Generation of Inducible Cell Lines for RIPK3 Study

To generate HCT116/Tet‐On‐GFP‐SUB/RIPK3‐2xFV cells, HCT116 cells were first transduced with lentiviral particles generated from pLenti‐CMV‐Blast plasmids carrying the Tet Repressor gene. During selection with 5 μg/ml blasticidin (Thermo Fisher A1113903), single clones were isolated, and clone C4 with high levels of Tet‐Repressor expression was used for the next steps.
HCT116/Tet‐On clone C4 was plated at a density of 5 × 10⁴ cells per well in 12‐well plates, and transduced with 3 μl precipitated lentiviral particles generated with pLVX‐tight‐puro plasmids encoding GFP, GFP‐K63‐SUB, or GFP‐M1‐SUB sequences (Hrdinka et al, 2016 (link)). After selection with 1 μg/ml puromycin (Invitrogen ant‐pr) in the presence of 5 μg/ml blasticidin, HCT116/Tet‐On‐GFP, HCT116/Tet‐On‐GFP‐K63‐SUB and HCT116/Tet‐On‐GFP‐M1‐SUB cells were transduced with retroviral particles produced from LZRS‐zeo‐RIPK3‐2xFV plasmids and selected with the combination of 5 μg/ml blasticidin, 1 μg/ml puromycin, and 250 ng/μl zeocin.