Fig. 5: TRIM24 is phosphorylated by AURKB at S1042 in CRC cells.

A Kinase prediction tools (KinasePhos 2.0, GPS 3.0, and iGPS 1.0) were used to identify potential kinases targeting TRIM24 at Ser1042. “+” indicates a positive prediction by the respective tool; “−” indicates no prediction. B DLD1 and SW620 cells were transferred with si-NC, si-AURKA, si-AURKB, si-CK2α, and si-AKT. After 72 h, whole cell lysates were derived and immunoblotted for indicated proteins, with Tubulin as a loading control. Experiments were conducted independently three times, consistently producing similar results. C DLD1 and SW620 cells were treated with hesperadin (AURK inhibitor, which has a particularly significant inhibitory effect on AURKB, 20 μM), CX-4945 (CK2 inhibitor, 20 μM), and AKT Inhibitor VIII (AKT1/AKT2 inhibitor, 20 μM) for 4 h and 8 h, respectively. Whole cell lysates were derived and immunoblotted for indicated proteins. Experiments were conducted independently three times, consistently producing similar results. D In vitro phosphorylation assays utilized the purified GST-fusion proteins and AURKB, incubated with hesperadin or DMSO vehicle control. The reaction products were immunoblotted using indicated antibodies. AKT was employed as a positive control. Data are representative of three independent experiments. E SW620 cells were treated with hesperadin for 72 h, whole cell lysates were derived and immunoblotted for indicated proteins. Data are representative of three independent experiments. F Cytosolic (Cyto) and nuclear (Nuc) fractions derived from DLD1 and SW620 cells stimulated with 20 μM of hesperadin for 8 h were immunoblotted for indicated proteins. PARP1 and Caspase-3 served as loading controls and nuclear/cytosolic markers, respectively. Shown are the representative results from three independent experiments. G DLD1-WT/DLD1-KO-1 and SW620-WT/SW620-KO-1 cells were co-transfected with Renilla luciferase and TOPFlash plasmids. The cells were harvested 48 h after transfection, and firefly and Renilla luciferase substrates were added successively. The ratio of the detection value of firefly luciferase to the detection value of Renilla luciferase is taken to compare the difference in the results between the two groups of cells. Data were expressed as means ± SEM, two-tailed Student’s t-test. n  =  3 biological replicates per group.