Figure 4. The role of HAUSP on the pathogenesis of neuroblastomas.

(a) Kaplan-Meier analyses for SEQC RNA-seq database (left) and Kocak database (right) of overall survival for only MYCN-amplified samples. (b) Correlation of USP7 expression versus N-Myc transcriptional activity in neuroblastoma patients; MYCN-amplification (red) MYCN non-amplification (grey). (c—e) Pre-clinical neuroblastoma mouse model (see methods) (c) Representative in vivo bioluminescence imaging of tumor-bearing mice from day 12. Luciferase activity: low = blue, high = red; scale bar 13 mm. (d; top) Representative images of tumor-bearing kidneys versus normal kidney at day 15; scale bar 10 mm. (d; bottom) Tumor weight from day 15; n = 8 mice per group; ** represents a P value < 0.005 using two-tailed Student's t-test. (e) Representative immunohistochemistry of SK-N-DZ tumors for HAUSP, N-Myc, and Ki-67 from day 15. Magnification 40 ×; scale bar 25 μm; n = 3. (f—i) Subcutaneous xenograft models (see methods); (f) SK-N-DZ tumor image and weight; scale bar 10 mm; n = 5 mice per group; * represents a P value < 0.05 using two-tailed Student's t-test. (g) Representative immunohistochemistry of tumors harvested at day 12 stained for HAUSP, N-Myc, and Ki-67; Magnification 40 ×; scale bar 12.5 μm; n = 3. (h) NB-16 tumor image and weight; scale bar 10 mm; n = 5 mice per group. (i) Representative immunohistochemistry of tumors harvested at day 12 stained for HAUSP, N-Myc, and Ki-67; Magnification 40 ×; scale bar 12.5 μm; n = 3. **Note: Because NB-16 derived tumors grow faster in the subcutaneous model compared with SK-N-DZ, treatment started two weeks post implantation while with SK-N-DZ derived tumors, treatment began three weeks after implantation to more closely account for tumor size. Both SK-N-DZ and NB-16 subcutaneous models were treated with the same dose and time period (See Supplementary Fig. 6g).