LXR Regulation of Epidermal Barrier Genes

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).

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Na T.Y., Kim G.H., Oh H.J., Lee M.H., Han Y.H., Kim K.T., Kim J.S., Kim D.D, & Lee M.O. (2017). The trisaccharide raffinose modulates epidermal differentiation through activation of liver X receptor. Scientific Reports, 7, 43823.

Publication 2017

PA1–332 sc-66192 sc-14719 sc-51130 ab18180 ab52617 I9018 NB400-135 ab125219 sc-1616 sc-74 sc-28310 sc-585 ab4441

Abca1 Abcg1 Actin Antibodies Biologicals Cdna Charcoal Chip assays Filaggrin Hacat cells Involucrin Isolation Loricrin Novus P300 Primers Steroid Synthesis α tubulin

Corresponding Organization :

Other organizations : Seoul National University

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Variable analysis

independent variables

Testing materials

dependent variables

Expression of LXRα, LXRβ, filaggrin, SCD1, loricrin, ABCA1, ABCG1, involucrin, ChREBP, AQP3
Binding of LXR, JunD, Fra1, p300, AcH3K9 to DNA

control variables

HaCaT cells
Culture medium (DMEM with 5% charcoal-stripped FBS, then DMEM with 1% charcoal-stripped FBS)
Isolation of total RNA, cDNA synthesis, and qRT-PCR procedures
Western blotting procedures
ChIP assay procedures

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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