S100A8/A9 expression induced cell cycle and mitotic arrest at G2/M.

<p>(A) Cell cycle analysis of KB, KB-EGFP and KB-S100A8/A9 cells post-synchrony. Cells cultured under standard conditions were serum-starved overnight, synchronized at G1/S with aphidicolin treatment and stimulated to re-enter cell cycle. Synchronized cells were stained with propidium iodide DNA staining solution and analyzed by flow cytometry for changes in DNA content following release from G1/S blockage. (B) Mitotic analysis of synchronized cells stained with phospho-Histone H3 (Ser10) and analyzed by flow cytometry. (C) Percentage of cells in G₂/M. KB, KB-EGFP and KB-S100A8/A9 cells were analyzed over time post-synchrony and reported as mean ±SEM; n = 2 independent experiments (each analysis performed in duplicate); *p<0.05. (D) Percentage of mitotic cells post-synchrony, representing the mean of two independent repeat experiments. KB-S100A8/A9 cells showed fewer mitotic cells as shown by lower phospho-Histone H3 (Ser10) staining.</p

Expression of S100A8 and S100A9 in human head and neck tissues and carcinoma cell lines.

<p>The mRNA expression levels of S100A8 and S100A9 in (A) normal adjacent (NAT) and HNSCC tissues and (B) TR146 HNSCC and KB cells were measured by qRT-PCR and normalized to GAPDH; dotted line shown as a threshold for detection. Total RNA extracted from matching NAT and HNSCC tissues from each of three patients was pooled separately for gene expression analysis. Cell lines cultured under standard conditions were harvested and analyzed at approximately 70% confluency. Data presented as mean ±SD (n = 2). Representative immunoblots of (C) S100A8 and S100A9 in KB-S100A8/A9 transfected cells and (D) TR146-shRNA-S100A8/A9 knockdown cells compared to wild type and negative transfection controls. β-actin was used as loading control for immunoblotting analysis separated in 10% SDS-PAGE gels.</p

S100A8/A9 suppressed anchorage-dependent and -independent growth of KB cells.

<p>(A) Growth curves of KB-S100A8/A9 cells compared to KB-EGFP and KB wild-type control cells. Cells were grown on non-pyrogenic polystyrene tissue flasks in fresh MEM supplemented with 10% FBS every three days. (B) Anchorage-independent growth in soft agar for KB, KB-EGFP and KB-S100A8/A9 cells (mean ±SEM, *p<0.03, n = 4). (C) Anchorage-dependent growth on tissue flasks in complete medium (mean ±SD, *p<0.02, n = 3) and (D) colony formation in soft agar (mean ±SEM, ***p<0.0005, n = 2) of TR146-shRNA-S100A8/A9KD cells compared to TR146 WT and TR146-shRNA-control cells.</p

S100A8/A9 interaction with PP2A phosphatase increases activity.

<p>(A) PP2A-Aα/β co-immunoprecipitated (IP) with either S100A8/A9 complex (IP with 27E10 antibody) or S100A9 subunit (left panel) and S100A9 (used as S100A8/A9 complex marker protein) co-IP with PP2A-Aα/β subunit as detected by immunoblotting (IB). (B) PP2A subunit protein expression, phosphorylation and methylation compared in KB, KB-EGFP and KB-S100A8/A9 cells (synchronized at 5 h post-G1/S) as detected using IB. Blot shown is a representative of multiple repeats (n≥3) of total protein lysates separated in 10% SDS-PAGE gels. β-actin was used as protein loading control. (C) The PP2A-C phosphatase activities in KB-EGFP and KB-S100A8/A9 cells were normalized to the detectable PP2A-Aα/β co-immunoprecipitated with S100A8/A9 (27E10 antibody) shown in A, and then further normalized to KB-EGFP levels. Data was shown as Mean ±SE (n = 2). ^#Error bars not visible. (D) Inhibition of S100A8/A9-dependent p-Cdc25C (Ser216) phosphorylation by 10 nM okadaic acid (OA). Expression of p-Cdc25C in the absence (left panel) and presence of OA (right) as detected by IB.</p

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.</p