Dispase solution

Prostatospheres were assayed as described (Lukacs et al., 2010a (link)). In brief,
3,000 Akt transformed prostate stem cells were resuspended in 100 μL of a 60:40 mixture of Matrigel (BD Bioscience,
NJ):PrEGM (Lonza, Basel, Switzerland) and plated in triplicate around the rim of a 12-well tissue culture plate. Matrigel was
allowed to solidify at 37°C, and 800 μL of PrEGM/well was added. Medium was changed twice weekly and spheres
were counted after 7 to 10 days. Each experiment was repeated 3 times. To passage the spheres, wells were treated with 1ml of
1 mg/ml Dispase solution (GIBCO, NJ). Spheres were digested with 0.25% trypsin with 2.21mM EDTA to obtain single cells, and
equal numbers of cells (2,000) were seeded in triplicate in Matrigel. The same protocol was used for all 6 passaged
generations.

Primary NHOKs and normal human oral fibroblasts (NHOFs) were obtained according to previously described methods (9 (link)) following the approval of the Institutional Review Board (IRB# 04-060-04). Briefly, discarded oral mucosal tissues were collected without identifiers from the normal patients who are undergoing routine dental procedures (e.g., gingivectomy for the crown-lengthening procedures). The obtained oral mucosal tissues were cut into 25 mm² × 0.5 mm sections and incubated in 2.5 mg/ml dispase solution (Cat. no. 17105; Gibco, Grand Island, NY, USA) in 37°C for 1 h, after which the epithelial layers and connective layers were separated. The separated epithelial layers were minced and subjected to trypsin digestion (Cat. no. 25200056; Gibco) in 37°C for 5 min to harvest the NHOKs. The separated connective tissue layers were minced and subjected to collagenase digestion (Cat. no. 17100017; Gibco) in 37°C for1 h to harvest the NHOFs. The NHOKs were cultured in EpiLife supplemented with human keratinocyte growth supplement (HKGS) (Cascade Biologics, Portland, OR, USA), and the NHOFs were cultured in DMEM supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA). ZOL and PAM were purchased from LKT Laboratories, Inc. (St. Paul, MN, USA). Throughout the study, 1, 2, and 4 µM of ZOL and 10, 20 and 50 µM of PAM were used to treat the cells for the indicated periods of time.

Biopsies were rinsed with PBS containing antibiotics and incubated in 0.25% Dispase solution (Gibco, Thermo Fisher Scientific, USA) overnight at 4°C. On the following day, the epidermis was peeled from the dermis and incubated in the solution with 0.25% Trypsin-EDTA (Gibco) at 37°C for 10–15 min, and then the digestion was terminated with DMEM solution (Gibco) containing 10% fetal bovine serum (Gibco). The epidermis was blown and filtered, and then the KCs suspension was collected. The psoriatic KCs were seeded in culture flask filled with defined KC serum-free medium supplemented with growth supplement and bovine pituitary extract (Gibco). Cells were grown in the incubator at 37°C with 50 ml/L CO₂ and 95% relative humidity. At flaky confluence, KCs passage was made. The third generational KCs were applied for later experiments. Each experiment described below was performed three times using the same lot of KCs.