Hes1 mediates the different responses of hematopoietic stem and progenitor cells to T cell leukemic environment

Figures & data

Figure 1. Analyses of SCF expression in T-ALL mice. To assess whether SCF expression was upregulated in the leukemic environment in our model, we performed ELISA to determine SCF concentration in PB serum and BM samples from leukemia and control mice 10 d after transplantation (A and B). The expression of SCF in BM, spleen and blood cells as well as CD45.1⁺ BM cells and GFP⁺ BM cells was analyzed by real-time PCR (C). The level of SCF protein in leukemia and normal cells was determined by western blot (D).

Figure 2. Gene profiling analyses of normal HSCs in leukemic environment. Normal HSCs were obtained from control and leukemia mice on day 10 after transplantation (A). The clustering map showed the genes differently expressed in normal HSCs between leukemia and control group. Red color meant the genes were upregulated while green color meant the genes were downregulated (B). Schematic representation of the analyses strategies integrating expression profiles obtained from two independent experiments (C).

Figure 3. Validation of the results of microarray by real-time PCR. Real-time RT-PCR was used to validate the expression pattern of eight genes using specific murine primers detailed in Table 1 (A). Quantitative analysis of Hes1 and p21 expression in normal HSCs and HPCs in leukemia and control mice was shown in (B and C). Data are shown as means ± SD *, p < 0.05, statistical analysis was performed using Student’s t-test.

Figure 4. In vitro CFC study on Hes1-transduced HSPCs and HSCs. Infection rates were assessed using transduction of 293T cells. After infection, the cells were analyzed with fluorescence microscope (A) and GFP⁺ cells were sorted for the expression of Hes1 and other related genes (B). Lin- cells (C) and LSK cells (D) of B6.SJL mice were transduced with Hes1 or control retrovector. Successfully transduced (GFP⁺) cells were enriched by FACS sorting and plated in standard methylcellulose-containing CFC assays. Data are shown as means ± SD ***, p < 0.0001, statistical analysis was performed using Student’s t-test.

Figure 6. HSCs/HPCs with Hes1 overexpression were preserved under leukemic environment. The Hes1-transduced mouse model is described in detail in Materials and Methods. On day 10 after transplantation, the absolute number and proportion of normal CD45.1⁺GFP⁺Lin^- cells (A), CD45.1⁺ GFP⁺ HSPCs (B), CD45.1⁺ GFP⁺ HSCs (C) were analyzed with BD FACS Aria II.

Figure 7. The function and cell cycle analyses of HSPCs in Hes1 and control model. On day 10 after transplantation, GFP⁺CD45.1⁺ cells were sorted for colony-forming cell (CFC) assay (A). The cell cycle of CD45.1⁺ GFP⁺ HSPCs was analyzed with Hoechst/PY (B).

Table 1. The sequences of primers

Primer	Sequence(5′-3′)		Size(bp)	Transcription ID
CXCR4	upper	TCT TCC TGC CCA CCA TCT AC	202	NM009911
	lower	TTC CCA AAG TAC CAG TCA GC
Hes1	upper	CTC CCG GCA TTC CAA GCT AG	164	NM008235
	lower	AGC GGG TCA CCT CGT TCA TG
IL18r1	upper	AGA TGG TTC AAA GGC AGT GC	152	NM008365
	lower	CGA CGA TCA TTT CCG ACT TG
Itgb3	upper	TCT CCC CAC CAC AGG CAA TC	114	NM016780
	lower	CAT TGA AGC GGG ACA CCT GG
Mmp2	upper	TGA CCT GGA CCC TGA AAC CG	131	NM008610
	lower	CCC AGC GTC CAA AGT TGA TC
Fbxw11	upper	TTT GTC CGC ACT CTG AAT GG	147	NM134015
	lower	TTC GTG CCC CTC TAG GAC TC
C-Kit	upper	GTG CCA ACC AAG ACA GAC AAG AG	176	NM021099
	lower	GCC CGT GAG TGA GGA GGA TAT TC
p21	upper	AAT CCT GGT GAT GTC CGA CC	422	NM007669
	lower	TTG CAG AAG ACC AAT CTG

Figure 5. Establishment of Hes1-transduced mouse model. Lin^- cells from B6.SJL mice at the age of 6–8 wk were transduced with either MSCV-Hes1-IRES-GFP or blank plasmid for 72 h. After transduction, Hes1-GFP⁺ or blank-GFP⁺ cells (CD45.1) were sorted and then injected into lethally irradiated C57BL/6J (CD45.2) by tail vein at a quantity of 2 × 10⁵ cells per mouse with 10⁶ cells of Notch1-induced leukemia cells (CD45.2).