Du145

The following cancer cell lines, MCF-7 (human ER-positive breast adenocarcinoma cells), HepG2 (human liver hepatocellular carcinoma), and DU145 and PC3 (human androgens independent prostate carcinoma) cells, were acquired from LGC Promochem (Wesel, Germany).
Culture of HepG2 cells was done using Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% of fetal bovine serum (FBS). RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) was used for the growth and subculture of MCF-7, DU145, and PC3 cells. All cell cultures were also supplemented with penicillin 100 U/mL, streptomycin 100 μg/mL, and HEPES 10 mM. The cell cultures were maintained at 37°C in a CO₂ 5% humidified atmosphere and pH 7.4. After every other day, 90% of the supernatant was replaced with fresh medium during the cells passage. The number of viable cells was assessed using the trypan blue method and Neubauer chamber was used to assess the number of viable cells and cell count, respectively, prior to performing all experiments.

Sublines of human adenocarcinoma cell line DLD1 A4 (with homologously deleted STAT3) and A4wt (A4 reconstituted with the wild-type variant of STAT3) were a kind gift of Zhenghe Wang, Genetics and Case Comprehensive Cancer Center, Case Western Reserve University. A4 and A4wt cell lines stably expressing a STAT3-inducible luciferase reporter were generated in the lab and were grown on 1 mg/mL of Hygromycin (Life Technologies AB Sweden). MCF7, MDA-MB468, PC3 and DU145 cell lines were all purchased from LGC Standards.
A4, A4wt, MCF7 and MDA-MB-468 cell lines were cultured in Dulbecco’s modified Eagle’s medium; PC3 and DU145 cells were cultured in RPMI 1640 medium; all supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 μg/mL streptomycin and 100 U/mL penicillin at 37°C in 5% CO₂. All media and reagents were from HyClone, Thermo Fisher Scientific Inc., Sweden. The cells were routinely tested against mycoplasma using Plasmotest (# REP-PT2) from InVivoGen (USA).

Branched low molecular weight (4–10 kDa) polyethylenimine PEI F25-LMW was prepared as described previously [18 (link)]. Cell Culture media, Dulbecco’s phosphate buffered saline (PBS) and trypsin were from Sigma Aldrich (Taufkirchen, Germany), and fetal calf serum (FCS) was purchased from Gibco Thermo Fisher (Darmstadt, Germany). Cell culture plastic and other disposable plastics were from Sarstedt (Nümbrecht, Germany). Chemically synthesized antimiRs (miRCURY LNA inhibitor; Exiqon/Qiagen, Hilden, Germany or 2′-OMe modified RNAs; Eurogentec, Seraing, Belgium) were used; see Additional file 3: Table S1 for additional information. Stable human prostate carcinoma cell lines PC3, DU145 and LNCaP as well as the human or mouse melanoma cell lines LOX, A375, SK-Mel-28, B16F10 and B16V were obtained from ATCC/LGC Promochem (Wesel, Germany) or the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany).