Figure 3
Reduced expression of antigen processing machinery genes is mediated by histone hypoacetylation and is increased by pharmacologic histone deactylase inhibition. For all experiments the indicated MCC cell lines were analyzed without treatment (grey) and after treatment with mithramycin A (MA, turquoise), vorinostat (V, light blue), or the combination thereof (V + MA, dark blue). (A) Global H3K9 acetylation of untreated, V, MA or V + MA treated MCC cell lines was determined by immunoblot with an AcH3K9 antibody; β-tubulin served as loading control. (B) Chromatin immunoprecipitation (ChIP) assay was performed with untreated or V + MA treated WaGa cells followed by a qRT-PCR using TAP1, TAP2, LMP2 or LMP7 promoter specific primers. CT values of AcH3K9 antibody or rabbit IgG isotype precipitated DNA were normalized to histone H3 antibody precipitated DNA as described in material and methods. Experiments were performed in triplicates twice and results are expressed as mean + SEM. (C) mRNA expression of HLA-A, B2M, TAP1, TAP2, LMP2 and LMP7 was determined by RT-qPCR in triplicates; CT values were normalized to RPLP0 and calibrated to the ΔCT value of untreated MKL-2 cells; relative mRNA expression is depicted + SEM for MKL-2, BroLi, MKL-1 and WaGa. (D) HLA class-I intracellular localization was determined via immunofluorescent staining using an HLA-A detecting antibody (clone EP1395Y) with a dylight 488 labeled secondary antibody (green). Wheat germ agglutinin (Alexa fluor 647, red) was used to stain cellular membranes and DAPI (blue) served as nuclear stain. Single cell images in the upper right corner of the overview image were acquired using confocal microscopy. (E,F) HLA class-I cell surface expression was determined by flow cytometry using a HLA-ABC specific antibody as exemplified for WaGa (E); the results for all cell lines analyzed are depicted as the geometric mean fluorescence intensity (gMFI) of HLA class-I (HLA-ABC) staining, + SEM in three independent experiments (F). Statistical analysis was performed using the Friedman test as indicated. (G) Viral protein expression was determined by immunoblot of whole cell lysates using an antibody specific for LT (clone CM2B4) depicting the different truncated LTs characteristically for MCC cells. β-tubulin served as loading control.
