Allstars sirna

Pre-coated growth-factor reduced Matrigel cell culture inserts (24-well, pore size 8 μm; BD Biosciences, San Jose, CA, USA) were seeded with serum-deprived 5 x 10⁵ PC3 or 22Rv1 vector-only cells (22Rv1-VO) or 22Rv1 EphB4 over-expressing cells (22Rv1-B4) and transfected with 100 nM ITGB8 siRNA or non-silencing AllStars siRNA (Qiagen) in 0.1% FCS-containing medium. Medium containing 10% FCS was used as chemo-attractant. Cells were incubated for 22 h and cells that had not invaded were removed from the upper chamber using a cotton swab. Membranes were excised and mounted on glass slides with ProLong Gold Antifade containing 4, 6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Life Technologies) for visualization of the nuclei of cells that had invaded through the Matrigel to the underside of the membrane. Nuclei were counted in five random fields at 20 X magnification using an Olympus epifluorescent microscope.

All cell lines were seeded at a density between 3–5 × 10⁴ cells/well and were transfected with siRNAs using Oligofectamine (Invitrogen, Karlsruhe, Germany) according to the manufacturer's protocol. In each transfection 72 nM of siRNAs were used in total. 12 nM of siRNA were applied per target gene, filled up with nonsense siRNA, in low-dose and combined transfections for simultaneous gene silencing (SGS). The target sequences shown in Table S3 were used with 3′-dTdT overhangs. Regarding BCLX and MCL1, the siRNAs target only the transcripts which code for the long forms of the proteins with anti-apoptotic functions [13 (link)]. As negative control we used Allstars siRNA (Qiagen, Hilden, Germany), as positive control siRNA targeting KIF11/Eg5, an essential cytoskeletal component. After incubation for 72 h the cells were harvested for flow cytometry and a caspase activity assay to determine the apoptosis rate. Gene silencing was confirmed by qRT-PCR and Western blot.

Animal studies were conducted in accordance with the German Animal Protection Law. Ten-week-old female NMRI^nu/nu mice, obtained from the animal facility of the University of Dresden, were held under standardized pathogen-free conditions with ad libitum access to food and water. 1 × 10₆ TB32047 or MiaPaCa2 cells, resuspended in 50 μl PBS, were injected bilateral into the flanks. Using a digital caliper the established tumors were measured and the tumor volumes were calculated with the following formula: V = 1/6 × π × length × width × 1/2 (length + width). After 12 – 28 d the mice were subdivided into four groups with the same tumor volume (TB32047: 150 – 200 mm^³, MiaPaCa2: 250 – 300 mm^³). The tumors were treated by intratumoral injections of 10 μg of in vivo-jetPEI (Polyplus, Strasbourg, France) complexed siRNAs. Additional to the group of SGS6 therapy, one group remained untreated, one received nonsense Allstars siRNA (Qiagen, Hilden, Germany) and one was treated with Eg5 siRNA as positive control for the local transfection effectiveness. Mice were sacrificed one day after the last treatment and the tumors were prepared for weight measurement and mRNA analysis. The experiments were performed twice with 11 to 14 tumors per group in total.

Temozolomide, carmustine, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Millipore-Sigma (St. Louis, MO, USA). Cisplatin was acquired from Tocris/Bio-Techne (Minneapolis, MN, USA). Lipofectamine RNAiMax, DNase-free RNase A, and 1 mg/ml stock solution of propidium iodide in water were from Thermo Fisher Scientific (Waltham, MA, USA). Cell culture components—fetal bovine serum (FBS), glutamine-containing Earl’s minimal essential medium (MEM, cat. # 10,293), OptiMEM, penicillin plus streptomycin, and the recombinant protease TrypLE Express, all of the Gibco brand—were from Thermo Fisher Scientific. Quantitative PCR primers for LRRC8A and the housekeeping genes RPL13a, RPS20, and GAPDH, gene-specific siRNAs and negative control Allstars siRNA were all purchased from Qiagen (Germantown, MD, USA). All other salts and reagents were purchased from Millipore-Sigma, and were of analytical grade, unless specified otherwise.
carmustine and TMZ were dissolved under sterile conditions in dimethyl sulfoxide (DMSO) to stock concentrations of 30 and 150 mM, respectively, and stored at −20°C until used in experiments. Cisplatin was dissolved in sterile Dulbecco’s phosphate-buffered saline (DPBS) to produce 3.3 mM stock solution, and stored at −20°C until used.

HeLa cells were incubated in serum-free DMEM for 4 h with siRNA (10 nM) against HIF-1α (Qiagen) or Lpin1[6] (link) in the presence of Lipofectamine™ RNAiMAX (Invitrogen). AllStars siRNA (Qiagen) was used as negative control.