Ca 074

Human colonic epithelial NCM460 cells were cultured in RPMI 1640 medium with no glucose (Invitrogen, Carlsbad, CA, USA, 11879020) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM glucose, and 10% fetal bovine serum (FBS). The cells were treated with 2.0% DSS (MP Biomedicals, Santa Ana, CA, USA, molecular weight of 36,000–50,000) in the presence or absence of mannose (25 mM) for 24 h. To eliminate the effect of the mannose transporter, cells were pretreated with ouabain (1 μM, Selleck, S4016) for 4 h before incubation. Rotenone (5 μM, Merck, HY-B1756) was added simultaneously with DSS and mannose to inhibit mitochondrial respiratory activity. In some cases, protease inhibitors, such as the cathepsin B inhibitor CA-074 (10 μM, Selleck, HY-103350), cysteine protease inhibitor aloxistatin (1 μM, Selleck, S7393), serine protease inhibitor nafamostat mesylate (5 μM, Selleck, S1386), and aspartic protease inhibitor pepstatin A (10 μM, Selleck, S7381), were added simultaneously with DSS and mannose. Bafilomycin A1 (1 μM, Selleck, S1413) was added simultaneously with DSS and mannose for 24 h. mito-tempo (2 μM, Merck, HY-112879), a mitochondrial-targeted antioxidant, was used to stimulate cells along with DSS plus mannose stimulation.

Antibodies against HDAC3 (#85057, 1:1000 for western blot; #3949, 1:100 for immunofluorescence staining), HDAC1 (#5356, 1:1000), HDAC2 (#5113, 1:1000), HDAC8 (#66042, 1:1000), CTSB (#31718, 1:1000 for western blot; 1:100 for immunofluorescence staining), CTSD (#69854, 1:1000), RIP1 (#3493, 1:1000), GAPDH (#2118, 1:2000), β-ACTIN (#4970, 1:2000), Phosphorylated-RIP1 (#38662, 1:1000), Phosphorylated-P65 (#3033, 1:1000), P65 (#8242, 1:1000 for western blot; 1:100 for immunofluorescence staining), H3K27ac (#8173, 1:50 for CHIPseq) were purchased from Cell Signaling Technology (CST). Leupeptin (#S7380), CA-074 (#S7420) were bought from Selleck. MG132 (#M8699) was bought from Sigma-Aldrich. TNFα (#410-MT-010) was bought from R&D systems. Lipo3000 (InvitroGen, #L3000015) was used for the transfection experiment. PCMV-C-EGFP (#D2626) was bought from Beyotime Biotechnology. The plasmids needed for the experiment were synthesized by GENEWIZ.