Pepstatin a

This study was performed in accordance with institutional ethical guidelines and was approved by the Ethics Committee of the Nanfang Hospital. Informed written consent was obtained from each patient. Specimens from 87 patients with laryngeal carcinoma (two women and 85 men, aged 40 to 86 years) were collected from the laryngeal carcinoma tissue bank of the Nanfang Hospital. The tissue specimens were routinely frozen in liquid nitrogen, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned according to routine procedures.
The American Type Culture Collection (ATCC) human laryngeal carcinoma cell line Tu212 was purchased from Guangzhou Juyan Biological Technology (Guangzhou, China) and Hep-2 was purchased from Shanghai Aolu Biological Technology (Shanghai, China). The cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin. The cells were maintained at 37 °C in a humidified 5% CO₂ atmosphere. Porcine pepsin (Sigma-Aldrich, St Louis, MO, USA) was used for pepsin exposure. The pepsin inhibitor pepstatin A and the interleukin-8 (IL-8) inhibitor SB225002 were synthetized by Selleckchem (Shanghai, China).

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.

The DK from E. cava was prepared in previous our studies [20 (link),23 (link),25 (link)]. The MTT was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The Z-VAD-fmk, 3MA, HCQ, E64D, and pepstatin A were purchased from Selleck Chemicals (Houston, TX, USA). The primary antibodies against caspase-3 (#2696), cleaved caspase-3 (#9664), PARP (#9542), cleaved PARP (#5625), Bcl-2 (#4223), Bax (#5023), Bim (#2933), Bak (#12105), LC3B (#3868) were purchased from Cell Signaling Technology (Danvers, MA, USA). Sequestosome 1 (SQSTM1, p62, sc-28359) and LAMP-1, (sc-20011) were purchased from Santa Cruz (St. Dallas, TX, USA), and actin (MAB1501) was purchased from Millipore (Billerica, MA, USA). All the remaining chemicals and reagents were purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany).