Supplemental table 1
Summary of DUSP26 IHC on neuroblastoma tissue specimens. Staining was scored according to a
DUSP26 immunohistochemistry (IHC) score determined by combining the % cells stained score plus
the staining intensity score determined by the study pathologist in a blinded fashion. Mean scores for
COG risk groups (RG) and INSS stages were compared by Student’s T-test. P-value <0.05 was
considered significant.
Supplemental Figure 1
DUSP26 inhibits the expression of doxorubicin-induced p53 target genes, such as Bax, Puma in IMR-32
and SK-N-SH cell lines. (a, b) Cells were seeded into 6-cm dishes for two days and then were either left
untreated or treated with 3 M of DOX for 4h, 6h or 8h. Then total RNAs were isolated from cells and
subjected to quantitative RT-PCR in IMR-32 cells (a) and SK-N-SH cells (b) for detecting the
expression of p53 target genes such as p53AIP1, Bax and Puma. (c, d) Whole cell lysates from IMR-32
cells (c) and SK-N-SH cells (d) were subjected to immunoblotting analysis with an array of antiphospho-p53, anti- p53AIP1, anti- Bax, anti- Puma and anti- p53 (DO-1) antibodies.
Supplemental Figure 2
DUSP26 promotes the resistance of SH-SY5Y neuroblastoma cell line to doxorubincin-induced
cytotoxicity. (a) Knockdown of DUSP26 expression in SH-SY5Y cells was detected by quantitative RTPCR. (b) The SH-SY5Y sh-Control, sh-DUSP26-1, and sh-DUSP26-2 cell lines were plated in 96-well
plates at 1 x 104 cells/well. After 24 hours of growth, cells were treated with the indicated concentration
of doxorubicin for 24 hours. Cell viability was determined with the CCK-8 cell viability assay relative to
the 0 M group. All experiments were performed in triplicate and statistical significance was determined
by Student’s T-test comparing each sh-DUSP26 to sh-Control group where * denotes p<0.05 and **
denotes p<0.001. (c) DUSP26 inhibits doxorubicin-induced p53 phosphorylation at Ser-20 and Ser-37
residues in SH-SY5Y. (d) DUSP26 inhibits doxorubicin-induced p53 target gene expression and
apoptosis in SH-SY5Y.
Supplemental Figure 3
DUSP26 inhibits VP-16-induced p53 phosphorylation at Ser-20 and Ser-37 residues and apoptosis. (a,
b) Cells were seeded into 6-cm dishes for two days and then were either left untreated or treated with 1
M (a) or 2.5M (b) of VP-16 for 0h, 4h, 6h or 8h. Then cells were harvested and subjected to
immunoblotting analysis with an array of anti-PARP, anti-phospho-p53 and anti-p21 antibodies in IMR32 cells (a) and SK-N-SH cells (b).
Supplemental Figure 4
DUSP26 binds to p53. (a) HEK293T were transfected with vector control (-) or FLAG-DUSP26 (+).
Endogenous p53 was immunoprecipitated with anti-p53 (DO-1) antibody and immunoblotted with antiFLAG antibody. (b) Recombinant GST-DUSP26-CS protein was incubated with lysate from HEK293T
transfected with GFP-p53. Samples were resolved on SDS-PAGE and immunoblotted with anti-p53
(DO-1) antibody.
Supplemental Figure 5
DUSP26 proteins can be immunoprecipitated by anti-DUSP26 antibodies. (a) Anti-DUSP26 antibody
was used to immunoprecipitate FLAG-DUSP26 transfected in HEK293T. Samples were resolved by
SDS-PAGE and immunoblotted with anti-FLAG antibody. (b) SH-SY5Y was plated in culture and
harvested for cell lysate after treatment with doxorubicin. Anti-DUSP26 antibody was used to
immunoprecipitate endogenous DUSP26 and immunoblotting for endogenous p53 was performed with
anti-p53 (DO-1) antibody.