Figure 3.
Figure 3. The CDK9/mLST8/RAPTOR nuclear complex regulates expression of genes important for leukemogenesis. (A) MV4-11 cells were crosslinked with 1% formaldehyde. Chromatin-protein complexes were immunoprecipitated with anti-CDK9, anti-mLST8, and anti-RAPTOR antibodies. Rabbit IgG and anti-RICTOR antibodies were used as negative controls. qPCR was performed on immunoprecipitated DNA with primers for the MYC and PIM1 promoters. Data are expressed as fold enrichment over the IgG control. Shown are means + standard error (SE) of 4 independent experiments. (B) Cells were treated with atuveciclib for 0, 1, 2, and 4 hours. Cells were then lysed, and proteins were resolved by SDS-PAGE, followed by transfer to PVDF membranes. Membranes were immunoblotted with the indicated antibodies. The immunoblots with antibodies against the phosphorylated forms of the proteins or against the total proteins were from lysates from the same experiments analyzed in parallel by SDS-PAGE. (C) Cells were treated with control (DMSO) or atuveciclib for 4 hours and lysed, and proteins resolved by SDS-PAGE followed by transfer to polyvinylidene fluoride membranes. Membranes were then immunoblotted with the indicated antibodies. *Nonspecific band in the c-MYC blot. (D) qRT-PCR analysis of the relative mRNA expression of MYC and PIM1 in U937 cells after atuveciclib treatment of 4 hours. GAPDH was used for normalization. Data are expressed as fold change over the control. Shown are means + SE of 3 independent experiments. **P < .01, ****P < .0001 using paired Student t test analysis. (E) qRT-PCR analysis of the relative mRNA expression of MYC and PIM1 in MV4-11 cells after atuveciclib treatment of 4 hours. GAPDH was used for normalization. Data are expressed as fold change over the control. Shown are means + SE of 4 independent experiments. **P < .01, using paired Student t test analysis. (F) qRT-PCR analysis of the relative mRNA expression of MYC and PIM1 in primary AML patient samples after incubation with atuveciclib for 4 hours. GAPDH was used for normalization. Data are expressed as fold change over the control. Shown are means + SE of 7 independent experiments using cells from 7 different AML patients. *P < .05, **P < .01 using paired Student t test analysis.

The CDK9/mLST8/RAPTOR nuclear complex regulates expression of genes important for leukemogenesis. (A) MV4-11 cells were crosslinked with 1% formaldehyde. Chromatin-protein complexes were immunoprecipitated with anti-CDK9, anti-mLST8, and anti-RAPTOR antibodies. Rabbit IgG and anti-RICTOR antibodies were used as negative controls. qPCR was performed on immunoprecipitated DNA with primers for the MYC and PIM1 promoters. Data are expressed as fold enrichment over the IgG control. Shown are means + standard error (SE) of 4 independent experiments. (B) Cells were treated with atuveciclib for 0, 1, 2, and 4 hours. Cells were then lysed, and proteins were resolved by SDS-PAGE, followed by transfer to PVDF membranes. Membranes were immunoblotted with the indicated antibodies. The immunoblots with antibodies against the phosphorylated forms of the proteins or against the total proteins were from lysates from the same experiments analyzed in parallel by SDS-PAGE. (C) Cells were treated with control (DMSO) or atuveciclib for 4 hours and lysed, and proteins resolved by SDS-PAGE followed by transfer to polyvinylidene fluoride membranes. Membranes were then immunoblotted with the indicated antibodies. *Nonspecific band in the c-MYC blot. (D) qRT-PCR analysis of the relative mRNA expression of MYC and PIM1 in U937 cells after atuveciclib treatment of 4 hours. GAPDH was used for normalization. Data are expressed as fold change over the control. Shown are means + SE of 3 independent experiments. **P < .01, ****P < .0001 using paired Student t test analysis. (E) qRT-PCR analysis of the relative mRNA expression of MYC and PIM1 in MV4-11 cells after atuveciclib treatment of 4 hours. GAPDH was used for normalization. Data are expressed as fold change over the control. Shown are means + SE of 4 independent experiments. **P < .01, using paired Student t test analysis. (F) qRT-PCR analysis of the relative mRNA expression of MYC and PIM1 in primary AML patient samples after incubation with atuveciclib for 4 hours. GAPDH was used for normalization. Data are expressed as fold change over the control. Shown are means + SE of 7 independent experiments using cells from 7 different AML patients. *P < .05, **P < .01 using paired Student t test analysis.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal