Flexitube sirna premix

For a transient transfection approach with the aim to inhibit or enhance the miR-218 function, RA-FLSs were transfected with synthetic precursor miRNA (Pre-miR), with inhibitors of miR-218 (anti-miR), or with scrambled controls (Pre-miR/Anti-miR Negative Control #1; Ambion/Applied Biosystems, Foster City, CA, USA) at a final concentration of 100 nM with the use of Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). In another set of experiments, RA-FLSs were transfected with specific small interfering RNA (siRNA) that target ROBO1 mRNA using FlexiTube siRNA Premix (Qiagen, Hilden, Germany) at a final concentration of 25 nM according to the manufacturer’s protocol. AllStars Negative control siRNA (siRNA-premix, Qiagen) served as a control. Transfection efficiency of pre/anti-mir-218 and siRNA were controlled by TaqMan-based real-time polymerase chain reaction (PCR).

The RNA interference analysis was performed by transfecting Huh7 cells with small interfering RNA (siRNA) (FlexiTube siRNA Premix, Qiagen). One day before transfection, 1 × 10⁶ Huh7 cells were seeded on 6‐well plates. Huh7 cells were transfected with 25 nmol/L TRAIL‐specific siRNA (SI00056154) or control siRNA (SI03650318).
For the migration assay, 48 h after transfection, cells were harvested and 1 × 10⁴ cells were seeded in transwell inserts with a pore size of 8 μm in serum‐free media with normal growth media in the outer chamber. After 24 h, the cells were fixed in 4% Roti^®‐Histofix (Carl Roth, Karlsruhe, Germany) and stained with crystal violet. The cells were counted with a light microscope (Advanced Microscopy Group, Bothell, Washington, USA).
For the colony‐formation assay performed 48 h after transfection, cells were harvested and 1 × 10⁴ cells were seeded in a growth medium containing 0.35% agarose in 12‐well plates. During the 21‐day cultivation period, cell culture medium was changed twice per week. Staining was performed with 0.005% crystal violet (Sigma) for 2 h before the colonies were counted with a light microscope (Advanced Microscopy Group).

RNA interference experiments to suppress Ubc9 expression in cells were performed using the FlexiTube siRNA Premix (Qiagen, Hilden, Germany) and processed according to the manufacturer’s instructions. The following target sequence was used: 5′-ACCACCATTATTTCACCCGAA-3′. Neuroblastoma cell lines were cultured in a 6-well plate at a density of 3 × 10⁵ cells/well for 24 h and transfected with 5.5 μg siRNA using Lipofectamine^tm. After 48 h, siRNA validation was determined using an assay for Ubc9 expression.
Viral particles were made in HEK-293 T cells (ATCC, cat. no. CRL-11268) by cotransfection of the lentiviral vector Ubc9 and the Packaging Plasmid Mix with Lipofectamine^tm 2000. The culture medium containing the packaged viruses was harvested 48 h after transfection and spun at 3,000 rpm for 20 min at 4 °C. The transduced cells were harvested after 72 h to check for RNA interference efficiency.

Human bronchial epithelial cell line, 16HBE14o was grown on fibronectin-coated 6 well plates (Corning, NY) to 60–65% confluence and transfected with FlexiTube siRNA premix (Qiagen, Germantown, MD) against MyD88 and Act1/CIKS. Scrambled siRNA was used as a control. The cells were transfected twice within 48 hours. Cells from replicate wells were used for verification of silencing with western blot and the experimental wells were stimulated with 1 μg/ml PL for 0, 0.5, 1 and 6 hours and IL-17 levels measured from the culture supernatant using ELISA (eBiosciences, San Diego, CA).

For VEGFR-1 and YAP-1 knockdown, HUVECs were seeded in 6-well plates and were transfected with human VEGFR-1 or YAP-1 siRNA (FlexiTube siRNA Premix, obtained from Qiagen. The siRNA transfection was carried out using an Oligofectamine transfection kit (Invitrogen) as per standard protocol. For each gene-targeted, two independent siRNA sequences were tested. The VEGFR-1 siRNA sequences are presented previously (Singh Angom et al., 2019 (link)), and the YAP-1 siRNA sequences are shown in Supplementary Table S2.