GS induces cell death in HNSCC cells. (A) SCC4 cells treated with 50 μM GS for 0 h, 24 h, and 48 h were fixed and stained with propidium iodide and cell cycle analysis was carried out using flow cytometry as described in Materials and Methods. Panel shows representative DNA histograms of GS-treated SCC4 cells in sub-G0, G0/G1, S- and G2/M phase of cell cycle after (i) 0 h, (ii) 24 h; (iii) 48 h; (iv) histograms showing mean % of cell population in each phase of cell cycle (* p < 0.05). (B) Annexin V assay. The panel shows % apoptotic cells on treatment with GS (50 μM) for (i) 0 h, (ii) 24 h and (iii) 48 h and (iv) histogram showing mean % of apoptotic cells (mean ± S.D., * p < 0.005). (C) Immunofluorescence analysis revealing M30 protein expression in SCC4 cells (i) untreated control cells; (ii) GS (50 μM) treatment for 12 h (original magnification × 400). (D) SCC4 cells were treated with GS (50 μM) for 6 h - 48 h followed by DNA extraction as described in materials and methods. Panel represents an agarose gel electrophoretogram showing DNA ladder confirming apoptosis.