Figure 4.

KCNN4 activated the RAS-MAPK/PI3K-BCL2A1 signaling pathway. A. RNA-seq pathway signatures between 231/KCNN4-OE and MDA-231/CON cells ranked by the -Log10 (P-value). B-D. Venn diagram for the interaction genes between ORF library screening and RNA-seq; the overlapped genes from ORF library and RNA-seq are listed. E, F. Western blot was conducted to detect MAPK and PI3K signaling pathway activity using phospho-AKT/MEK/ERK and their downstream molecule, BCL2A1. G, H. Western blot analysis of the variation of MAPK and PI3K signaling pathway activity with or without treatment with LY294002, a highly select inhibitor of phosphatidylinositol 3 (PI3) kinase, or U0126, a selective inhibitor of MEK, in MDA-231/KCNN4-OE cells and HS578T/KCNN4-OE cells. I. MDA-231 cells were retrovirally transduced with BCL2A1-OE or control vector. The protein level was detected by western blot and the IC50 values by cytotoxicity are presented. J. Western blot assays for BCL2A1 after stable knockdown with shRNAs; the corresponding cells were subjected to cytotoxicity analysis to determine the IC50. Experiments were performed in triplicate and representative results are shown.