*2.4. Cellular Viability Assay*

CRC cells were seeded at a density of 1–2 × 104 cells in 96-well plates. A total of 24 h after seeding, cells were treated with 0, 25, 50, or 75 μg/mL of deflamin. Every day, for 5 consecutive days, 1:10 of AlamarBlue reagent (Invitrogen, Waltham, MA, USA) was added to each well, and fluorescence was measured 2 h after (excitation 560 nm; emission 590 nm) in Infinite M200 Plate Reader (Tecan).

#### *2.5. Cellular Apoptosis Assay*

For apoptosis analysis, cells were seeded at a density of 1–2 × <sup>10</sup><sup>4</sup> cells in 96-well plates. 24 h after seeding, cells were treated with 0, 25, 50, or 75 μg/mL of deflamin for 48 h. The measurement of caspase 3/7 activity was performed using the Apo-ONE® Homogeneous Caspase-3/7 Assay kit (G7790, Promega, Madison, WI, USA) following manufacture instructions.

#### *2.6. Zymography*

In order to determine the anti-MMP role of deflamin in cancer cells, a zymographic analysis was performed as previously described [22]. Briefly, 12.5% polyacrylamide-SDS gels (*v*/*v*) were co-polymerized with 1% gelatin (*w*/*v*). CRC cell lysates (without and with deflamin treatment at 20 μg/mL, 40 μg/mL, 80 μg/mL) were treated with a nonreducing buffer containing 62.6-mM Tris–HCl pH 6.8, 2% (*w*/*v*) SDS, 10% (*v*/*v*) glycerol and 0.01% (*w*/*v*) bromophenol blue were loaded into each well of the SDS-gel. Electrophoresis was carried out vertically at 100 V and 20 mA per gel. Subsequently, gels were washed three times in 2.5% (*v*/*v*) Triton X-100 for 90 min each, to remove the SDS and incubated with a solution of 50-mM Tris–HCl pH 7.4, 5-mM CaCl2, 1-μM ZnCl2, and 0.01% *w*/*v* sodium azide, for 48 h at 37 ◦C. After incubation, gels were stained with Coomassie Brilliant Blue G-250 0.5% (*w*/*v*) in 50% (*v*/*v*) methanol and 10% (*v*/*v*) acetic acid, for 30 min, and destained with a solution of 50% (*v*/*v*) methanol, 10% (*v*/*v*) acetic acid. The gelatin degradation bands (white bands against a blue background), denoting relative MMP gelatinolytic activity, were analyzed according to their intensity with UN-SCAN-IT gelTM 6.1 software (Silk Scientific Corporation, Orem, UT, USA) and the relative values expressed in % of untreated lysate control.
