P65 inhibitor bay11 7802

RARα (sc-773), MEK1/2 (sc-436), NF-κB-p65 (sc-109), IκB-α (sc-1643), p62 (sc-28359) and actin (sc-47778) antibodies were obtained from Santa Cruz Biotechnology (TX, USA); The antibodies to CD11b (C0551) and ICAM-1 (C2969) were from Sigma(St. Louis, MO, USA); Antibody to LC3 (#NB600-1384) was from Novus (Littleton, CO, USA); The p-ERK1/2 (#9101), ERK1/2 (#4695), p-JNK (#9251), JNK (#9252), p-P38 (#9215), P38 (#9212) and PCNA (#2586) antibodies were from Cell Signaling Technology (MA, USA); ATRA, ATO, DMSO, JNK inhibitor (SP600125), p38 inhibitor (SB203580) and ERK inhibitor (U0126) were from Cell Signaling Technology (MA, USA); p65 inhibitor (BAY11-7802) was from Merck Millipore (Darmstadt, Germany); The recombinant HMGB1 protein was kindly provided by Dr. Rui Kang (University of Pittsburgh, USA).

APL cell line NB4 was generously provided by Prof. Tong Jian-hua (Shanghai Ruijin Hematology Institute, China), which originated from Dr. M. Lanotte, INSERM Unit 301, St. Louis Hospital (Paris) [15] (link). This cell line was established from a patient with APL, who harbors characteristic chromosome abnormality, i.e., t(15∶17), and responds to ATRA with granulocytic differentiation. NB4 cells were seeded at a density of 5×10⁵ cells/mL and maintained in RPMI-1640 culture medium supplemented with 10% fetal calf serum, 2 mM of L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin stored at 37°C in a 5% CO₂ humidified atmosphere.
For experimentation, NB4 cells were plated at 2×10⁵ cells/ml in complete RPMI- 1640 medium and pre-treated with celastrol or protein inhibitors for 30 min, and then incubated in the presence of ATRA (1 µmol/L) for the indicated times. ATRA at 1 µM was used to induce NB4 maturation to granulocytes. P38 inhibitor SB203580, JNK inhibitor SP5600125, and MEK inhibitor U0126 were purchased from Cell Signaling Technology (Danvers, MA, USA). P65 inhibitor BAY11-7802, ERK inhibitor FR180204, and STAT1 inhibitor AG490 were purchased from Merck Millipore (Calbiochem, Darmstadt, Germany).