I9018

NHEK cells were seeded in 60-mm dishes and incubated overnight. After treatment with raffinose, the cells were washed twice with phosphate-buffered saline. The culture medium was changed every 48 h for raffinose treatment. Nile-red staining including fluorescence microscopy was performed as previously described39 (link). Immunocytochemistry was performed essentially as previously described using specific antibodies against involucrin (I9018, Sigma-Aldrich), loricrin (sc-51130, Santa Cruz Biotechnology), filaggrin (sc-66192, Santa Cruz Biotechnology), AQP3 (ab125219, Abcam), and LXR (PP-PPZ0412-10, Perseus Proteomics, Tokyo, Japan)39 (link).

HaCaT cells were seeded in DMEM with 5% charcoal-stripped FBS, and the medium was changed to DMEM with 1% charcoal-stripped FBS for treatment of the testing materials to avoid any interference caused by steroid-like substances present in FBS. Isolation of total RNA, cDNA synthesis, and qRT-PCR were performed as previously described using specific primers as shown in Table S2. Western blotting was performed using specific antibodies against LXRα and LXRβ (PA1–332, Thermo Scientific, Waltham, MA), filaggrin (sc-66192, Santa Cruz Biotechnology, CA), SCD1 (sc-14719, Santa Cruz Biotechnology), loricrin (sc-51130, Santa Cruz Biotechnology), ABCA1 (ab18180, Abcam, Cambridge, UK), ABCG1 (ab52617, Abcam), involucrin (I9018, Sigma-Aldrich), ChREBP (NB400-135, Novus Biologicals, Littleton, CO), AQP3 (ab125219, Abcam), Actin (sc-1616, Santa Cruz Biotechnology), and α-tubulin (05-829, Millipore, Billerica, MA), as previously described39 (link). ChIP assays were performed using antibodies against LXR (PA1-332, Thermo Scientific), JunD (sc-74, Santa Cruz Biotechnology), Fra1 (sc-28310, Santa Cruz Biotechnology), p300 (sc-585, Santa Cruz Biotechnology), and AcH3K9 (ab4441, Abcam). Immunoprecipitated DNA was amplified by PCR with specific primers as described previously (Table S2)39 (link).

After 15 days of culture, RHE were fixed with a 4% paraformaldehyde solution for 24 h and kept in 30% sucrose for 48 h at 4°C. RHE on membranes were separated from the insert and embedded in OCT embedding matrix (Leica, Wetzlar, Germany). Immediately, specimens were snap-frozen in isopentane on dry ice and then kept at -20°C. RHE were cut into 7 μm thick slices with a cryomicrotome (Leica). Before immunostaining, cryosections were incubated in citrate buffer (0.01 M and pH 6) at 90°C for 45 min and saturated with TBS containing 1% bovine serum albumin, 3% goat serum, and 0.3% Triton X-100, for 1 hour. The immunohistofluorescent staining was performed using primary antibodies specific to Ki67 (550609, BD Pharmingen, Le Pont-de-Claix, France), involucrin (I9018, Sigma-Aldrich), loricrin (PRB-145P, Covance ref.), catalase (12980, Cell Signaling), SOD1 (ab183881, Abcam), and SOD2 (ab13533, Abcam) which were incubated overnight at 4°C. Cryosections were subsequently incubated with the corresponding secondary antibodies (Alexa Fluor 568 goat anti-rabbit IgG, A-21069, or Alexa Fluor 488 goat anti-mouse IgG, A-21121, Invitrogen) for 1 h at room temperature. Before mounting, nuclei were stained with a solution of 50 μM bisbenzimide (Sigma-Aldrich) for 10 min at room temperature. Finally, cryosections were mounted using Fluoromount-G mounting medium (Thermo Fisher Scientific).