Tweak

Appropriate cell culture conditions were established as the controls needed in the expression of genes with and without peptides and with and without TWEAK activation, both as pools and individually (see Supplementary Materials Figure S2). The protocol started with the seeding of 125,000 cells/well in 500 µL of culture media supplemented with 10% fetal bovine serum (CM+FBS10%) in 24-well dishes for 24 h before removing FBS (CM-FBS). After overnight starvation in CM-FBS culture, different peptide treatment conditions (at final concentration of 450 µM) were established in the presence or absence of TWEAK (25 ng/mL final concentration) (Peprotech EC, Ltd., London, UK). Some cells were treated with peptides at a single, unique, initial dose (time 0) while other were treated with repeated doses to account for the effect cell-culture degradation. These include a repeated treatment every hour (i.e., time 0, 1, 3, 4, 5), every two hours (time 0, 2, and 4) and every three hours (time 0 and 3)—see Supplementary Materials Figure S2 for diagrammatic depiction. At time 6 (after six hours) cells were lysed and RNA extracted for transcriptomic analyses (see next). Cell were culture for six hours as this was the time needed to achieve the highest upregulation of the TWEAK-Fn14 axis upon TWEAK treatment as shown in previous time-course RT-PCR experiments (see Figure 2).

Human recombinant TWEAK was from PeproTech (Hamburg, Germany). All experiments were performed under serum-free conditions. Cells were then either left untreated or treated with 10–100 ng/mL TWEAK alone for various time periods. Cell culture experiments for RNA isolation were performed for 6 hours and those for protein isolation after 24 hours, if not indicated otherwise. Cardiomyocytes were transfected with replication deficient adenoviral vectors carrying TNFSF12 (Vector BioLabs, Philadelphia, USA) or LacZ at a multiplicity of infection (MOI) of 10 and 50. Analyses were performed after 48 hours of adenoviral infection. The ADP/ATP ratio was measured after 6 hours according to the manufacturer's instructions (EnzyLight ADP/ATP Ratio Assay Kit, ELDT-100, Bioassay Systems, Hayward, USA). Metabolic experiments and ADP/ATP assay kit as functional readout were performed in both NRVCMs and ARVCMs since immature and mature cardiomyocytes differ in terms of metabolism, gene expression, and receptor composition. Total RNA was isolated using the TRIzol method (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions and resuspended in DEPC-treated water (Sigma, München, Germany). RNA integrity and purity were verified by measurement of OD260/OD280 absorption ratios. TUNEL assay (Roche Cell death detection kit) was performed after 24 hours.

Primary mouse hepatocytes were isolated from 10- and 20-week-old mice using collagenase perfusion as previously described [26 (link)]. Fresh primary mouse hepatocytes were cultured or co-cultured in 5% FBS–Williams’ Medium E and subsequently treated with 100 μM conjugated BAs or 100 ng/ml TWEAK (Peprotech, NJ, USA) for 12 or 24 h. Finally, whole cell lysates were collected as described previously [26 (link), 29 (link)] for subsequent real-time qPCR and Western blot analysis.

For generating enteroids, the small intestine or colon were cut longitudinally and incubated for 15 min at room temperature in DPBS containing 2.5 μg/mL amphotericin B, 25 μg/mL gentamicin and 50 μg/mL normocin. The intestines were incubated in 10 mM DTT for 15 min at room temperature and followed by gentle rotation in 8 mM EDTA at 4 °C for 75 min. After three washes with DPBS, the crypts were separated by snap shaking. The crypts were washed with DPBS containing antibiotics and then pelleted by spinning at 40 × g for 2 min at 4 °C. The pellet was suspended in a solution of 66% Matrigel (Corning, Corning, NY), 33% LWRN medium, and 10 μM Rock inhibitor (Y27632; Miltenyi, San Diego, CA) at a concentration of 2 crypts/μL. A volume of 250 μL of crypt suspension were plated in a 6-well plate. On the fourth day of culture, enteroids were treated with either vehicle or 100 ng/mL human recombinant RANKL (ProSpec, East Brunswick, NJ), 100 ng/mL Tweak (PeproTech Inc., Rocky Hill, NJ), 10 ng/mL Ltα/β for 24- or 72-h and then processed for RNA or protein or for histological analysis. For UEA staining, frozen enteroid sections were stained with FITC-conjugated-UEA1 for 1-h in dark at room temperature and then washed with PBST thrice before imaging.