Analysis of p18^INK4c protein expression and promoter hypermethylation. (A) Immunohistochemical detection of the p18^INK4c protein. In reactive lymphoid tissue (left panel: a benign lymph node), p18^INK4c is expressed mainly by lymphocytes in germinal centers and interfollicular plasma cells, but not by mantle cells. Middle and right panels: Examples of HL cases showing absence and presence, respectively, of p18^INK4c protein expression by RS cells. (B) MSP analysis of the methylation status of the p18^INK4c promoter in HL-derived cell lines (top), NHL-derived cell lines (middle), and HL tumors (bottom). IVMD indicates in vitro–methylated DNA; NS, DNA from a sample of normal spleen, used as a negative (unmethylated) control; U, unmethylated; and M, methylated. (C) p18^INK4c protein expression in HL-derived cell lines. Top: Western blot analysis of p18^INK4c expression in total protein extracts from the HL cell lines. The multiple myeloma cell line RPMI-8226 shows total absence of p18^INK4c expression and was included as a negative control. The expression of α-tubulin was analyzed as a loading control. Bottom: Immunohistochemical staining for p18^INK4c on cytospin preparations of the HL-derived cell lines. Original magnification × 1000 for all electron microscopy except left panel of A (× 200).