Gelred nucleic acid

PCR was performed using the GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA). The PCR mixture contained 5 μL of template ssDNA (~15 ng), 2.5 μL of each primer (10 μM), 25 μL of the PCR master mix solution (AmpliTaq Gold Fast PCR Master Mix, Applied Biosystems, Barcelona, Spain), and 15 μL of autoclaved water. The PCR conditions for ssDNA amplification were 95 °C for 5 min for denaturation, followed by 8 cycles of denaturation at 95 °C for 30 s, annealing at 56.3 °C for 30 s, and extension at 72 °C for 10 s, and a final extension of 72 °C for 5 min for the next round of STC-SELEX selection. After PCR, the reaction products were confirmed by gel electrophoresis using 2.0% agarose gel in 1× TAE (Tris-acetate-EDTA) buffer at 100 V for 40 min. The gels were stained with 0.5 mL of GelRed Nucleic Acid (Biotium, Inc., Hayward, CA, USA) in water and observed under UV light.

Genomic DNA (gDNA) from cell lines was isolated according to the protocol by GeneJET Genomic DNA Purification Kit (K0721; Thermo Fisher Scientific, Inc.). DNA samples were quantified by spectrophotometer BioSpec-nano (Shimadzu Scientific Instruments, MD, USA) and the integrity was analysed by horizontal 1.2% agarose gel electrophoresis stained with GelRed Nucleic Acid (Biotium, Inc., USA).

The cells were treated with GAL, NF-κB inhibitor, AP-1 inhibitors, and LPS as indicated in individual experiments. The total RNAs were isolated with Trizol reagent (Invitrogen, CA, USA) and converted into corresponding cDNAs using SuperScript III reverse transcriptase and random hexamer primers (Thermo, Waltham, MA, USA). The mRNA expression was detected with specific primers as follows: COX-2 mRNA (NM_011198), sense: 5′-TGATCGAAGACTACGTGCAAC-3′, antisense: 5′-TCATCTCTCTGCTCTGGTCAA-3′; 5-LOX mRNA (NM_009662), sense: 5′-CCCCCGAAGCTCCCAGTGACC-3′, antisense: 5′-TCCCGGGCCTTAGTGTTGATA-3′; iNOS mRNA (NM_010927), sense: 5′-AAAGTGACCTGAAAGAGGAAAAGGA-3′, antisense: 5′-TTGGTGACTCTTAGGGTCATCTTGTA-3′; β-actin mRNA (NM_007393), sense: 5′-ATGGATGACGATATCGCTGC-3′, antisense: 5′-TTCTGACCCATTCCCACCATC-3′. PCR amplifications were performed according to the following program: denaturation at 94°C for 3 min, 25 cycles, (94°C for 30 sec, 57°C for 30 sec, and 72°C for 1 min) and extension at 72°C for 10 min. PCR products were analyzed by gel electrophoresis in 1.5% agarose containing GelRed nucleic acid (Biotium, Hayward, CA, USA) and visualized under UV light.

PCL (MW 60 kDa), sodium cacodylate, sucrose, acetonitrile, propidium iodide (PI), Hoechst 33342 (HOE), hexafluoroisopropanol (HFIP), doxorubicin, and bovine serum albumin (BSA) were obtained from Sigma Aldrich (Steinheim, Germany), and ethanol from VWR Chemicals Prolab (Fontenay-sous-Bois, France). Bleomycin was from Abcam (Cambridge, UK).
The HCC1954 cell line was provided by ATTC (Guernsey, UK). Fetal bovine serum (FBS), culture media, and supplements were obtained from Corning (Mediatech Inc., Manassas, VA, USA). The PrestoBlue™ Cell Viability Reagent and secondary antibody goat anti-mouse Alexa 488 were purchased by Thermo Fisher Scientific (Eugene, OR, USA), whereas paraformaldehyde, Masson’s trichrome, and Alcian blue staining kits by BioOptica (Milan, Italy). The TriReagent and the qPCRBIO SyGreen 1-Step Go Lo-ROX were purchased from PCRBIOSYSTEMS (London, UK), and Gel Red Nucleic Acid staining from Biotium (Hayward, CA, USA). Anti-CD44 antibody was provided by AbD Serotech (Kidlington, UK), and Fluoroshield with DAPI was from Vector Laboratories (Peterborough, UK). Tissue-Tek^® OCT was purchased from Thermo Fisher Scientific (Waltham, MA, USA).