Blood, Vol. 113, Issue 11, 2375-2385, March 12, 2009

HOXA9 is required for survival in human MLL-rearranged acute leukemias
Blood Faber et al. 113: 2375

Supplemental materials for: Faber et al

Files in this Data Supplement:

• Document 1. Supplemental methods and results (PDF, 83.7 KB)
  
  
• Figure S1. Determination of GFP control shRNA transduction efficiency in 17 different MLL-rearranged and MLL-wildtype leukemia cell lines by puromycin selection (JPG, 80 KB) -
  When viability of puromycin selected cells was compared to transduced cells without puromycin selection (3 days after puromycin selection / 4 days after transduction) a transduction efficiency > 80% was confirmed for each cell lines tested.
  
  
  
  
  
  
• Figure S2. Analysis of expression of the interferon response genes OAS1, IFITM1, and MX1 (JPG, 62.9 KB) -
  By quantitative real-time PCR 72h after transduction with the four HOXA9 shRNA constructs, the non-targeting GFP control shRNA or empty pLKO.1 vector. Compared to untreated MOLM-14 cells all lentiviral constructs used showed normal interferon response gene expression levels and no induction whereas pI:C transfected cells revealed significant induction confirming that the interferon response pathway is inducible in this cell line.
  
  
  
  
  
  
• Figure S3 (JPG, 52.2 KB) -
  Quantitative real-time PCR analysis of HOXA9 expression 24 hours after transduction of murine leukemia stem cells (L-GMP) with the 1F3-HOXA9shRNA or 1F2-HOXA9shRNA construct demonstrated efficient HOXA9 suppression as compared to GFP-shRNA transduced controls.
  
  
  
  
  
  
• Figure S4. Analysis of the proliferative capacity as assessed by MTT assay in MOLM-14 cells after transduction with the 1F3-HOXA9shRNA construct (JPG, 53.8 KB) -
  Inhibition of the proliferative capacity was observed starting 48h after transduction.
  
  
  
  
  
  
• Figure S5. HOXA9 knockdown by lipofection of a 21-mer siRNA oligomer in t(9;11) THP-1 cells (JPG, 54.2 KB) -
  (A) Efficient HOXA9 suppression (75–80%) similar to HOXA9shRNA mediated suppression levels was confirmed by quantitative real-time PCR 72h after transfection. (B) Time course analysis of viability after HOXA9siRNA transfection revealed a progressive induction of cell death with an extent and kinetic similar to the effects observed after shRNA mediated suppression. (C) Ectopic overexpression of a non-targetable HOXA9 construct significantly rescued the phenotype (Annexin V staining).