Phosphor jnk

The human gastric cancer cell lines AGS and SGC-7901 were prepared from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cultivation of the cells was regularly performed in RPMI-1640 media with the supplementation of 10% fetal bovine serum (Gibco, USA). Moreover, streptomycin (100 U/mL) and penicillin (100 U/mL) were inserted in the above-mentioned culture system which was set at 37 °C in a moistened air under 5% CO2.
Melatonin was bought from (St. Louis, MO, Sigma Aldrich, USA), dissolved at a concentration of 1M as a stock solution in DMSO, and diluted with culture medium to suitable concentrations prior to use. Antibodies against β-actin, LC3A/B, Beclin-1, Ki-67, JNK, and phosphor-JNK were obtained from Cell Signaling Technology (Danvers, MA, USA). The P62 antibody was from Proteintech (Rosemont, IL, USA).Antibodies consist of IRE1α, GRP78, Caspase-3, Bax, and Bcl-2 were obtained from Abcam (Cambridge, Massachusetts, USA). The antibody was from 4-PBA was bought from Sigma Aldrich. 3-methyladenine(3-MA) and STF-083010 were purchased from Med Chem Express.

Cells were lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton-X 100, 1 mM each MgCl₂, MnCl₂ and CaCl₂, 1 mM PMSF and 10 mM sodium fluoride) [23 (link),24 (link)]. Tissues were homogenized in T-PER tissue protein extraction reagent (Thermo, 78510), in which protease inhibitor cocktail (Biotool, B14001) was added. Insoluble material was removed by centrifugation at 10,000 g for 10 min. Proteins were separated by SDS-PAGE under reducing condition, followed by blocking in phosphate-buffered saline/Tween-20 containing 1% BSA. The membrane was incubated with antibodies for CTHRC1 (Huabio), TGFBR1 (Cell Signaling, 3712S), TGFBR2 (Cell Signaling, 11888S), TGFBR3 (Cell Signaling, 2519S), Endoglin (Cell Signaling, 4335S), phospho-Smad2 (Cell Signaling, 3108P), Smad2 (Cell Signaling, 5339P), phospho-Smad3 (Cell Signaling, 9520P), Smad3 (Cell Signaling, 9523P), Smad4 (Cell Signaling, 9515P), phospho-TAK1 (Cell Signaling, 4508S), TAK1 (Cell Signaling, 5206S), phospho-p38 (Cell Signaling, 4511S), p38 (Cell Signaling, 8690S), phosphor-JNK (Cell Signaling, 4668S), JNK (Cell Signaling, 9252S), Wnt5a (Cell Signaling, 2392S), and GAPDH (Huabio, M1211-1), followed by incubation of species-specific secondary antibodies. Bound secondary antibodies (LI-COR, 926-32213; 926-68051) were revealed by Odyssey imaging system (LI-COR).

DMAS (purchased from Tokyo Chemical Industry) was dissolved in DMSO, and the DMSO content in all groups was 0.1%. MTT, DAPI and SP600125 were obtained from Calbiochem (San Diego, CA, USA). RPMI 1640 medium and FBS were obtained from Gibco Life Technologies (Grand Island, NY, USA). Pan‐caspase inhibitor (Z‐VAD‐FMK) was purchased from Beyotime Biotechnology Corporation (Shanghai, China). 3‐Methyladenine (3‐MA), chloroquine (CQ), 4‐phenylbutyrate (4‐PBA), and monodansylcadaverine (MDC) were obtained from Sigma‐Aldrich (St Louis, MO, USA). Bafilomycin A1 was obtained from Selleck Chemicals (Houston, TX, USA). Primary antibodies (ie poly ADP ribose polymerase [PARP], cleaved caspase‐3, cleaved caspase‐8, cleaved caspase‐9, LC3B, Atg5, Beclin‐1, Bip, phospho‐eIF2α, ATF4, CHOP, IRE1α, and phosphor‐JNK) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA).

Gefitinib was purchased from J&K Chemical (Beijing, China). Sorafenib, regorafenib, MK2206 (AKT inhibitor), and PD98059 (ERK inhibitor) were obtained from MedChem Express (Princeton, NJ, USA). The primary antibodies against Sox2, Oct4, Nanog, STMN1, E-cadherin, N-cadherin, vimentin, ERK, phosphor–ERK, Akt, phosphor–Akt, JNK, phosphor–JNK, MMP-9, Histone3, and β-actin were obtained from Cell Signaling Technology. The primary antibodies against FOXM1, E2F1, and MMP2 were purchased from Abcam. Silencer Select Validated siRNAs against STMN1 and FOXM1 were obtained from Life Technologies, Carlsbad, CA, USA.