Nb400 135

AGS and SGC-7901 cells were cultured on a confocal laser dish, which were then washed, fixed, permeabilized. Afterwards it was incubated with anti-CHREBP (1:100, #NB400-135, Novus Biologicals, Germany) and anti-cyclin D1 (1:250, #55506, Cell Signaling Technology, USA) antibodies at 4 °C for 24 h. Samples were then blocked, washed, and incubated with a FITC-conjugated goat anti-rabbit or Cy3-conjugated goat anti-mouse IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA). In addition, DAPI (1:1000, RiboBio, China) was employed to stain cell nuclei for 3 min. TCS SP8 confocal microscope (Leica, Wetzlar, Germany) was utilized to microscopically analyze cells.

Immunohistochemistry was carried out following an established protocol^{40 (link)}. Briefly, colon tissue microarray or paraffin tissue sections were dewaxed, rehydrated through graded ethanol and incubated with 3% hydrogen peroxide for 30 min. Antigen retrieval was performed by heating the sections to 95 °C in 0.01 mol/l citrate buffer (pH6.0) for 15 min. Slides were then washed in PBS for 15 min and treated with 10% normal horse serum for 30 min and incubated with primary antibody at 4 °C overnight. The reaction products were detected with the 3-amino-9-ethylcarbozole (AEC) substrate-chromogen kit after incubating with the secondary antibody (Dako REAL EnVision Detection Kit (Dako, Carpinteria, CA)) for 30 min and washing in 0.1 M PBS at room temperature. Staining with AEC resulted in red signals. The primary rabbit-derived polyclonal antibody for anti-ChREBP (Novus biologicals, NB400-135, USA) was used at a 1:200 dilution. For negative controls, primary antibodies were replaced with PBS.

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).

Immunohistochemistry was carried out following an established protocol [31 (link)]. Briefly, liver tissue microarray or paraffin tissue sections were dewaxed, rehydrated through graded ethanol and incubated with 3% hydrogen peroxide for 30 min. Antigen retrieval was performed by heating the sections to 95 °C in 0.01 mol/l citrate buffer (pH 6.0) for 15 min. Slides were then washed in PBS for 15 min and treated with 10% normal horse serum for 30 min and incubated with primary antibody at 4 °C overnight. The reaction products were detected with 3-amino-9-ethylcarbozole (AEC) substrate-chromogen kit after incubating with the secondary antibody of Dako REAL EnVision Detection Kit (Dako, Carpinteria, CA) for 30 min and washing in 0.1 M PBS at room temperature. Staining with AEC resulted in red signals. The primary antibody for GLUT2 (Novus Biologicals, NBP2-22218SS, USA), GLUT1 (Abcam, ab115730, USA), were used at a 1:500 dilution and anti-ChREBP (Novus Biologicals, NB400–135, USA) was used at a 1:200 dilution. The antibodies against GLUT2 and ChREBP were rabbit-derived polyclonal and the antibody against GLUT1 was rabbit-derived monoclonal. For negative controls, the primary antibodies were replaced with PBS.