Fig. 5: Expression of USP5 and MYCN are linked in a forward feedback loop, which can be inhibited by combination therapy effects on Lys48-specific polyubiquitin chains.

a Kelly and SK-N-BE(2)-C neuroblastoma cell lines were transiently transfected with control siRNA or two USP5 siRNAs #10 or #11 for 24 and 48 h and immunoblot analysis was used to measure protein expression of USP5 and MYCN with USP5 and MYCN antibodies in total cell protein lysates. b Representative immunoblots from cycloheximide (CHX) chase assays 48 h after USP5 knockdown using control siRNA or USP5-specific siRNAs #10 or #11 in SK-N-BE(2)-C cells. Cells were then treated with 100 µg/ml CHX for up to 40 min followed by immunoblotting to detect USP5 and MYCN protein expression using USP5 and MYCN antibodies. The ratio of MYCN protein/Actin protein was artificially set at 1.0 for control samples and then half-life of MYCN protein was obtained from the line graph. c HEK293T cells were transfected with either control vectors, an expression vector encoding HA-tagged MYCN, Flag-tagged USP5, and/or Flag-FBXW7 as indicated. Lysates were extracted 48 h after transfection and precipitated. USP5, MYCN, and FBXW7 protein expression were detected using Flag or HA antibodies in immunoblots (left panel). Densitometric analysis was used to determine the protein expression levels of MYCN in different samples, and the p values were normalised against the MYCN level in lane 2 (right panel). d SHEP-tet21N and shMYCN SK-N-BE(2)-C cells were treated with 2 µg/ml of Doxycycline (Dox) or DMSO for 24, 48, or 72 h. Protein was subjected to immunoblot analysis of USP5, MYCN, and Actin (left panel). Densitometric analysis was used to determine the protein expression level of USP5, while Actin expression was used as a loading control (**p < 0.005 vs control) (right panel). e Schematic representation of the USP5 gene promoter containing the MYCN binding E-Boxs. TSS refers to the transcription start site (top panel). ChIP assays were performed with a control IgG or MYCN antibody, followed by PCR with primers targeting a remote negative control region (Control) or the E-Box regions near the TSS (Amplicon A and B) of the USP5 gene in SK-N-BE(2)-C and Kelly cells. Fold enrichment of the USP5 gene promoter region was calculated as the difference in cycle thresholds obtained with the MYCN antibody compared to the control IgG, relative to input (bottom panel). f HEK293T cells were transfected with constructs expressing Flag-tag-USP5, HA-MYCN, or empty vector (EV) for 24 h. Proteins from the cells were immunoprecipitated with control IgG or an anti-Flag antibody for USP5, then immunoprecipitated (IP) products were analyzed by immunoblot with a Flag or HA antibody. g Co-IP using IgG or MYCN antibodies of total protein from Kelly and SK-N-BE(2)-C cells, followed by immunoblotting with USP5 and MYCN antibodies. h Immunoblot analysis of cell protein lysates collected from Kelly cells treated for 48 h with DMSO, single agents, or combination therapy (1 µM SAHA + 5 µM SE486-11) and detection of Lys48 (K48)-linked polyubiquitin chains using Lys48-linkage-specific ubiquitin antibody. i Immunoblot analysis of protein lysates from Kelly neuroblastoma cells transiently transfected with either empty vector control or a USP5 expression vector for 24 h, followed by 48 h of treatment with DMSO, single agents, or combination therapy (1 µM SAHA + 5 µM SE486-11). The level of Lys48-linked polyubiquitin chains is detected using a Lys48-linkage-specific ubiquitin antibody. A Flag antibody was used to confirm overexpression of USP5 in the protein lysates.