Hur sirna

Actinomycin D (ActD) was purchased from Sigma-Aldrich (St. Louis, USA). Lipofectamine 2000 (11668–027) and Opti-MEM I reduced serum medium were obtained from Invitrogen (Carlsbad, USA). MK2 siRNA, HuR siRNA and negative control siRNA were received from GenePharma (Shanghai, China). TNF-α (300-01A) was purchased from PeproTech (Rocky Hill, USA). Phosphatase Inhibitor Cocktail (04906845001) was from Roche (Mannheim, Germany). An enhanced chemiluminescent (ECL) kit were obtained from Pierce (Rockford, USA). The protein assay kit, nuclear and cytoplasmic protein extraction kit (P0027) and the RIPA Lysis Buffer (P0013C) were purchased from Beyotime Institute of Biotechnology (Nantong, China). Antibodies raised against MK2 (#3042), phospho-MK2 (Thr334, #3041), ICAM-1 (#4915) was obtained from Cell Signaling (Danvers, USA); a monoclonal antibody against HuR (ab14371) was received from Abcam (Cambridge, USA); antibodies against β-actin (P30002) and histone (P30266) were from Abmart (Shanghai, China);IL-8 ELISA Kit (BMS204/3) was received from eBioscience (San Diego, USA). RevertAid First Strand cDNA Synthesis Kit (#K1622) was purchased from Fermentas UAB (Vilnius, Lithuania). FastStart Universal SYBR Green Master (Rox) (04913914001) was purchased from Roche (Mannheim, Germany). Endothelial cell medium and HPMECs were purchased from ScienCell (Carlsbad, USA).

SGC7901 and BGC823 cells were transfected with specific siRNAs using Lipofectamine 2000 (Invitrogen, USA). Three individual LINC00324 siRNAs (si-LINC00324 1#, 2#, 3#) and scrambled negative control siRNA (si-NC) were purchased from Invitrogen. HUR siRNA and FAM83B siRNA were synthesized by Genepharma(Shanghai, China). The nucleotide sequences of LINC00324 siRNAs, HUR siRNA, and FAM83B siRNAs are listed in Supplementary Table 3. Cells were harvested for qRT-PCR or western blot analysis 48 h after transfection.