The effect of the GSK3 inhibitor Azakenpaullone (Azak) and the differentiation agent retinoic acid (RA) was studied in low and high MYCN levels in the MYCN inducible neuroblastoma cell line SH-SY5Y/6TR(EU)/pTrex-Dest-30/MYCN (SY5Y-MYCN).
Azak was dissolved in DMSO in order to apply it to the cells. Therefore a vehicle control consisting of SY5Y-MYCN cells treated with 24h 1µl/ml DMSO only was used in duplicates. Doxycycline (Sigma) dissolved in water was used at a final concentration of 1μg/ml to induce MYCN expression in SY5Y-MYCN. A co-treatment study with Dox and Azak was conducted. SY5Y-MYCN cells were treated with 24h Azak, 24h Azak & 48h Dox and 48h Dox, with biological duplicates.
1 μM RA (dissolved in DMSO) and 1 μg/mL Doxycycline were given individually and in combination. SY5Y-MYCN cells were treated with 24h RA, 24h RA & 48h Dox, and 48h Dox and RNA was extracted in biological duplicates. For the 24h RA & 48h Dox co-treatment cells were treated with Dox for 24h and then with RA and fresh Dox for a further 24h.