Tip39 peptides

Normal human epidermal keratinocytes (NHEK) were cultured as previously described (9 ). 3D in vitro skin constructs were prepared as described (10 ). TIP39 peptides (10μM; Bachem Americas, Torrance, CA) or control peptides (10 μM; GenScript, Piscataway, NJ) were added to keratinocyte growth media for 6days.

TIP39 peptides were obtained from Bachem Americas (Torrance, CA). Goat anti-decorin (R&D, Minneapolis, MN), rabbit anti-CREB (Abcam, Cambridge, MA), rabbit anti-pCREB (Abcam), rabbit anti-laminB1 (Abcam), goat anti-perilipin (Abcam), goat anti-actin (Santa Cruz, Dallas, TX), and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (Abcam) were used as primary antibodies for immunoblotting or immunostaining. Alexa Fluor 488- or 594-conjugated donkey IgG (Thermo Fisher Scientific, Waltham, MA) were used as secondary antibodies for immunostaining.

NHEK were cultured as previously described (MacLeod et al., 2015 (link); Morizane et al., 2010 (link); Yamasaki et al., 2011 (link)). Cells at 70% to 80% confluence were stimulated with 0.06 mM or 1.2 mM CaCl₂ (Life Technologies, NY) from 12 hours to 120 hours. Three-dimensional in vitro skin constructs were prepared as described (Borkowski et al., 2013 (link)). TIP39 peptides (10–1000 nM; Bachem Americas, Torrance, CA; Phoenix Pharmaceuticals, Burlingame, CA), HYWH-TIP39 peptides (0.1–10 μM; gift from Dr Usdin, NIMH, MD), or control (phosphate buffered saline) was added in culture media from 24 to 120 hours. All culture media and chemicals were replaced every other day.