Accugene water

For total protein isolation, colonic biopsy samples were minced into small pieces with a razor blade in a Petri dish and were transferred into 1 ml RIPA lysis buffer [Millipore, 20-188]. The 10 × RIPA lysis buffer was diluted in AccuGene water [Lonza, 51200] and completed with EASYpack Protease Inhibitor Cocktail [Roche, 05892970001]. The biopsy samples were homogenised with a Branson Sonifier SFX150 on ice [four cycles, 10 s sonication, and 10 s break per cycle]. After that, the samples were centrifuged at 3500 rpm for 10 min at 4°C. The supernatant was transferred into a new 1.5 ml tube, snap-frozen, and stored at -80°C until use. For total protein isolation from stool samples, 100 mg stool was vortexed in 300 µl RIPA lysis buffer with protease inhibitor for 5 min. After that, the samples were centrifuged at 4000 g for 10 min at 4°C. The supernatant was transferred to a new, sterile tube, and the centrifuge step was repeated at 12000 g for 10 min at 4°C. The supernatant was transferred into a new 1.5 ml tube, snap-frozen, and stored at -80°C until use.

At the end of the induction or culture period, the medium was removed, and the cells were washed with PBS. Then, 1.5 mL of extraction buffer (7M Urea, 1% SDS, 0.35 M NaCl, all chemicals from Sigma-Aldrich Inc., St. Louis, MO) was added to the cell culture vessel, and the lysate was collected with the help of a cell spatula (TPP, Trasadingen, Switzerland). As quickly as possible, the lysate was transferred into a 5 mL ED Eppendorf tube (Eppendorf, Hamburg, Germany) containing 1.5 mL of phenol/chloroform/isoamyl alcohol 25:24:1 solution (all chemicals from Sigma-Aldrich Inc., St. Louis, MO, USA) and mixed vigorously. The aqueous phase was then transferred to a new ED tube containing 3.5 mL of 100% EtOH (Sigma-Aldrich, St. Louis, MO, USA), mixed, and centrifuged at 2200× g for 10 min at room temperature (RT). The supernatant was discarded, and the pellet was first washed with 100% EtOH and then resuspended in 100 µL of Accugene water (Lonza, Basel, Switzerland). Finally, the pellet was processed as indicated in the RT-qPCR analysis section (Section 2.5.2).