TMZ treatment activates the WNK1/OSR1/NKCC1 signaling pathway in GC#99 and GC#22. A and C. Representative immunoblots showing increased expression of p-WNK1, p-OSR1 and p-NKCC1 proteins in response to TMZ exposure in GC#99 and GC#22. GCs were exposed to control medium (Con), 10 μM bumetanide (BMT), 100 μM TMZ, or 100 μM TMZ plus 10 μM BMT (T + B) for 4 h. B and D. Summary data of immunoblotting. Expression of each phosphorylated protein was first normalized by its total protein, and relative expression levels with different treatments were then normalized to Con. Data are mean ± SEM. n = 4–5, *p < 0.05 vs. Con.