Figure 6

5-aza-dC involvement in apoptosis, cell proliferation, migration and differentiation of RMS cells. (A) FACS analysis. Increased number of apoptotic RH30 cells after treatment with 20 μM 5-aza-dC along with paired negative controls (DMSO treatment). Results are the means ± SD of three separate experiments. (B) Migration assay. 5-aza-dC treated RH30 cells, compared with control, showed significantly impaired ability to cross through Boyd chamber membranes. Representative fields of migrated cells through the membrane, stained with crystal violet dye (20× magnification). (C) MTT assay. Time-dependent inhibition of cell proliferation after 5-aza-dC treatment resulted in 80% decrease at 72 h. Data are shown as means ± SD of sextuplicate points of two separate experiments. (D) FACS analysis. Cell cycle distribution of 20 μM 5-aza-dC RH30 treated cells showed a marked arrest in G2-phase after 72 h. Results are means ± SD of three separate experiments. (E) Immunofluorescence analysis. Five-aza-dC exposure led to marked changes of RH30 cell morphology, to more organised actin bundles (TRITC-phalloidin staining) and to MyHC positivity (100× magnification). Asterisks denote data in which miR-378a-3p was significantly different from control (**, p < 0.01; ***, p < 0.001).