5.

KCTD12 knockout enhances A375 cells’ spheroids forming capacity and survival capacity in hostile environment. (A) KCTD12 knockout by CRISPR/Cas9, along with upregulation of CD271, was confirmed by Western blot. (B) Apoptotic cells were decreased in A375 cells after KCTD12 knockout when cultured in the basal medium without FBS for 8 days detected by Calcein-AM/PI kit. Scale bars: 200 μm. (C) KCTD12 knockout enhanced A375 cells’ spheroids forming ability (8 days). Scale bars: 500 μm. (D) Apoptotic cells were decreased in A375 cells after KCTD12 knockout when cultured under a hypoxia condition for 4 days detected by Calcein-AM/PI kit. Scale bars: 200 μm. (E) KCTD12 knockout decreased the doxorubicin uptake in A375 cells (24 h). Scale bars: 200 μm. (F) KCTD12 knockout reduced the cytotoxicity of doxorubicin to A375 cells (24 h). Data are mean ± SD (n = 3 - 5); **, P < 0.01, determined by one-tailed unpaired t-test.