Sirna oligonucleotides

SiRNA oligonucleotides with two thymidine residues (dTdT) at the 3-end of the sequence were purchased from Nippon Gene. The sequence of the siRNA oligonucleotide targeting human MYCN was as follows: sense 5′-CGGAGATGCTGCTTGAGAA-3′ [30 (link)]. Transient transfections of 2.0 × 10⁵ cells were performed with siRNA (15 pmol) in Opti-MEM (Life Technologies) using Lipofectamine RNAiMAX (ThermoFisher Scientific). In this step, we used the reverse transfection method [31 (link)]. To study the specific effect of MYCN silencing, we prepared the following four groups (one experimental group and three different types of control): group 1 (MYCN-siRNA group; experimental), cells transfected with siRNA against human MYCN; group 2 (NC-siRNA group; control), cells transfected with a universal negative control siRNA that shows no homology to any eukaryote genes; group 3 (mock control group), cells treated with the transfection reagent and Opti-MEM without any siRNAs, to check the effect of the transfection reagent; group 4 (full control), cells received no treatment. After 2 days of siRNA treatment, cells were used for immunostaining and microfluidic culture.

chABC, heparinase I/III, heparinase II, CS-A/C, and CS-B were purchased from Sigma–Aldrich. cRGD peptide was purchased from Peptide Institute, Inc. Anti-NG2 CS proteoglycan (AB5320) and anti-CS proteoglycan (MAB2029) antibodies were purchased from Millipore. Antibodies targeting NG2 (catalog no.: 4235), GFAP (catalog no.: 3670), p44/42 MAPK (Erk1/2) (catalog no.: 9102), and phospho-p44/42 MAPK (Erk1/2) (catalog no.: 9101) were purchased from Cell Signaling Technology. Anti-β-actin (catalog no.: 013-24553) was purchased from Wako Pure Chemical Industries. Antibodies targeting CS (ab11570) and ITGAV (ab179475) were purchased from Abcam. Antihuman integrin α5/CD49e antibody was purchased from R&D Systems. Anti-XYLT1 (catalog no.: 55061-1-AP) was purchased from Proteintech. Alexa Fluor 546-labeled goat anti-rabbit IgG, Alexa Fluor 546-labeled goat antimouse IgG, Alexa Fluor 488-labeled goat anti-rabbit IgG, and Alexa Fluor 488-labeled goat antimouse IgG were purchased from Invitrogen. Antimouse and anti-rabbit horseradish peroxidase–linked secondary antibodies were purchased from GE Healthcare. All siRNA oligonucleotides were purchased from Nippon Gene.