P53 sirna

p53 siRNA, Parkin siRNA, ATG7 siRNA and the plasmid encoding Parkin were obtained from Genepharma (Shanghai, China). Transfection was performed using Lipofectamine 2000 reagent (Invitrogen) according to the instructions provided by the manufacturer.

Human p53-targeted (p53-siRNA), JNK1-targeted (JNK1-siRNA), JNK2-targeted (JNK2-siRNA) and negative control (NC-siRNA) siRNAs were designed by GenePharma (Shanghai, China). siRNA transfection was performed on SK-Hep-1 cells using Lipofectamine 2000 (Thermo, USA) according to the manufacturer’s instruction. Transfection efficacy was determined via qPCR and WB assays. The siRNA targeting sequences of P53, JNK1, and JNK2 were shown in Table 1.

Z‐VAD‐FMK was purchased from Selleck (Selleckchem, Houston, TX, USA). Nutlin‐3, tenovin‐1, and doxorubicin were commercially available from MedChem Express (Shanghai, China). The DAPK siRNA and p53 siRNA were purchased from GenePharma (Shanghai, China). LV‐DAPK1‐RNAi was purchased from GeneChem (Shanghai, China). The miR‐34a‐5p mimics, inhibitor, antagomiR, and negative control duplex were synthesized by Shanghai GenePharma. The antibodies and related reagents used in this study were obtained as follows: anti‐DAPK1 (3008S for immunoblot; Cell Signaling, Danvers, MA, USA), anti‐poly ADP‐ribose polymerase (PARP) (9532S; Cell Signaling), anti‐E‐cadherin (3195T; Cell Signaling), anti‐vimentin (5741T; Cell Signaling), anti‐pDAPK‐Ser308 (D4941; Sigma, St. Louis, MO, USA), anti‐GAPDH (G9545; Sigma), anti‐p53 (sc‐126; Santa Cruz, Dallas, TX, USA), Apoptosis Western Blot Cocktail (ab136812; Abcam, Cambridge, MA, USA), anti‐Ki67(ab15580; Abcam), anti‐DAPK1 [BA3712‐1 for immunohistochemistry(IHC); Boster Biological Technology, Wuhan, China], and anti‐N‐cadherin(CDH2) (ab76011; Abcam).

BTG2 siRNA, p53 siRNA, and siNC were designed and synthesized by GenePharma (China).
Cell transfection was performed using Lipofectamine 3000 (Invitrogen Life
Technologies) according to the manufacturer's instructions.

NR4A3 siRNA (5′‐GCGUACAGAUAGUCUGAAATT‐3′) and negative control siRNA were obtained from Sangon. P53 siRNA (5′‐GAAGAAAATTTCCGCAAAA‐3′) and its negative control siRNA were purchased from GenePharma. Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) and Opti‐MEM (Gibco) were used to introduce siRNA into chondrocytes according to the manufacturer's protocol. NR4A3 siRNA‐transfected cells and negative control siRNA‐transfected cells were defined as siNR4A3 and siControl. And P53 siRNA‐transfected cells and its negative control siRNA‐transfected cells were defined as siP53 and siNC.

The sequence of Crispr‐cas9 and Crispr‐sgRNA were designed and synthesized by Genechem (Genechem Co, Ltd), while ISYNA1siRNA, p53siRNA and siRNA control were purchased from GenePharma (GenePharma Co, Ltd). Firstly, Capan‐2 and SW1990 cells were infected with Crispr‐cas9, next these cells were filtrated by puromycin (Sigma). Then the stable sublines were infected with MSI2‐sgRNA (sgMSI2‐1/sgMSI2‐2) and sgRNA control (scramble) to specifically silence target genes. Capan‐2, SW1990 and Miapaca‐2 cells were transiently transfected with ISYNA1siRNA, p53siRNA and siRNA control for 48‐72 hours with lipofectamine 3000 (Invitrogen) according to the manufacturer. The silencing efficiency of MSI2, ISYNA1 and p53 was verified by WB. All of target sequences were seen in Table S1.