Knockdown of G9a or inhibition of its enzymatic activity decreases HNSCC cell growth in vitro. (A) FaDu and SAS cells were infected with lentivirus containing two different G9a-shRNA plasmids to knockdown G9a expression. (B) Cells were treated with different doses of BIX-01294 to inhibit the enzymatic activity of G9a. We used MTT assay to determine the five-day proliferation rate of G9a-knockdown cells compared with luciferase-knockdown controls, and BIX-01294 treated cells compared with vehicle (*, p < 0.05). The MTT value at Day 0 was considered as 100% of the proliferation rate for each experimental group. (C and D) The clonogenic cell survival assay of cells infected with lentivirus containing luciferase shRNA, G9a shRNA, vehicle and BIX-10294 treated cells (**, p < 0.01). (E and F) Examination of anchorage-independent growth ability of cells and quantification results of soft agar assay. Scale bar, 500 μm. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).