Fig. 4. Selectivity of C28 toward LMTK3.

(A) Selectivity profile of C28 (1 μM) against 140 kinases using radioactive filter binding assay. The data are displayed as percent activity remaining of assay duplicates with an SD. Only kinases with >50% decrease in their activity are shown. The relative IC₅₀ values are also presented. (B) Treespot interaction map depicting the kinome phylogenetic grouping, with kinases interacting with C28 (5 μM) represented as red circles. The larger the diameter of the circle, the higher the C28 binding affinity to the respective kinase. Kinases whose binding was inhibited by C28 to less than 10% of the control (DMSO) are shown. Lower numbers indicate most probable hits to bind to C28. Overlapping kinases identified in assays (radioactive filter binding and site-directed competition binding) are highlighted in yellow. (C) Western blots of LMTK3 and ERα in BC cell lines treated with increasing concentrations of C28 at different time points. Mean densitometry values of three independent experiments were calculated using ImageJ. GADPH, glyceraldehyde 3-phosphate dehydrogenase. (D) Effects of C28 on LMTK3 protein half-life in MDA-MB-231 cells. Cells were treated with CHX (cycloheximide) (100 μg/ml) and 10 μΜ C28 (or DMSO) for different time points. The relative LMTK3 protein levels (−/+ C28) were calculated and plotted against the time of treatment with CHX. (E) Western blots of total and phospho-HSP27 (normalized versus total) in MCF7, T47D, and MDA-MB-231 cell lines treated with 10 μΜ C28 for different time points. Mean densitometry values of three independent experiments are shown. (F) Western blots of total and phospho-HSP27 (normalized versus total) in BC cell lines treated with either control or LMTK3 small interfering RNAs (siRNAs) for 72 hours. Mean densitometry values of three independent experiments are shown. (G) Western blots of different kinases in BC cell lines treated with C28 (10 μΜ) at different time points.