Ab125219

The colon of the rat was fixed in 4% paraformaldehyde for 24 h and embedded in paraffin, and tissue sections (5 μm) of the colon were prepared for immunofluorescence analysis. For in vitro experiments, HT-29 cells were plated in 12-well plates and treated with drug serum or PMA (1 μM; Beyotime, Shanghai, China) for 24 h. After washing with PBS twice, cells were fixed in 4% paraformaldehyde for 10 min at room temperature and prepared for immunofluorescence analysis.
For immunofluorescence analysis, tissue sections (5 μm) of the colon or HT-29 cells were permeabilized with 0.1% Triton X-100, blocked with 5% BSA, and then incubated with primary rabbit anti-AQP3 antibody (ab125219; Abcam, CA, USA) overnight at 4°C. Then, the tissue and cells were washed twice with PBS/0.1% Tween-20 and incubated with a secondary goat anti-rabbit antibody conjugated with Alexa Fluor 488 for 1 h at room temperature. Then, the tissue and cells were reacted with 4, 6-diamidino-2-phenylindole (DAPI) solution (Beyotime) in PBS at room temperature for 5 min and then observed under a fluorescence microscope (Olympus Corporation, Tokyo, Japan) and photographed. The expression level of AQP3 in rat colon or HT-29 cells was analyzed by ImageJ software.

An ice cold RIPA lysis buffer (Sigma, USA) containing 10% PMSF (Sigma, USA) was used to lyse the cells for 30 minutes. The protein solving liquid was centrifuged at 12000 × g for 20 minutes at 4°C and the supernatants were collected. Equal amount of proteins were separated through electrophoresis utilizing 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred to a polyvinylidene fluoride membrane (PVDF; Millipore, USA). After blocking with 5% fat‐free milk in TBST buffer at room temperature for one hour, the blots were then incubated with the primary antibodies at 4°C overnight. The primary antibodies were against AQP3 (1:1000; cat. ab125219, Abcam, Cambridge, USA), E‐cadherin (1:1000; cat. ab1416, Abcam), N‐cadherin (1:1000; cat. ab18203, Abcam), Vimentin (1:1000; cat. ab8978, Abcam), p‐PI3K (1:1000, cat. BS4605, Xinle Biology, Shanghai, China), PI3K (1:1000, cat. MA5‐26514, Thermo Fisher Scientific, Waltham, MA, USA), p‐AKT (1:1000, cat. 4060, Cell Signaling), AKT (1:1000, cat. 4691, cell signaling) and GAPDH. The membranes were subsequently incubated with anti‐rabbit or mouse HRP‐conjugated secondary antibody for two hours at room temperature. Finally, the signals were evaluated by enhanced chemiluminescence (ECL, Pharmacia Biotech, Arlington, USA).

Each TMA contained 17 patients’ tissues including 2 cores of tumor tissue and 1 core of paracancerous tissue for one patient. The core diameter of TMA was 1.6 mm. The immunohistochemical staining was performed on a Leica BOND-MAX autoimmunostainer (Leica Instrument Co., Ltd.). The tissue chips were first cut into 4-μm-thick slices using a microtome, attached to slides, dried, and baked at 60°C for 24 h before use. The prepared slices were dewaxed with xylene, rehydrated with different concentrations of ethanol (100% and 95%), rinsed with distilled water, and repaired with heat-induced antigen. Finally, the antigen was detected by an automatic staining machine (Benchmark) and Bond Polymer Purification Detection Kit (LeicaInstrumentCo., Ltd.). The primary antibodies (Abcam) were diluted as follows: anti-AQP1 (ab168387, 1:1,000), anti-AQP3 (ab125219, 1:500), and anti-AQP5 (ab92320, 1:200).

The AQP3 and beclin-1 expression were measured by adding the primary antibodies (AQP3: ab125219, Abcam, Cambridge, UK; beclin-1: GTX31722, GeneTex, Irvine, CA, USA), followed by the secondary antibody of goat Alexa Fluor 568-conjugated anti-mouse IgG (H + L) (A11004, Invitrogen, Waltham, MA, USA). Beclin-1 expression reflects autophagy. Nuclei were stained with DAPI (Fluoromount-G; Sigma, St. Louis, MO, USA). Fluorescence images were acquired by Olympus BX53 microscope and analyzed using ZEN software (Zeiss, Oberkochen, Germany)^{31 (link)}.

Total cell proteins were extracted using a 99:1 mixture of cell lysate and protease inhibitor, added to loading buffer and then boiled in boiling water for 5 min. Denatured protein samples were electrophoresed on 10% SDS/polyacrylamide gel (SDS/PAGE), and then the proteins on the gel were transferred to PVDF membrane. The PVDF membranes were closed with 5% milk powder and incubated with primary antibodies for N-cadherin (United Kingdom, Abcam,ab125219,1:500), vimentin (United Kingdom, Abcam, ab125219, 1:500) and β-actin (China, Bioss, AH11286402), followed by incubation with secondary antibody (China, absin, abs20040ss, 1:5,000). Finally, exposure development was performed using an enhanced chemiluminescence kit (ECL; China, Meilunbio, MA0186).

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).