*2.2. Lentiviral Infection*

For transduction of HCT116, SW480 and HT29 with tdTomato fluorescent protein, cells were infected with lentiviral particles containing FUdtTW plasmid. 2 × <sup>10</sup><sup>5</sup> cells were seeded in 6-well plates and incubated overnight at 37 ◦C. The next day, a mixture containing complete growth medium with 5 μg/mL polybrene, and lentiviral particles was added to the cells. The medium was replaced 24 h after infection and cells were expanded and then sorted in BD FACSAria III with a 98% purity.

#### *2.3. Deflamin Purification*

Deflamin was isolated from mature and dried white lupin seeds (*Lupinus albus*) as described previously [20] Briefly, lupin seeds were milled to a fine powder and extracted using 50 mM of Tris-HCl buffer, pH 7.5 (1:10, *w*/*v*). The homogenate was centrifugated at 12,000× *g* for 30 min at 4 ◦C. The supernatant was collected, boiled for 10 min, and centrifugated at 12,000× *g* for 20 min at 4 ◦C. The supernatant was then made to pH 4.0 and centrifugated at 12,000× *g* for 20 min at 4 ◦C. The resulting pellet was resuspended in 40% (*v*/*v*) ethanol containing 0.4 M NaCl, and centrifugated at 13,500× *g*, 30 min, 4 ◦C. The supernatant was made to 90% (*v*/*v*) ethanol and left overnight at −20 ◦C. The following day, the mixture was centrifugated at 13,500× *g* for 30 min at 4 ◦C and the pellet, containing isolated deflamin, was resuspended in the smallest possible volume of milli-Q water. Desalting was performed with PD-10 Columns (GE Healthcare Life Science, Uppsala, Sweden) according to manufacturer recommendations and the final solution was collected, frozen at −20 ◦C, and lyophilized. The obtained deflamin reached 98% purity and the integrity of the protein was detected by polyacrylamide gel electrophoresis and subsequent Coomassie brilliant blue staining [20]. Deflamin was diluted in PBS for in vitro and in vivo experiments.
