Figure 4. BEND3 knockout dampens TAK-243 effects and reduces the intracellular transport of TAK-243 into AML cells.

(A and B) Control and BEND3-knockout OCI-AML2-Cas9 cells were treated with DMSO or TAK-243 (30 nM) for 24 hours. After treatment, whole cell lysates were prepared, and levels of UBA1, UBA3, UBA6, UBA2, poly-ubiquitylated proteins, activating transcription factor 4 (ATF4), poly (ADP-ribose) polymerase (PARP), cleaved PARP (C. PARP), DNA-damage inducible transcript 3 (CHOP), phospho-JNK (p-JNK), and Ser¹³⁹ phosphorylated H2AX (γH2AX) were measured by immunoblotting. GAPDH and β-actin were used as loading controls. (C) Control and BEND3-knockout OCI-AML2-Cas9 cells were treated with DMSO or increasing concentrations of TAK-243 at 15–120 nM for 1 hour followed by heating the intact cells at 54°C. After heating, whole cell lysates were prepared, and levels of UBA1 and GAPDH were measured by immunoblotting. (D) Control and BEND3-knockout OCI-AML2-Cas9 cells were washed, seeded in equal numbers, and lysed. Luminescence was then measured after adding an ATP-dependent luciferase reagent. Relative luminescence obtained from BEND3-knockout OCI-AML2-Cas9 cells was calculated by normalizing to control cells. Data points represent means ± SEM of 3–5 independent experiments. (E) Control and BEND3-knockout OCI-AML2 cells were treated with increasing concentrations of TAK-243 (300–1200 nM) for 1 hour and washed, and pellets were then extracted with acetonitrile. TAK-243 concentrations were then measured by LC-MS. Data points represent means ± SEM of triplicate data from a representative experiment (n = 2). **P ≤ 0.01; ****P ≤ 0.0001 using 2-way ANOVA and Sidak’s multiple comparisons test.