Fig. 2
Global CGI hypermethylation is functionally relevant for Alb-R26Met tumorigenesis. a Scheme reporting demethylating treatments (Decitabine; 0.3 μM) used for in vitro and in vivo experiments with Alb-R26Met HCC cells. Cells were pre-treated (48 h) before using them for experiments with or without subsequent Decitabine pulses every 3 days (top; pink). Untreated cells were exposed to pulses of demethylating treatments during the assay (bottom; green). b Effect of Decitabine treatment (48 h) on cell viability of mouse liver progenitor MLP-29 cells and of Alb-R26Met HCC cells (HCC13 and HCC14). c Anchorage-independent growth (soft agar) assay using 3 different Alb-R26Met HCC cell lines (HCC3, 13 and 14) showing effects of demethylating treatments described in a. d Effect of Decitabine treatment in anchorage-dependent growth (foci formation) assay of 2 different Alb-R26Met HCC cell lines (HCC13 and 14). Graphs report number and size of colonies. e Decitabine (pre-treatment and treatment) reduces numbers of tumour spheres derived from Alb-R26Met HCC cells (HCC13 and HCC14). f Left: Graph reporting the tumour volume of mice injected either with untreated cells (No treat), with Decitabine pre-treated Alb-R26Met HCC cells (Pre-treat), or with untreated cells following pulses of Decitabine in vivo treatments (Pulses). Note that tumour volume was significantly reduced in mice injected either with Decitabine pre-treated Alb-R26Met HCC cells or with untreated cells following in vivo Decitabine pulses. Right: Mouse weight of the indicated groups was measured before and after xenograft experiments. No significant changes were observed, indicating that the dose of Decitabine used in vivo was not toxic. Significant differences between groups are indicated on the top. Not significant (ns): P > 0.05, *P < 0.05, **P < 0.01; ***P < 0.001
