Mouse normal colon intestinal epithelial cells (BALB-5047, USA) were obtained from the RIKEN Cell Bank (Ibaraki, Japan). The cells were cultured in 96-well culture plates (5 × 106 cells/mL) and 12-well culture plates (5 × 105 cells/mL). Some LPS-treated cells (10 nM, Solarbio Ltd., Beijing, China) were treated with 2 μM TWS119 (a selective GSK-3β antagonist; MCE, Nanjing, USA; LPS + TWS-cells), 100 μM PDTC (an NF-κB antagonist; MCE, Nanjing, USA; LPS + PDTC-cells), or 5 mM butyrate (Sigma-Aldrich, St. Louis, MO, USA; LPS + butyrate cells). After butyrate supplementation for 30 min, some LPS + butyrate cells were sequentially treated with 50 μM ITSA-1 (a nonselective HDAC3 agonist; MCE, New Jersey, USA; LPS + butyrate + ITSA-1-cells) or 5 nM Chetomin (a nonselective HIF-1α antagonist; Abcam, Cambridge, MA, USA; LPS + butyrate + Chetomin-cells). Each plate of treated cells was incubated for 24 h.
The cells from the 96-well culture plates were assessed for proliferation activity using an MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide; Sigma, St. Louis, MO, USA) assay. The optical density was determined using a microplate reader (Model 680, Bio-Rad, St. Louis, MO, USA) equipped with a 570 nm wavelength filter. The cells from the 12-well culture plates were collected for LDH assessment and Western blotting. Each assay used a repeat of 8 wells.
The cells from the 96-well culture plates were assessed for proliferation activity using an MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide; Sigma, St. Louis, MO, USA) assay. The optical density was determined using a microplate reader (Model 680, Bio-Rad, St. Louis, MO, USA) equipped with a 570 nm wavelength filter. The cells from the 12-well culture plates were collected for LDH assessment and Western blotting. Each assay used a repeat of 8 wells.