human methylcellulose based medium  (R&D Systems)

Journal: Clinical Cancer Research

Article Title: Synergistic Activity of Combined FLT3-ITD and MDM2 Inhibition with Quizartinib and Milademetan in FLT3 -ITD Mutant/ TP53 Wild-type Acute Myeloid Leukemias

doi: 10.1158/1078-0432.CCR-24-2764

Figure Lengend Snippet: Combination of Q/M-induced proapoptotic activity is p53-dependent, and the combination treatment impairs clonogenicity in FLT3 mutant/ TP53 WT leukemia blasts. A, MOLM-13-p53-Vec or MOLM-13-p53-KD cells were treated with increasing concentrations of single-agent milademetan or quizartinib or Q/M combination for 48 hours. Apoptosis was measured by FCM with annexin V staining. CIs were calculated using CalcuSyn software (version 2.0, PREMIER Biosoft). B, MOLM-13-p53-Vec or MOLM-13-p53-KD cells were treated with MOLM-13 and milademetan (60 nmol/L) or quizartinib (3 nmol/L) or the combination for 24 hours. Correlated signaling pathway proteins were measured with immunoblotting. C, Primary AML samples (three cases; harboring FLT3 mutant/ TP53 WT) and two normal BM samples from normal donors were treated with indicated concentrations of quizartinib and/or milademetan for 2 weeks in methylcellulose medium. CFU-GM colonies were counted, and the percentage of colony forming against the control group was calculated. The data were obtained from triplicated wells, and error bars are presented as mean ± SD. Asterisks indicate the level of statistical significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001 as determined using a two-tailed unpaired t test. BAX, Bcl-2–associated X; CFU-GM, colony-forming unit granulocyte–macrophage; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NS, no statistical significance.

Article Snippet: The cells (0.2 × 10 6 ) were plated in methylcellulose medium (1 mL/well; cat.: 04435; STEMCELL Technologies Inc.) for 2 weeks, exposing them to the indicated drugs (in triplicate per condition).

Techniques: Activity Assay, Mutagenesis, Staining, Software, Western Blot, Control, Two Tailed Test