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Blood, Vol. 110, Issue 6, 2075-2083, September 15, 2007
Pharmacologic inhibition of CDK4/6: mechanistic evidence for selective activity or acquired resistance in acute myeloid leukemia
Blood Wang et al.
110: 2075
Supplemental materials for: Wang et al, Vol 110, Issue 6, 2075-2083
Files in this Data Supplement:
- Figure S1. A 24-hour incubation with THRX-165724 and SU14813 is not sufficient to induce apoptosis (JPG, 83.5 KB)
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Apoptosis assay: MOLM13 and MV4-11 cells were incubated with DMSO (vehicle control), THRX-165724 (300 nM), or SU14813 (100 nM). After 24 hours the cells treated with THRX-165724 or SU14813 were washed with media 3 times and subsequently split into two groups. One group was incubated with media containing no inhibitors (“wash-out” group), whereas in the second group the 2 respective inhibitors were added again. At the 72-hour time point the cells were stained with 7-AAD and Annexin-V PE followed by flow cytometric analysis.
- Figure S2. The G1 cell cycle block induced by THRX-165724 and SU14813 is reversible (JPG, 68 KB)
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Cell cycle analysis:. MOLM13 and MV4-11 cells were incubated with DMSO (vehicle control), THRX-165724 (300 nM), or SU14813 (100 nM). After 24 hours the cells treated with THRX-165724 or SU14813 were washed with media 3 times and subsequently split into 2 groups. One group was incubated with media containing no inhibitors (“wash-out” group), whereas in the second group the 2 respective inhibitors were added again. At the 72-hour time point the cells were stained with propidium iodide followed by flow cytometry.
- Figure S3. The inhibition of Flt3 ITD in MOLM13 leads to the rapid downregulation of Cyclin D2 and D3 as well as pRb dephosphorylation (JPG, 29.1 KB)
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Western blot: MV4-11 cells were treated with THRX-165724 for 0, 1, 2, 4, 8, and 12 hours. Lysates were resolved by SDS/PAGE and immunoblotted with the indicated antibodies. Probing with antitubulin was used to assure equal loading.
- Figure S4. PD0332991 enhances the pro-apoptotic activity of SU14813 in MV4-11 and MOLM13 cells but not in a primary AML sample with Flt3 ITD (JPG, 207 KB)
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MV4-11, MOLM13, THP-1, and U937 cells as well as primary AML cells from patient 17 (Flt3 ITD) and patient 75 (Flt3 wt) were incubated for 3 days with DMSO (control), PD0332911 (250 nM), SU14813 (100 nM), or PD0332991 (250 nM) plus SU14813 (100 nM). The cells were stained with Annexin V-PE and 7-AAD followed by flow cytometric analysis.
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