Fig. 4.

HOTS is lost in LOH and LOI WT and suppresses tumor cell growth in vitro and in vivo. (A) Quantitative real-time expression analysis of HOTS in WT samples shows loss of expression in LOI (WT5–14) and LOH (WT15–21) cases, but not in non-LOH, non-LOI cases (WT1–4). Error bars (SD) are plotted but are too small to see. (B) Tetracycline-inducible knockdown of HOTS by HOTS shRNA in HeLa cells. Induction with tetracycline activates the HOTS shRNA-expressing plasmid, leading to silencing of HOTS. (C) Western blot showing siRNA knockdown of HOTS with HOTS siRNA but not with scrambled siRNA (S. siRNA). β-Actin was used as a loading control. (D–F) HeLa tumor cell growth assayed on soft agar, showing that growth is inhibited by HOTS expressed from a tetracycline-inducible vector after Zeocin selection. (D) Nontransfected HeLa cells, selected with 0.5 mg/mL Zeocin. No visible colonies were observed due to cell death from Zeocin drug selection. (E) HeLa cell colonies from a culture that was not induced to express anti-HOTS RNAi. (F) Fifteen-fold more HeLa cell colonies occur when cells are induced by tetracycline to express anti-HOTS RNAi. (Scale bar, D–F: 0.3 mm.) n = 3. (G) Increased tumor cell growth in nude mice upon RNAi knockdown of HOTS. n = 6. Mean tumor volume was plotted against time. Statistically significant difference in tumor area between the HOTS knockdown animals (Induced, •) and the HOTS-expressing animals (Non-Induced, X-dotted line) was scored (P < 0.01 at 3 wk and P < 0.02 at 4 wk, Student's t-test). As controls, we included scrambled siRNA (S.siRNA) tetracycline-induced (▲, w/tet) and Non-Induced S.siRNA (■, wo/tet) HeLa transfected cells.