Figure 6.

Analysis of Xevinapant target proteins cIAP1 and XIAP using immunostaining: (A) Representative images of untreated cells from three different cell lines (CLS-354, HSC4, 01-GI-SBL) stained with DAPI (blue), cIAP1 (green) and XIAP (red)—fluorescence microscopy image. (B) Background expression levels of cIAP1 and XIAP in untreated cells of seven HNSCC cell lines and one healthy cell line. Gray bar represents protein intensity in the cytoplasm, and black bar represents protein intensity in the nucleus of the cells. (C) Change in cIAP1 and XIAP expression levels in (a) cells treated with Xevinapant (13.3 µM) compared with untreated cells (negative control), (b) cells treated with IR (2 Gy) compared with untreated cells or (c) cells treated with a combination of Xevinapant (13.3 µM) and IR (2 Gy) compared with treatment with IR alone. In each experiment, the average protein intensity of all counted cells (n > 400) was determined. Each value in the graph represents the total mean ± SD of at least 3 independent experiments. Significance was determined by Mann–Whitney U test * p ≤ 0.05. (D) Representative images of Cal33 cells stained with DAPI (blue), cIAP1 (green) and XIAP (red)—fluorescence microscopy image. Top: cells after treatment with IR (2 Gy); bottom: cells treated with combination of IR (2 Gy) and Xevinapant (13.3 µM).