of ADAR1-dependent nanoluciferase signals in CD34+ cells from primary, high-risk MF samples (*untreated patient) after in vitro transduction with the ADAR1 nanoluc-GFP reporter and treatment with vehicle control (DMSO) or Rebecsinib (72 h). Relative luciferase signals were normalized to cell viability for each condition. Data are represented as mean ± SEM. p < 0.05 Rebecsinib compared with DMSO controls by pairwise t test. (G) Intracellular flow cytometry-based quantification of STAT3 phosphorylation (expressed as mean fluorescence intensity [MFI], values within HPC populations) after in vitro treatment with vehicle control (DMSO) or Rebecsinib (1 mM, 72 h). p < 0.05 Rebecsinib compared with DMSO controls by pairwise t test. See also Figure S1.

Article Snippet: In vitro culture of cell lines The following cell lines were used for in vitro lentiviral reporter assays and biomarker development: human KG-1a cells (AML cells derived from a 59 y/omale, cultured in Iscove’sModified Dulbecco’sMedium, Catalog No. 30-2005, 20% fetal bovine serum), human K562 cells (blast crisis chronic myeloid leukemia cells isolated from a 53 y/o female, cultured in Iscove’s Modified Dulbecco’s Medium, Catalog No. 30-2005, 10% fetal bovine serum), human TF-1a cells (erythroleukemia cells isolated from a 35 y/o male, cultured in RPMI-1640 Medium, ATCC 30-2001, 10% fetal bovine serum), humanMOLM-13 (sAML cells isolated from a 20 y/o male, cultured in RPMI-1640 Medium, ATCC 30-2001, 10% fetal bovine serum), human HL-60 cells (acute promyelocytic leukemia cells isolated from a 36 y/o female, cultured in Iscove’s Modified Dulbecco’s Medium, Catalog No. 30-2005, 20% fetal bovine serum), 293T cells (human kidney epithelial cells from human fetus, cultured in Dulbecco’s Modified Eagle’sMedium (DMEM) (ATCC 30-2002, 10% fetal bovine serum), and rat RBL-1 cells (leukemia cells isolated from the Wistar strain, cultured in Eagle’s Minimum Essential Medium, Catalog No. 30-2003, 10% fetal bovine serum).

Techniques: Sequencing, Expressing, Transfection, Mutagenesis, Control, Luciferase, Imaging, Transduction, Plasmid Preparation, In Vivo, In Vitro, Cytometry, Phospho-proteomics