Fig 1: The role of monopolar spindle 1 (MPS1) in the anticancer activity of VU‐0365114. (A) The inhibitory activity of VU‐0365114 on 89 kinases (SelectScreen Kinase Profiling Services, ThermoFisher Scientific). A kinome tree was generated using KinMap (http://www.kinhub.org/kinmap/). Illustration reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com). An enlarged image was provided in Fig. S12. (B) HCT116 cells were treated with 10 μm VU‐0365114 (VU) for 1 h, and then a cellular thermal shift assay (CETSA) was performed to examine the thermal stability of MPS1 and CAMK1D. Data represent three independent experiments. (C) HCT116 cells were treated with 100 ng mL−1 nocodazole for 18 h, and then exposed to various concentrations of VU‐0365114 (VU) for 1 h. Next, protein expressions were examined by Western blotting. (D) HCT116 cells were treated with VU‐0365114 (VU) for 24 h, and protein expressions were examined by Western blotting. Data represent three independent experiments. (E) Correlations of MPS1 or CAMK1D mRNA with VU‐0365114‐induced growth inhibition in NCI‐60 cell panel. The MPS1 and CAMK1D mRNA levels in NCI‐60 cancer cells were obtained from the CellMinerCDB database. Statistical significance was determined using the Pearson's correlation coefficient (r) and the corresponding P value. (F, G) HCT116 cells were transfected with MPS1 siRNA in F or plasmid in G for 48 h, and then exposed to VU‐0365114 for 72 h. MPS1‐knockdown in F or MPS1‐overexpression in G was confirmed by Western blotting (left panel). Cell viability was examined by an MTT assay (right panel). The error bars are the mean ± SD (n = 5). Statistical significance, compared to the transfection control group at each dose (*P < 0.05 and ***P < 0.001), was determined using a two‐tailed paired Student's t‐test. si‐NC, negative control siRNA.