Fig. 3: Combination therapy significantly reduced MYCN protein expression and stability, and, increased ubiquitination.

a Top panel: cell viability was measured using the Alamar Blue assay across a panel of MYCN non-amplified and MYCN-amplified cell lines after 48 h of combination therapy (1 µM SAHA + 5 µM SE486-11). Results are represented as the mean ± SD for at least three independent experiments. Bottom panel: Immunoblot analysis with MYCN, c-Myc, and actin antibodies against whole cell protein lysates from both MYCN non-amplified and MYCN-amplified cell lines without combination treatment. b Kelly and SK-N-BE(2)-C neuroblastoma cell lines were treated with either DMSO, single agents, or combination therapy (1 µM SAHA + 5 µM SE486-11) for 24 h. Cell protein lysates were collected, and representative immunoblots of MYCN expression using a MYCN antibody. c Representative immunoblots from cycloheximide (CHX) chase assays after 24 h of DMSO or combination treatment (1 µM SAHA + 5 µM SE486-11) of Kelly cells. Cells were then treated with 100 µg/ml CHX for up to 60 min followed by immunoblotting and densitometric analysis to determine the protein half-life of MYCN relative to no CHX. d Immunoblot analysis with a MYCN antibody against whole cell protein lysates from Kelly and SK-N-BE(2)-C cells treated with combination therapy for 24 h, and then treated with 30 µM of the proteasome inhibitor, MG-132, for 4 h. e Representative immunoblot of Kelly cells treated with DMSO, single agents, or combination therapy (1 µM SAHA + 5 µM SE486-11) for 48 h, before cell protein lysates were collected and endogenous MYCN was immunoprecipitated using a MYCN antibody. Ubiquitination was detected in each pull-down using a Ubiquitin antibody.