Figure 4

GRP78 inhibition impaired in vitro and in vivo tumorigenic properties of HN-CICs. (A) To elucidate the capability of migration of GRP78 shRNA knockdown and sh-Luc HN-CICs, single cell suspension of GRP78-specific shRNA or control sh-Luc HN-CICs were plated onto transwell and analyzed as described in Materials and Methods. Results are means ± SD of triplicate samples from three experiments (**, p < 0.01). (B) Single cell suspension of GRP78-specific shRNA or control sh-Luc HN-CICs were plated onto transwell coated with matrigel and analyzed as described in Materials and Methods. Data are means ± SD of triplicate samples from three experiments (**, p < 0.01; ***, p < 0.001). (C) To elucidate the anchorage independent growth, single cell suspension of stable GRP78-specific shRNA or control sh-Luc HN-CICs (SAS (upper panel), OECM1 (lower panel)) were plated onto soft agar and analyzed as described in Materials and Methods. Results are means ± SD of triplicate samples from three experiments (*, p < 0.05; **, p < 0.01). (D) Summary of the in vivo tumor growth ability of different numbers of GRP78-knockdown or control (sh-Luc) HN-CICs examined by xenotransplantation analysis. (E) Representative tumor growth of 10000 control and 10000 GRP78-knockdown HN-CICs was generated in the subcutaneous space of recipient mice (upper panels).Tumor volume was measured after inoculation of GRP78-knockdown shRNA and sh-Luc-expressing HN-CICs (Yellow arrows: sh-Luc-expressing HNSCCs; Red arrows: sh-GRP78-expressing HNSCCs) (lower panel). Error bars correspond to SD (*, p < 0.05; **, p < 0.01).