Sigenome non targeting control sirna

RNAi target sequences (sense strand): TRIM41 siRNA #2: AAGGCGTGCTGTGGAAATAAA; TRIM41 siRNA #3: TTCAATAGGTGTGAAGAGGTA. siGENOME Non-Targeting Control siRNA (Dharmacon, # D-001210-02-05) was used as the control siRNA. Five pmol of siRNA duplexes were transfected into A549 cells using Lipofectamine RNAiMAX Transfection Reagent (Life Technologies) according to the manufacturer’s protocol.

On day 1, 1 × 10⁵ BV2 cells were seeded in a culture medium in a six-well plate. On day 2, the cell medium was changed to Gibco opti-MEM reduced-serum medium (Thermo Fisher Scientific, MA, USA) and incubated for 2 h. siGENOME mouse SOX21 siRNA or siGENOME non-targeting control siRNA (Dharmacon, Lafayette, CO, USA) was mixed with 4 µL X-tremeGENE siRNA Transfection Reagent (Sigma-Aldrich Corporation, USA) for 15 min at room temperature. The mixture was then added to the cells in the six-well plate. After incubation for 5 to 6 h, the opti-MEM reduced-serum medium was replaced with DMEM medium. On day 3, the BV2 cells were exposed to 5 Gy gamma irradiation. On day 4, the cells were harvested for experiments.

Human breast adenocarcinoma cell lines, MCF7 cells and MDA-MB-231 cells were purchased from American Type Culture Collection (ATCC). Each cell line was maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 1% penicillin/streptomycin. Transfection reagent, Effectene or lipofectamine 3000 was from Qiagen, Inc. (Valencia, CA) or Invitrogen (Grand Island, NY), respectively. ON-TARGETplus SMARTpool MMP9 siRNA and siGENOME Non-targeting control siRNA was from GE Dharmacon (Lafayette, CO). Lipopolysaccharide and b-actin antibody was purchased from Sigma (St. Louis, MO). Antibodies against p-p38 (Thr180/Tyr182), p-JNK (Thr183/Tyr185), p-ERK1/2 (Thr202/Tyr204) and p-IkBa (Ser-32) were from Cell Signaling Technology, Inc. (Beverly, MA). Antibodies against TOPK, TLR4, MMP9 or p-serine/threonine, and breast cancer tissue array paired with metastatic tumors or MMP9 inhibitor were purchased from Abcam (Cambridge, MA). Luciferase assay system was from Promega (Madison, WI). IkBa antibody or protein A/G plus-agarose bead was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). TOPK inhibitor, HI-TOPK-032 was from R&D system (Minneapolis, MN). SuperScript III reverse transcriptase was from Invitrogen (Grand Island, NY). GST-IkBa protein was from EMD Millipore (Billerica, MA).

RNAi target sequences (sense strand): PKP2 siRNA #1: 5′- ACGGCTCATGTTAATGAGTTA-3′; PKP2 siRNA #2: 5′- TCCGTGGGCAACGGAAATCTT-3′; PKP2 siRNA #3: 5′-ATCCAGCGAAATGAATCTACA-3′. ZMPSTE24 siRNA: 5′-CAATCTATGCTGATTATAT-3′. EIF2B4 FlexiTube siRNA (Qiagen, # GS8890). TRIM41 FlexiTube siRNA (Qiagen, # GS90933). FKBP8 FlexiTube siRNA (Qiagen, # GS23770). siGENOME Non-Targeting Control siRNA (Dharmacon, # D-001210-02-05) was used as the control siRNA. siRNA duplexes were transfected into A549 (5 pmol) and primary tracheal cells (5 pmol) using Lipofectamine RNAiMAX Transfection Reagent (Life Technologies, # 13778030) according to the manufacturer's protocol.