Fig. 4: USP5 is a target of combination therapy in neuroblastoma cells.

a Kaplan–Meier analyses for overall survival (OS) and event-free survival (EFS) of neuroblastoma patients with MYCN amplification (MA) and MYCN non-amplification (MNA), subdivided around the median USP5 mRNA expression level in tumour samples using the publically available SEQC (n = 498) and Kocak (n = 476) patient datasets. b Kelly and SK-N-BE(2)-C neuroblastoma cell lines were treated with either DMSO, single agents, or combination therapy for 48 h. Cell protein lysates were collected, and representative immunoblots of USP5 expression detected using a USP5 or MYCN antibody and a control Vinculin antibody. c Representative immunoblots from cycloheximide (CHX) chase assays after DMSO or combination treatment (1 µM SAHA + 5 µM SE486-11) of Kelly cells for 24 h. Cells were then treated with 100 µg/ml CHX for up to 24 h followed by immunoblotting and densitometric analysis to determine the protein half-life of USP5 relative to no CHX. d Left panel, overexpression of USP5 in Kelly cells following transient transfection of empty vector or USP5 expression vector for 24 h, followed by 48-h treatment with DMSO, single agents, or combination therapy and measurement of cell viability using the Alamar Blue assay. Significance (**p < 0.001) was determined from at least three independent experiments. Right panel, a representative immunoblot confirming overexpression of USP5 detected by a Flag antibody. e–g SK-N-BE(2)-C and Kelly neuroblastoma cell lines were transfected with two USP5 siRNAs (siRNA#10 and #11) for 24, 48, and 72 h and the cell viability was measured over time using the Alamar Blue assay (e). Cell growth was measured using BrdU incorporation (f), and clonogenic colony formation (g) after 14 days of treatment. Data are represented as the mean ± SD for three independent experiments and was compared to the control siRNA.