Trim32 targets MYCN for inducing asymmetric cell division

Trim32 facilitates degradation of MYCN on spindle poles and induces asymmetric cell division in human neuroblastoma cells

Hideki Izumi & Yasuhiko Kaneko

Division of Cancer Therapeutics, Research Institute for Clinical Oncology, Saitama Cancer Center, 818 Komuro, Ina, Saitama 362-0806, Japan

Supplementary figures legends

Supplementary Figure S1. Proteasome activity is involved in spindle pole localization of MYCN in human neuroblastoma cells. A, Representative images of MYCN localization to spindle poles during mitosis in SK-N-BE and CHP-212 cells with or without exposure of 10M MG132 for 5h. 0.1% DMSO treated cells served as a control. NuMA is green, MYCN is red, and DAPI (DNA) is blue. Arrows show spindle poles. Scale bars indicate 10 m. B, Proteasome activity in human neuroblastoma and control (Jurkat) cells.

Supplementary Figure S2. c-Myc does not localize to spindle poles in SK-BR-3, HeLa and U251MG cells. These cell lines were treated with or without MG-132 for 5h. NuMA is green, c-MYC is red, and DAPI (DNA) is blue. Scale bars indicate 10 m.

Supplementary Figure S3.Phospho-T58-MYCN accumulates to spindle poles in SK-N-DZ cells. Representative images of phospho-T58-MYCN but not phospho-S62-MYCN accumulation to spindle poles during mitosis in SK-N-DZ cells. MYCN is red, phospho-T58-MYCN or phospho-S62-MYCN is green, and DAPI (DNA) is blue. Arrows show spindle poles. Scale bars indicate 10 m.

Supplementary Figure S4.AGSK-3 inhibitor IX (BIO) suppresses spindle pole localization of MYCN under MG132 treatment. Percent of TGW (left) or SK-N-DZ (left) cells with MYCN accumulation to spindle poles. Error bars represent SEM from three experiments,P<0.0001 for TGW and P=0.006 for SK-N-DZ.

Supplementary Figure S5. Fbxw7 and Huwe1 do not localize to spindle poles during mitosis. A, Representative images of Fbxw7-stained SH-SY5Y, TGW and SK-N-DZ cells in mitosis. A centrosome marker, -tubulin is red, Fbxw7 is green, and DAPI (DNA) is blue. Arrows show spindle poles. B, Representative images of Huwe1-stained SH-SY5Y, TGW and SK-N-DZ cells in mitosis. -tubulin is red, Huwe1 is green, and DAPI (DNA) is blue. Arrows show spindle poles. Scale bars indicate 10 m.

Supplementary Figure S6.Evaluation of different doses Cdk1/ Cyclin B inhibitor (RO-3306) treatment in neuroblastoma cells. Cells (SK-N-DZ) were treated with 50ng/ml of nocodazole (Noc) with or without low or high concentration (5M or 10M) of RO-3306 for 3h. The treated samples were then subjected to immunoblot analysis. RO-3306 treatment reduced Cyclin B and phospho-histone H3 (a mitosis marker) levels in a dose dependent manner as reported previously(1).

Supplementary Figure S7.The Cdk1/cyclin B inhibitor (RO-3306) suppresses spindle pole localization of Trim32. Percent of TGW (upper panels) or SN-N-DZ (lower panel) cells with intense, weak, or no spindle pole localization of Trim32 with or without RO-3306 treatment. Error bars represent SEM from three experiments. Localization statuses of Trim32 tospindle poles was categorized with three types (intense, weak or no localization).

Supplementary Figure S8.Colocalization of T58-phosphorylated-MYCN and Trim32 to spindle poles in TGW (A) and SK-N-DZ (B) cells under MG132 treatment for 5h. Trim32 is red, MYCN-T58-P or MYCN is green, and DAPI (DNA) is blue. Arrows show spindle poles. Scale bars indicate 10 m.

Supplementary Figure S9.TP58 phosphorylation in MYCN is dispensable for interaction with Trim32 in vivo. Each 1mg of lysates from SH-SY5Y cells transfected with the MYCN or MYCN-T58A expression vector was immunoprecipitated with an anti-MYCN antibody. As a control, immunoprecipitates with preimmune mouse immunoglobulin-G (IgG) were used. The immunoprecipitates were then subjected to immunoblotting using an anti-Trim32 antibody. Right panel shows 5% input.

SupplementaryFigure S10.Trim32 ubiquitinylates MYCN in vitro. Trim32 in TGW cells with or without nocodazole treatment (30ng/ml for 16h) was immunoprecipitated by an anti-Trim32 antibody, andmixed with recombinant GST-MYCN. Then,anin vitro ubiquitinylation assay was performed. Ubiquitinylation ability of Trim32 was active both in interphase and mitosis.Since the ubiquitin ligase activity of Trim32 was inhibited by phosphorylation of PKA in interphase as reported recently(2), Trim32 may be more active in mitosis rather than in interphase.

SupplementaryFigure S11. ACD and distribution of Trim32 and MYCN. Representative images of Flag- and MYCN-stained SK-N-DZ cells transfected with the Flag-trim32 or Flag-Trim32/3A expression vector. Flag is green, MYCN is red, and DAPI (DNA) is blue. Arrows show Trim32-positive daughter cells. Arrowheads show MYCN-positive daughter cells. Dashed outlines show a pair of daughter cells.

Supplementary Figure S12.Cotransfection of Trim32/3A and MYCN shRNA increased the percentage of neuroblastoma cells showing NuMA-ACD. A, Immunoblot of Trim32/3A and MYCN expressing SK-N-DZ cells transfected with both Trim32/3A expression vector and control shRNA or MYCN shRNA. Immunoblot of -tubulin served as a loading control. B, Percent of SK-N-DZ cells in NuMA-, sympatric, or complete ACD transfected with both Trim32/3A expression vector and control shRNA or MYCN shRNA. Error bars represent SEM from three experiments.NuMA-ACD was more frequent in SK-N-DZ cells treated with Trim32/3A and MYCN shRNA than in those treated with Trim32/3A shRNA only (P=0.0068).

SupplementaryFigure S13. Relapse-free survival curves for 20 patients with high Trim32 expression in neuroblastoma and 68 patients with low Trim32 expression. Data was obtained from Public microarray database (R2);

Supplemental references

1.Vassilev LT, Tovar C, Chen S, Knezevic D, Zhao X, Sun H, et al. Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1. Proceedings of the National Academy of Sciences of the United States of America. 2006;103:10660-5.

2.Ichimura T, Taoka M, Shoji I, Kato H, Sato T, Hatakeyama S, et al. 14-3-3 proteins sequester a pool of soluble TRIM32 ubiquitin ligase to repress autoubiquitylation and cytoplasmic body formation. Journal of cell science. 2013;126:2014-26.

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