T84 cells

T84 cells (ATCC, Manassas, VA) were maintained at 37°C/5% CO₂ in complete Dulbecco’s modified Eagle Medium/F12 (cDMEM/F12): DMEM/F12 (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT), 100 U/ml Penicillin/Streptomycin (Gibco, Gaithersburg, MD), and 2 mM L-glutamine (Gibco). T84 cells were passaged at no greater than 80% confluency. Caco-2 cells (ATCC) were maintained at 37°C/5% CO₂ in complete DMEM (cDMEM): low glucose DMEM (Invitrogen) supplemented with 10% heat-inactivated FBS, 100 U/ml Penicillin/Streptomycin, and 2 mM L-glutamine.

The T84 cell assay was performed essentially as described previously [26 (link)]. Briefly, T84 cells (ATCC, Rockville, MD, USA) were seeded and grown to confluence on 48-well plates (Nunc, Roskilde, Denmark) in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM-F12) (Gibco life technologies, Paisley, UK), supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 0.2% gentamicin (LONZA, Walkersville, MD, USA). The cells were washed 3 times with 250 µL DMEM-F12 and pre-incubated with 40 µL DMEM-F12 containing 1 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich) for 10 min at 37 °C. A volume of 40 µL 1 µM native or mutant STh peptide was added to each well in duplicate (final peptide concentration 0.5 µM) and incubated for 30 min at 37 °C. Following incubation, the reaction medium was aspirated, and the cells were lysed with 0.1 M HCl at 20 °C for 20 min. Subsequently, the lysates were centrifuged at 16,000× g for 10 min, and the supernatants were collected to estimate cGMP levels using a cGMP enzyme immunoassay kit (Enzo Life Sciences, Inc., Farmingdale, NY, USA). The analysis was conducted according to the manufacturer’s instructions.

Authenticated Caco-2 cells and T84 cells were purchased from the ATCC. Authenticated Vero cells were kindly provided by William Goins. Caco-2 cells were cultured in 1× MEM (minimum essential medium) with Earle’s salts and l-glutamine (Corning, Cellgro) as well as 20% heat-inactivated fetal bovine serum (FBS; Sigma). Vero cells were cultured in DMEM (Dulbecco’s modification of Eagle’s medium) with 4.5 g/liter glucose, l-glutamine, and 5% FBS. T84 cells were grown in a 1:1 mixture of DMEM–F-12 medium with l-glutamine, HEPES, and 5% FBS. Cultures were grown to confluence in a humidified incubator at 37°C with 5% atmospheric CO₂.

IEC6 rat neonatal IECs, HT29 cells, and T84 cells (all from ATCC, Manassas, VA) were cultured under standard conditions [17 (link)] and were treated with all-trans retinoic acid (atRA; Sigma, St. Louis, MO). All chemical inhibitors used in this study were purchased from Santa Cruz Biotechnology, Santa Cruz, CA. SB203580, a pyridinyl imidazole, inhibits p38 mitogen-activated protein kinase (MAPK); Wortmannin is a selective inhibitor of the phosphatidylinositide 3-kinases (PI3K); SP600125 is a selective inhibitor of the c-Jun N-terminal kinases (JNK); PD98059 is a selective, cell-permeable inhibitor of the mitogen-activated protein kinase kinase 1 (MEK1); and Y-27632 dihydrochloride inhibits the Rho-associated, coiled-coil-containing protein kinases (ROCK).

HA (high molecular weight sodium hyaluronate 1.2 X 10⁶ Da), CAS No: 9067‐32‐7 purchased from Lifecore Biomedical, USA. Phosphate buffered saline (Lot # SLBP8218V) purchased from Sigma–Aldrich, USA. PEG (Mw 2000 Da‐CAS No.: 25322–68‐3, purity >95%) purchased from JenKem Technology, USA (Allen, TX). DMTMM (4‐[4.6‐Dimethoxy‐1,3,5‐triazin‐2‐yl]‐4‐methylmorpholinium chloride; Mw 276.72), CAS No: 3945‐69‐5. Na₂SO₄ (CAS No. 7757‐82‐6), EtOH (CAS No. 64–17‐5), NaCl (CAS No. 7647‐14‐5), dialysis membrane (Spectra Por, Mol 6–8 kDa), and 2‐(N‐morpholino) ethanesulfonic acid (MES salt) (CAS No. 4432‐31‐9) purchased from Sigma–Aldrich (Arklow, Ireland). HTB‐2 and T84 cells were purchased from ATCC (Manassas, VA). Cell culture reagents, Dulbecco's Modified Eagle's Medium (DMEM) (D5796) Fetal bovine solution (FBS) (F0804), penicillin–streptomycin (P4333), trypsin–EDTA (T4049), Hank balanced salt solution (HBSS) (H8264), protamine sulfate (P3369) and 0.9% NaCl solution (S8776) were purchased from Sigma–Aldrich (Arklow, Ireland). Iron‐enriched FBS for culturing T84 cells (SV30160.03) was purchased from Biosciences (Dublin, Ireland). Recombinant human TNF‐alpha (300‐01A) was purchased from Pepro Tech (London, UK).