Appendix S1. Supplemental material.

Figure S1. ChIP-qPCR primer design based on ChIP-chip data. SignalMap software (Roche NimbleGen) was employed to visualize enrichment at individual probes in our ChIP-chip data and as a basis for primer design for ChIP-qPCR validation. Shown here are positive binding primers for PTEN (A) and negative control binding primers for CCNA1 (B), black arrows. Panels under each chromosome are listed in order: probe enrichment (peaks), coding region (lines), and promoter region (blocks).

Figure S2. Effects of down-regulation of SALL4 in normal CD34+ cells on non-direct SALL4 target genes. (A) CD34+ cells were treated with lentiviruses with GFP-scrambled shRNA. GFP positive cells (about 25%) were sorted to be evaluated for gene expression changes upon the down-regulation of SALL4. (B) The expression level of CCNT1 was decreased upon down-regulation of SALL4A and SALL4B, but no change was observed on DAXX and DTGT. Fold enrichment was calculated after normalization of both control and SALL4 knock down samples to GAPDH (n > 3, Error Bars: ±SD). *p < 0.05.

Figure S3. Impaired in vivo engraftment upon SALL4 knock down evaluated by lentiviruses shRNA with GFP approach. CD34+ cells were treated with lentiviruses with GFP-scrambled or SALL4 shRNA prior to transplants into sublethally irradiated (1.0 Gy) NOD/SCID/IL2rγ-null mice by tail vein injection. Chimerisms were monitored at 8 weeks post transplantation by human CD45 and GFP co-expression Down-regulation of SALL4 (SALL4shRNA) group led to decreased in vivo engraftment. p values are listed as indicated.

Figure S4. There was no difference observed on the lineage marker expression between GFP positive or negative cells. CD34+ cells were treated with lentiviruses with GFP-scrambled shRNA. The expression of cell lineage marker was evaluated and compared by FACS on GFP positive and negative cells. The -GFP negative cells showed similar CD11b or CD14 or Glycophorine A (Gpa) expression when compared to those were GFP positive.

Figure S5. SALL4 and HOXA9 shares similar effects on erythroid differentiation in human primary CD34+ cells. CD34+ cells were either infected with scrambled shRNA or shRNAs against SALL4, HOXA9 and cultured in methylcellulose medium supplemented with SCF, IL-3, GM-CSF and EPO, with 1 ug/mL puromycin selection. Eyeloid colony (CFU-E) number on CFC plates was scored on day 14. Data depict average and standard deviation of 3 independent experiments. The p value was obtained by comparing to the control using a paired two-tailed distribution t-test. **p < 0.01.

Table S1. ChIP validations.

Table S2. ChIP-chip target list.

Table S3. The effect(s) of SALL4 or HOXA9 on CD34+ cell differentiation.

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