Cultured cells were seeded out one day prior to transfection to allow the cells to adhere. After 24 h the cells were washed once in Opti-MEM (Gibco) and transfected using a mixture of Lipofectamine 2000 (5 µg/mL (Sk-Hep-1), 10 µg/mL (Hep3B), 10 µg/mL (HEK293) 10 µg/mL (THLE-3) ThermoFisher), Opti-MEM and the ASOs at a final concentration of 1, 5 or 25 nM. Four hours after transfection, the mixture was carefully removed from the cells and fresh medium was added to each well. HepG2 cells were reverse-transfected. A mixture of Lipofectamine 2000 (2.5 µg/mL, ThermoFisher), Opti-MEM combined with the ASOs at a final concentration of 1, 5 or 25 nM were incubated at RT, and the HepG2 cells were added after 15 min of incubation. Knockdown was assessed 48 h after transfection. The cells were harvested, total RNA isolated and RNA expression measured using RT-qPCR as described.
In the experiments in which the effect of PURPL knockdown was assesses in combination with Doxorubicin or Nutlin treatment, cells were transfected as described above with ASOs at a final concentration of 25 nM for 24 h before DMSO (0.5%), Doxorubicin (Sigma; 5 µM) or Nutlin (Sigma; 5 µM) was added to the appropriate wells. Both Doxorubin and Nutlin were reconstituted in DMSO (Sigma).
In the experiments in which the effect of PURPL knockdown was assesses in combination with Doxorubicin or Nutlin treatment, cells were transfected as described above with ASOs at a final concentration of 25 nM for 24 h before DMSO (0.5%), Doxorubicin (Sigma; 5 µM) or Nutlin (Sigma; 5 µM) was added to the appropriate wells. Both Doxorubin and Nutlin were reconstituted in DMSO (Sigma).