S100A8/A9 modulates G₂/M cell cycle checkpoint regulating molecules in SCC.

<p>Expression and phosphorylation (activation/inactivation) status of cell cycle regulators are shown by immunoblotting analysis of G2/M synchronized cells. Data shown are representative of multiple independent repeat experiments. Goat anti-β-actin polyclonal IgG was used to detect β-actin as protein loading control. (A) Expression of G1/S regulating proteins, cyclin A, cyclin E, p21 and Rb, Chk2 and Cdc25B, in KB, KB-EGFP and KB-S100A8/A9. Phosphorylation of p-Chk2 (Thr68) was not detectable in any cell, with or without S100A8/A9 expression (not shown). (B) Expression and phosphorylation status of G2/M regulators, Chk1/p-Chk1 (Ser345), mitotic active p-Cdc25C (Thr48), Cdc25C/p-Cdc25C (Ser216), Cdc2/p-Cdc2 (Thr14/Tyr15), and cyclin B1. (C) Immunoblotting (IB) of Cdc25C protein co-immunoprecipitated (IP) with 14-3-3β captured with rabbit anti-14-3-3β polyclonal IgG. Immunoblotting of total 14-3-3β and Cdc25C proteins are also shown. (D) Protein levels of S100A8, S100A9, Cdc2 and p-Cdc2-(Thr14/Tyr15) in wild-type TR146 (TR146 WT), control shRNA transfectant (TR146-shRNA-control) and shRNA-induced S100A8/A9 knockdown (TR146-shRNA-S100A8/A9KD) cells relative to the levels in KB cells. Protein levels are also shown in several other HNSCC cell lines, SCC-58, OSCC-3 and UMSCC-17B with different levels of S100A8/A9 expression.