Fig 2: Western blot analysis for NEK6 and NEK1 on a panel of human cancer cell lines. Western blot showing NEK6 and NEK1 expression levels on a panel of cancer cell lines representative of four solid tumors: MDA-MB231 and MCF7 (breast cancer), PEO1 and COV318 (ovarian cancer), NCI-H1299 and NCI-H1975 (lung cancer), HCT-15 and SW948 (colon cancer). Actin immunoreaction was used as loading control. The cropped blots are used in the figureand full-length blots are presented in Supplementary Figure S7.

Fig 3: Identified inhibitors of NEK6 activity. Curves represent the percentage of NEK6 activity as a function of inhibitor concentration by LANCE-Ultra NEK6 kinase assay. (A) Compound 8 (ZINC05007751) inhibits NEK6 activity with IC50 value of 3.4 ± 1.2 µM; (B) compound 21 (ZINC04384801) inhibits NEK6 activity with IC50 value of 2.6 ± 0.05 µM. IC50 value derives by mean ± SEM of IC50 of 4 independent experiments. Each point of curve represents the mean of one triplicate ± SEM of one representative experiments.

Fig 4: Screening of the selected twenty-five compounds at a single concentration by LANCE technology. Bar chart showing the percent of NEK6 activity, following treatment with the vehicle (NEK6 + vehicle) or the tested compounds at a concentration of 30 µM, using LANCE-Ultra NEK6 kinase assay. Quercetin was used as positive control of inhibition.

Fig 5: Predicted binding mode of compound 8. (A) Solid surface of the ATP binding pocket of NEK6. (B) Three-dimensional representation. (C) Two-dimensional interaction plot. The N-lobe is coloured in blue, the C-lobe in light-blue, the activation loop in green, the DLG motif in pink, the Gly-rich loop in yellow and the hinge-region in red.