798592.fig.001a
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798592.fig.001b
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798592.fig.001c
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Figure 1: (a) Flow chart of the separation and sorting of the cells used for generating the microarray data. Cells from healthy bone marrows and CML patients went through a round of Percoll (to separate the mononuclear cells) and Ficoll (to eliminate dead cells). Highly enriched CD34+ cells were isolated from the mononuclear cells by two rounds of immunomagnetic separation using a Myltenyi kit, and the CD34+ cells were then stained it with Hoechst and Pyronin and sorted by FACS. The G0 cells are in the region of low fluorescence intensity for both dyes, and the G1/S/G2/M cells have a high level of Hoechst and PyroninY. (b) BrdU staining of a CML CD34+/G1/S/G2/M and CD34+/G0 quiescent fraction. To further validate our separation procedure, we pulsed labeled CML CD34+/G0 and G1/S/G2/M cells with BrdU. After 1 hour’s exposure, 22% of the cells from the CML cycling fraction were labeled with BrdU compared to 0.68% of the quiescent cells. The BrdU positive cells are fluorescent (green) while the blue are Dapi stained nuclei without BrdU incorporation. Left panel: CML G1/S/G2/M cells. Right panel: CML G0 cells. (c) Gene expression array data validation by qPCR. To confirm the differences in gene expression found by our microarray data analysis between the CML/G0 and BM/G0, we performed an RT-qPCR on nine up- and five downregulated genes. Thirteen out of fourteen genes (the exception being CPI-17) have been confirmed to be up-, or downregulated by this second technique. For each sample, we ran four reactions for each gene and the average value has been used for the calculation of relative expression using the ct method and GAPDH as a calibrator. -axis: gene expression fold change: CML relative to bone marrow. -axis: gene tested by RT-qPCR.