Transfection and infection of cell lines Cells were transiently transfected with the indicated mimic-miRNA or with the siRNA by using the Interferin reagent (Polyplus, New York, NY, USA), according to the manufacturer's instructions. SUV39H1 and TRF2 over-expressing HCT116 cells were obtained by transient transfection of human HCT116 by using the JetPEI reagent (Polyplus) according to the manufacturer's instructions. All the analyses were performed 72h after cell transfection. Stable TRF2 over-expressing HCT116 and HT29 cells and their control (pBabe-Empty) were infected with retroviral particles produced into Phoenix-AMPHO cells transfected with retroviral vectors (pBabe-puro-Empty and pBabe-puro-mycTRF2) [29]; using the JetPEI reagent (Polyplus, New York, NY, USA), according to the manufacturer's instructions. To establish stable suppression of TRF2 gene, HCT116 and SW620 were infected with lentiviral particles produced into HEK293T cells, transfected with the packaging pCMVR8.74 and the envelope pMD2.G vectors in combination with the vectors encoding either for a scramble short hairpin sequence (shSCR; N2040 targeting E.coli DNA polymerase) or for one of the two short hairpin sequences directed against TRF2 (shTRF2_N1; N2573 TRCN0000004813 or shTRF2_N2; N2571 TRCN0000004811, which were a gift from Prof Shoeftner S. (University of Trieste, Trieste, Italy). CRC organoids were resuspended in cultrex reduced growth factor BME type 2 and drops of 100 μl were plated into chamber slides (15 μ- Slide 4 well glass bottom, Ibidi). Then, organoids were transfected with 50 nM of the indicated mimic-miRNAs (following the protocol already reported for 2D cell lines). The day after, 4-well slides were loaded into a specific chamber (5% CO2 and 37 °C) and phase-contrast images were acquired at time 0 and after 96h using the microscope Leica DMi8 (20× objective). The software used to set the parameters was LasX (Leica).