Figure 6
Figure 6. CDK6/kinase activity is required for repression of CD25 and GATA3. (A-B) The relative mRNA expression for CD25 (A) and GATA3 (B) between different mutants and their corresponding controls. LK indicates purified stem cell/progenitors; and N+, cells cocultured on OP-9DL1 for 4 days. RNA isolated from LK or N+ cells was used for quantitative RT-PCR according to the manufacturer's protocol. “KO/WT (LK)” indicates the ratio of LK cells derived from BM of KO compared with LK cells from WT, without OP9-DL1 stimulation. Similarly, “KO-N+/LK” indicates the ratio of LK cells from KO compared with the same cells after 4 days culture on OP9-DL1 stroma. Data are mean ± SE (N = 3). *P < .05, significantly different from the control levels (denominator), which were arbitrarily defined as 1 unit. +P < .05, significantly different from bar 3. All the mRNA expression levels were normalized to the expression level of 26S RNA. (C) Quantitative RT-PCR analysis of indicated gene expression after treatment of WT-LK cells with γ-secretase inhibitor (DAPT: 5 μm) for 4 days on OP9-DL1. Sorted GFP−PI− cells were used for quantitative RT-PCR. All the mRNA expression levels were normalized to the expression level of 26S RNA and calculated as values relative to DMSO-treated cells. Data are the mean ± SE (N = 3). *P < .05, significantly different from the control levels, which were arbitrarily defined as 1 unit. (D-E) Quantitative RT-PCR for GATA3 (D) and CD25 (E) gene expression. Sorted WT-LK cells were infected with control vector (pLKO.1) or shRNA GATA3 (shGATA3) and then cocultured with OP9-DL1 for 4 days. The optimal puromycin concentration used for selection is 0.5 μg/mL. Sorted GFP−PI− cells were used for quantitative RT-PCR. All the mRNA expression levels were normalized to the expression level of 26S RNA and calculated as values relative to WT-LK cells. Data are the mean ± SE (N = 3). *P < .05, significantly different from the WT-LK control level, which was arbitrarily defined as 1 unit. +P < .05, significantly different from the pLKO.1 level.

CDK6/kinase activity is required for repression of CD25 and GATA3. (A-B) The relative mRNA expression for CD25 (A) and GATA3 (B) between different mutants and their corresponding controls. LK indicates purified stem cell/progenitors; and N+, cells cocultured on OP-9DL1 for 4 days. RNA isolated from LK or N+ cells was used for quantitative RT-PCR according to the manufacturer's protocol. “KO/WT (LK)” indicates the ratio of LK cells derived from BM of KO compared with LK cells from WT, without OP9-DL1 stimulation. Similarly, “KO-N+/LK” indicates the ratio of LK cells from KO compared with the same cells after 4 days culture on OP9-DL1 stroma. Data are mean ± SE (N = 3). *P < .05, significantly different from the control levels (denominator), which were arbitrarily defined as 1 unit. +P < .05, significantly different from bar 3. All the mRNA expression levels were normalized to the expression level of 26S RNA. (C) Quantitative RT-PCR analysis of indicated gene expression after treatment of WT-LK cells with γ-secretase inhibitor (DAPT: 5 μm) for 4 days on OP9-DL1. Sorted GFP−PI− cells were used for quantitative RT-PCR. All the mRNA expression levels were normalized to the expression level of 26S RNA and calculated as values relative to DMSO-treated cells. Data are the mean ± SE (N = 3). *P < .05, significantly different from the control levels, which were arbitrarily defined as 1 unit. (D-E) Quantitative RT-PCR for GATA3 (D) and CD25 (E) gene expression. Sorted WT-LK cells were infected with control vector (pLKO.1) or shRNA GATA3 (shGATA3) and then cocultured with OP9-DL1 for 4 days. The optimal puromycin concentration used for selection is 0.5 μg/mL. Sorted GFP−PI− cells were used for quantitative RT-PCR. All the mRNA expression levels were normalized to the expression level of 26S RNA and calculated as values relative to WT-LK cells. Data are the mean ± SE (N = 3). *P < .05, significantly different from the WT-LK control level, which was arbitrarily defined as 1 unit. +P < .05, significantly different from the pLKO.1 level.

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