Figure 3

Suppression of GRP78/^memGRP78 expression diminished spheres-forming capability, stemness genes expression, and side population cells of HN-CICs. (A) Down-regulation of GRP78 in HN-CICs (SAS (left panel) and OECM1 (right panel) mediated by shRNAi was validated by western blotting) (B) The percentages of ^memGRP78⁺ cells in sh-GRP78 knockdown and sh-Luc HN-CICs were compared by flow cytometry analysis, respectively. (C) Single cell suspensions of sh-GRP78 and sh-Luc-expressing HN-CICs incubated with Hoechst 33342 were examined for side population by flow cytometry. (D) HNSCCs-enriched sphere cells were first infected with Sh-GRP78-1, Sh-GRP78-2 or Sh-Luc lentivirus, and further cultivated under the serum-free defined selection medium. The tumor sphere formation capability and cellular morphology of enriched HN-CICs (Upper panel, SAS; Lower panel; OECM1) treated with either sh-Luc or GRP78- shRNA lentivirus were examined with microscope. (E) Total proteins from figure 3d were isolated and immublotted by using antibodies against, anti-Oct-4, anti-Nanog, anti-Nestin or anti-GAPDH antibodies as indicated. The amount of GAPDH protein of different crude cell extracts was referred as loading control. (F) Protein level of epithelial specific differentiation markers, CK18 and invoclurin in enriched HN-CICs cells infected with sh-Luc, or sh-GRP78 lentivirus was assessed by western blot. (G) Single cell suspension of spheres prepared from figure 3d transduced with sh-Luc or sh-GRP78 lentivirus were stained with Annexin V and examined by flow cytometry. The experiments were repeated three times and representative results were shown. Results are means ± SD (*, p < 0.05).