P53 sirna

Cells were transfected with p53 siRNA (Cell Signaling Technology, #6231) or NF-κB p65 siRNA (Cell Signaling Technology, #6261) via LipofectAMINE RNAi max (Invitrogen, 13778150) according to the manufacturer's instructions.

The NF-κB and p53-dependent luciferase reporter plasmids, the constructs expression FLAG-IKKα and FLAG-IKKα-KM were described in our previous reports^{14 (link),17 (link)}. The following primary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA): Beclin-1, LC3B, SQSTM1, ATG5, phospho-p53-Ser15, p53, phospho-IKKα-Ser176/180, IKKα, IKKβ, IκBα, phospho-CHK1-Ser345, CHK1, FLAG, and ACTB. The following primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): IKKγ, GADD45α, and DRAM1. IKKα siRNA, p53 siRNA, and ATG5 siRNA were purchased from Cell Signaling Technology (Beverly, MA, USA). DRAM1 siRNA and CHK1 siRNA were purchased from Riobo Technology (Guangzhou, China). 3-MA, BafA1, and MG132 were ordered from Sigma-Aldrich (St. Louis, MO, USA).

Dehydroandrographolide (DA) of ≥98% (HPLC) purity was purchased from ChemFaces. Stock solution of DA was made at 25, 50 and 100 mM concentration in dimethyl sulfoxide (DMSO) and stored at −20°C. The final concentration of DMSO for all treatments was less than 0.2%. Other chemicals and rapamycin were obtained from Sigma Chemical Co. Caspases inhibitor (Z-VAD-FMK), autophagy inhibitors (3-methyladenine, bafilomycin A1, wortmannin, choloquine) and autophagy inducer (tamoxifen) were obtained from LC Laborstories, St. Woburn, MA, USA. Hispolon, ≥98% (HPLC) powder, ERK1/2 inhibitor (U0126) and JNK1/2 inhibitor (SP600125) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The Muse Annexin V & Dead Cell Assay Kit (MCH100105) was obtained from Meck Millipore. Control siRNA, p53 siRNA, beclin-1 siRNA and antibodies were obtained from Cell Signaling. Wild-type p53 expression plasmid was obtained from Sino Biological, Inc (pCMV3-GFPSpark-TP53).