Fig. 2. STYK1 alters the anchorage-independent growth of EGFR TKI-treated NSCLC cells.

A–D PC9 cells were stably transduced with control or STYK1 shRNA plasmids. A, B STYK1 knockdown efficiency in three independent experiments as determined by qRT-PCR (mean ± SEM) (A) and Western blotting (representative images shown) (B). β-actin was used as a loading control. C The cells were seeded in soft agar medium containing afatinib (5 nM) or osimertinib (10 nM). Representative images of individual wells stained with nitro blue tetrazolium chloride after 21 days in culture are shown. D The average number of colonies (mean ± SEM) of 2 independent experiments is shown as calculated by the OpenCFU software. Statistics are shown for STYK1 shRNA compared to ctrl shRNA for the respective treatments. E–G PC9 cells with stable overexpression of empty vector (EV), STYK1-eGFP wild-type (WT), or STYK1-eGFP catalytically inactive mutants Y191F and K147A as confirmed by Western blotting (E). α-tubulin was used as the loading control. F The STYK1 overexpressing cells were grown in soft agar in the presence of afatinib (5 nM). Representative whole-well images of nitro blue tetrazolium chloride-stained colonies are shown. G Quantified results of 3 (or 5 in case of STYK1-WT) independent experiments (mean ± SEM).