Disrupting USP13 inhibited GSC tumor growth and prolonged animal survival. (A and B) In vivo bioluminescent imaging of GBM xenografts derived from luciferase-labeled GSCs expressing shUSP13 or shNT control. GSCs (T387) were transduced with shUSP13 (shUSP13-50 or shUSP13-52) or shNT and then transplanted into brains of immunocompromised mice (5 × 103 cells per animal). Mice bearing the intracranial xenografts were monitored after GSC transplantation. (A) Representative bioluminescent images at the indicated days are shown. (B) Quantification of bioluminescence indicated that disrupting USP13 significantly attenuated GSC tumor growth in mouse brains. n = 5. ***, P < 0.001; shNT versus shUSP13. Student’s t test was used to assess the significance. Five mice per group were used. (C) Representative images of mouse brain cross sections from mice intracranially implanted with the GSCs transduced with shUSP13 or shNT. GSCs (T387) were transduced with shUSP13 or shNT through lentiviral infection for 48 h and then transplanted into mouse brains. Cross sections (hematoxylin and eosin stained) of the mouse brains harvested on day 21 after injection are shown. (D) Kaplan-Meier survival curves of mice intracranially implanted with GSCs expressing shUSP13 or shNT control. Disrupting USP13 significantly increased survival of mice bearing GSC-derived xenografts. ***, P < 0.001; shNT versus shUSP13. Log-rank analysis was used to assess the significance. Five mice per group were used. (E) IB analysis of USP13 and c-Myc in GSCs (T387) transduced with the Tet-on–inducible shUSP13 (pTRIPZ-shUSP13) and treated with doxycycline (Dox) or vehicle control (Ctrl) for 3 d. Inducible knockdown of USP13 decreased c-Myc protein levels in GSCs. Mass is shown in kilodaltons. (F) Growth curves of GSCs (T387) transduced the inducible shUSP13 (pTRIPZ-shUSP13) and incubated with doxycycline or control. Then, cells were measured for cell growth over a time course (day 0 to day 8). Disrupting USP13 significantly inhibited the growth of GSCs. n = 5. **, P < 0.01; ***, P < 0.001. Student’s t test was used to assess the significance. Data are from three independent experiments. (G and H) In vivo bioluminescent imaging of GBM xenografts derived from luciferase-labeled GSCs expressing inducible shUSP13 and treated with doxycycline or vehicle control. GSCs (T387) were transduced with the Tet-on–inducible shUSP13 (pTRIPZ-shUSP13) and then transplanted into brains of immunocompromised mice (104 cells per animal). Mice bearing the intracranial xenografts were closely monitored. 7 d after GSC transplantation, mice were treated with the vehicle control or doxycycline (2 mg/ml in drinking water) to induce expression of shUSP13 in xenografts. (G) Representative images at the indicated days are shown. (H) Bioluminescent quantification at day 21 indicated that induced disruption of USP13 by doxycycline attenuated GSC tumor growth in mouse brains. n = 5. **, P < 0.01. Student’s t test was used to assess the significance. Five mice per group were used. (I) Kaplan-Meier survival curves of mice intracranially implanted with the GSCs (T387) transduced with the pTRIPZ-shUSP13 for 7 d and then treated with doxycycline or the vehicle control. Induced disruption of USP13 significantly increased the survival of mice bearing the GSC-derived xenografts. **, P < 0.01. Log-rank analysis was used. Five mice per group were used. Data are mean ± SD.
