Jsh 23

At the end of the incubation for cell attachment, the culture supernatants were replaced with growth medium containing 0 or 60 μM JSH-23 (i.e., 4-methyl-N1-(3-phenylpropyl)-1,2-benzenediamine, catalog no. 15036; Cayman Chemical, Ann Arbor, MI), an NF-κB transcription factor inhibitor, and the cells were incubated 1h under cell culture conditions. LPS (500 ng/mL final concentration) was then added to each well, and the cells were incubated 6h under cell culture conditions. At the end of this incubation, the culture supernatants were replaced with growth medium containing 0 or 60 μM JSH-23, and the cells were incubated an additional 1h. Cr³⁺ (300 ppm final concentration), nigericin (5 μM; positive control), or cell culture-grade water (negative control) was then added to the culture supernatants and the cells were incubated an additional 18 to 24h. In total, four JSH-23 conditions were tested: no JSH-23, JSH-23 present exclusively during the priming incubation with LPS, JSH-23 present exclusively during the activation incubation with Cr³⁺ or nigericin, and JSH-23 present during both incubations. At the end of the 18 to 24h incubation, supernatants were collected, centrifuged at 300 g for 10 min at 4°C, snap-frozen in liquid nitrogen, and stored at -80°C for IL-1β measurements by ELISA, as described above.

PAM212 cells (Lonza Walkersville, Inc.) were seeded in a medium composed of Eagle's minimal essential media, 10% FBS, l-glutamine, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, sodium pyruvate, nonessential amino acids, and penicillin-streptomycin (Gibco, Waltham, MA, USA). First, cells were treated with 24 h of starvation in a 2% FBS-added medium. Then, UVB irradiation (5.5 mJ/cm²) was performed with the use of an UVB device (302 nm) supplied by Rayminder, Ottawa Hills, OH, USA. Following this, cells in the stimulation groups were given 100 ng/ml recombinant IL-33 (ACROBiosystems) or 100 ng/ml bovine serum albumin (Gibco, USA) intervention. After 2 h of NF-κB inhibitor JSH-23 (purity ≥ 98%, 20 μM; Cayman), wortmannin (WM; 25 μg/ml; Sigma), and U0126 (10 μM; Promega) intervention, UVB and IL-33 interventions were carried out.