Figure 9. T58A–c-Myc mutant rescued the phenotype caused by USP13 knockdown or FBXL14 overexpression. (A) IB analysis of Flag-T58A–c-Myc mutant, endogenous c-Myc (WT), USP13, FBXL14, GFAP, and SOX2 in the T387 GSCs transduced with Flag-T58A–c-Myc or vector (Vec) control in combination with shUSP13 or shNT control. USP13 knockdown decreased the endogenous wild-type c-Myc but not the ectopic Flag-T58A–c-Myc mutant. (B) IB analysis of Flag-FBXL14, HA-T58A–c-Myc mutant, endogenous c-Myc (WT), USP13, GFAP, and SOX2 in the GSCs transduced with HA-T58A–c-Myc or vector control in combination with Flag-FBXL14 or vector control. Ectopic expression of FBXL14 decreased endogenous wild-type c-Myc but not the HA-T58A–c-Myc mutant. (A and B) Mass is shown in kilodaltons. (C–E) Tumorsphere formation of GSCs transduced with Flag-T58A–c-Myc or vector control in combination with shUSP13 or shNT control. (C) Representative images (phase contrast) of tumorspheres are shown. Ectopic expression of Flag-T58A–c-Myc mutant in GSCs rescued the impaired tumorsphere formation caused by USP13 disruption. (D and E) Quantification indicated that ectopic expression of the T58A–c-Myc mutant in GSCs rescued the decreased tumorsphere size (D) and number (E) caused by USP13 disruption. Data are mean ± SD. n = 4. ***, P < 0.001. Student’s t test was used to assess the significance. Data are from three independent experiments. (F–H) Tumorsphere formation of GSCs transduced with HA-T58A–c-Myc or vector control in combination with Flag-FBXL14 or vector (VEC) control. (F) Representative images (phase contrast) of tumorspheres are shown. Ectopic expression of the T58A–c-Myc mutant in GSCs rescued the reduced tumorsphere formation caused by FBXL14 overexpression. (G and H) Quantification indicated that ectopic expression of the T58A–c-Myc mutant in GSCs rescued the decreased tumorsphere size (G) and number (H) caused by FBXL14 overexpression. Data are mean ± SD. n = 4. ***, P < 0.001. Student’s t test was used to assess the significance. Data are from three independent experiments. (I and J) In vivo bioluminescent imaging of GBM xenografts derived from the luciferase-labeled GSCs transduced with Flag-T58A–c-Myc mutant or vector control in combination with shUSP13 or shNT control. The luciferase-labeled GSCs (T387) were transduced with Flag-T58A–c-Myc or vector control and shUSP13 or shNT through lentiviral infection and then transplanted into the brains of immunocompromised mice (5 × 103 cells per animal). Mice bearing the intracranial xenografts were monitored after GSC transplantation. (I) Representative images at the indicated days are shown. (J) Quantification of luminescence indicates that ectopic expression of the T58A–c-Myc mutant restored the GSC tumorigenic potential impaired by USP13 disruption. Data are mean ± SD. n = 5. ***, P < 0.001; VEC + shUSP13 versus other groups. Student’s t test was used to assess the significance. Five mice per group were used. (K and L) In vivo bioluminescent imaging of GBM xenografts derived from luciferase-expressing GSCs transduced with HA-T58A–c-Myc mutant or vector control in combination with Flag-FBXL14 or vector control. GSCs (T387) were transduced with HA-T58A–c-Myc or vector control in combination with Flag-FBXL14 or vector control through lentiviral infection and then transplanted into immunocompromised mouse brains (5 × 103 cells per animal). Mice bearing the intracranial xenografts were monitored after GSC transplantation. (K) Representative images at the indicated days are shown. (L) Luminescence quantification shows that ectopic expression of the c-Myc–T58A mutant restored GSC tumorigenic potential attenuated by ectopic expression of FBXL14. Data are mean ± SD. n = 5. ***, P < 0.001; VEC + FBXL14 versus other groups. Student’s t test was used to assess the significance. Five mice per group were used. (M) Kaplan-Meier survival curves of mice implanted with GSCs transduced with Flag-T58A–c-Myc or vector control in combination with shUSP13 or shNT control. Mice implanted with the GSCs were maintained until the development of neurological signs or for 60 d. Ectopic expression of the T58A–c-Myc mutant in GSCs significantly attenuated the increased survival of mice caused by USP13 disruption in the GSC-derived tumors. ***, P < 0.001; VEC + shUSP13 versus other groups. Log-rank analysis was used. Five mice per group were used. (N) Kaplan-Meier survival curves of mice implanted with GSCs transduced with HA-T58A–c-Myc or vector control in combination with Flag-FBXL14 or vector control. Mice implanted with the GSCs were maintained until the development of neurological signs or for 60 d. Ectopic expression of the T58A–c-Myc mutant in GSCs significantly attenuated the increased survival of mice caused by FBXL14 expression in the GSC-derived tumors. ***, P < 0.001; VEC + FBXL14 versus other groups. Log-rank analysis was used. Five mice per group were used.
Figure 9.

T58A–c-Myc mutant rescued the phenotype caused by USP13 knockdown or FBXL14 overexpression. (A) IB analysis of Flag-T58A–c-Myc mutant, endogenous c-Myc (WT), USP13, FBXL14, GFAP, and SOX2 in the T387 GSCs transduced with Flag-T58A–c-Myc or vector (Vec) control in combination with shUSP13 or shNT control. USP13 knockdown decreased the endogenous wild-type c-Myc but not the ectopic Flag-T58A–c-Myc mutant. (B) IB analysis of Flag-FBXL14, HA-T58A–c-Myc mutant, endogenous c-Myc (WT), USP13, GFAP, and SOX2 in the GSCs transduced with HA-T58A–c-Myc or vector control in combination with Flag-FBXL14 or vector control. Ectopic expression of FBXL14 decreased endogenous wild-type c-Myc but not the HA-T58A–c-Myc mutant. (A and B) Mass is shown in kilodaltons. (C–E) Tumorsphere formation of GSCs transduced with Flag-T58A–c-Myc or vector control in combination with shUSP13 or shNT control. (C) Representative images (phase contrast) of tumorspheres are shown. Ectopic expression of Flag-T58A–c-Myc mutant in GSCs rescued the impaired tumorsphere formation caused by USP13 disruption. (D and E) Quantification indicated that ectopic expression of the T58A–c-Myc mutant in GSCs rescued the decreased tumorsphere size (D) and number (E) caused by USP13 disruption. Data are mean ± SD. n = 4. ***, P < 0.001. Student’s t test was used to assess the significance. Data are from three independent experiments. (F–H) Tumorsphere formation of GSCs transduced with HA-T58A–c-Myc or vector control in combination with Flag-FBXL14 or vector (VEC) control. (F) Representative images (phase contrast) of tumorspheres are shown. Ectopic expression of the T58A–c-Myc mutant in GSCs rescued the reduced tumorsphere formation caused by FBXL14 overexpression. (G and H) Quantification indicated that ectopic expression of the T58A–c-Myc mutant in GSCs rescued the decreased tumorsphere size (G) and number (H) caused by FBXL14 overexpression. Data are mean ± SD. n = 4. ***, P < 0.001. Student’s t test was used to assess the significance. Data are from three independent experiments. (I and J) In vivo bioluminescent imaging of GBM xenografts derived from the luciferase-labeled GSCs transduced with Flag-T58A–c-Myc mutant or vector control in combination with shUSP13 or shNT control. The luciferase-labeled GSCs (T387) were transduced with Flag-T58A–c-Myc or vector control and shUSP13 or shNT through lentiviral infection and then transplanted into the brains of immunocompromised mice (5 × 103 cells per animal). Mice bearing the intracranial xenografts were monitored after GSC transplantation. (I) Representative images at the indicated days are shown. (J) Quantification of luminescence indicates that ectopic expression of the T58A–c-Myc mutant restored the GSC tumorigenic potential impaired by USP13 disruption. Data are mean ± SD. n = 5. ***, P < 0.001; VEC + shUSP13 versus other groups. Student’s t test was used to assess the significance. Five mice per group were used. (K and L) In vivo bioluminescent imaging of GBM xenografts derived from luciferase-expressing GSCs transduced with HA-T58A–c-Myc mutant or vector control in combination with Flag-FBXL14 or vector control. GSCs (T387) were transduced with HA-T58A–c-Myc or vector control in combination with Flag-FBXL14 or vector control through lentiviral infection and then transplanted into immunocompromised mouse brains (5 × 103 cells per animal). Mice bearing the intracranial xenografts were monitored after GSC transplantation. (K) Representative images at the indicated days are shown. (L) Luminescence quantification shows that ectopic expression of the c-Myc–T58A mutant restored GSC tumorigenic potential attenuated by ectopic expression of FBXL14. Data are mean ± SD. n = 5. ***, P < 0.001; VEC + FBXL14 versus other groups. Student’s t test was used to assess the significance. Five mice per group were used. (M) Kaplan-Meier survival curves of mice implanted with GSCs transduced with Flag-T58A–c-Myc or vector control in combination with shUSP13 or shNT control. Mice implanted with the GSCs were maintained until the development of neurological signs or for 60 d. Ectopic expression of the T58A–c-Myc mutant in GSCs significantly attenuated the increased survival of mice caused by USP13 disruption in the GSC-derived tumors. ***, P < 0.001; VEC + shUSP13 versus other groups. Log-rank analysis was used. Five mice per group were used. (N) Kaplan-Meier survival curves of mice implanted with GSCs transduced with HA-T58A–c-Myc or vector control in combination with Flag-FBXL14 or vector control. Mice implanted with the GSCs were maintained until the development of neurological signs or for 60 d. Ectopic expression of the T58A–c-Myc mutant in GSCs significantly attenuated the increased survival of mice caused by FBXL14 expression in the GSC-derived tumors. ***, P < 0.001; VEC + FBXL14 versus other groups. Log-rank analysis was used. Five mice per group were used.

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