Figure 3. Targeting USP13 disrupted the maintenance of GSCs. (A) IB analysis of USP13, the GSC markers (c-Myc and SOX2), and the differentiation markers (GFAP and TUJ1) during serum-induced GSC differentiation. GSCs isolated from T4121 xenografts were cultured in DMEM containing 10% FBS to induce differentiation and harvested for IB analysis on the indicated days. USP13, c-Myc, and SOX2 gradually decreased, whereas the differentiation markers GFAP (for astrocytes) and TUJ1 (for neurons) increased during the differentiation. (B) IB analysis of USP13, c-Myc, SOX2, OLIG2, TUJ1, and GFAP in the GSCs (T387) transduced with USP13 shRNA (shUSP13-50 and shUSP13-52) or shNT (02) for 2 d. USP13 disruption by shRNA for a short time rapidly induced a dramatic decrease of c-Myc protein level in GSCs. (C) IB analysis of USP13, c-Myc, GFAP, and SOX2 in the GSCs transduced with Flag-USP13 expression or vector control through lentiviral infection in two GSC populations derived from T4121 and T387 xenografts. Forced expression of USP13 increased c-Myc protein levels in GSCs. (A–C) Mass is shown in kilodaltons. (D–F) The effect of USP13 knockdown on GSC tumorsphere formation. Disrupting USP13 by shUSP13-50 or shUSP13-52 impaired tumorsphere formation of GSCs (T387). (D) Representative images of GSC tumorspheres are shown. (E and F) Quantification showing that USP13 knockdown significantly reduced GSC tumorsphere number (E) and size (F). n = 4. (G and H) Growth curves of GSCs expressing shUSP13 or shNT control. GSCs derived from T387 xenograft (G) or T4121 xenograft (H) were transduced with shUSP13 (sh50 and sh52) or shNT and then measured for cell growth over a time course (day 0 to day 8). Disrupting USP13 significantly inhibited the growth of GSCs. n = 6. (I and J) Annexin V–FITC staining to detect apoptosis in GSCs expressing shUSP13 or shNT. GSCs (T387) were transduced with shUSP13 or shNT through lentiviral infection for 48 h, stained with annexin V-FITC and PI, and then analyzed by flow cytometry. (I) Representative FACS data are shown. The upper right dots represent late apoptotic cells (positive for both annexin V and PI). (J) Quantifications showing that disrupting USP13 significantly increased apoptosis of GSCs. n = 3. (K) RT-PCR analysis of expression of c-Myc downstream targets including cyclin B (CCNB), prohibitin (PHB), and DNA mismatch repair protein (MSH2) in the GSCs expressing shUSP13 (sh50 or sh52) or shNT. GSCs (T387) were transduced with shUSP13 shNT for 48 h through lentiviral infection, and the RNA samples were isolated for RT-PCR analysis with specific primers for CCNB, PHB, and MSH2. Disruption of USP13 also reduced expressions of c-Myc downstream targets in GSCs. n = 3. Data are mean ± SD. **, P < 0.01; ***, P < 0.001; shNT versus shUSP13. Student’s t test was used to assess the significance. Data are from three independent experiments.
Figure 3.

Targeting USP13 disrupted the maintenance of GSCs. (A) IB analysis of USP13, the GSC markers (c-Myc and SOX2), and the differentiation markers (GFAP and TUJ1) during serum-induced GSC differentiation. GSCs isolated from T4121 xenografts were cultured in DMEM containing 10% FBS to induce differentiation and harvested for IB analysis on the indicated days. USP13, c-Myc, and SOX2 gradually decreased, whereas the differentiation markers GFAP (for astrocytes) and TUJ1 (for neurons) increased during the differentiation. (B) IB analysis of USP13, c-Myc, SOX2, OLIG2, TUJ1, and GFAP in the GSCs (T387) transduced with USP13 shRNA (shUSP13-50 and shUSP13-52) or shNT (02) for 2 d. USP13 disruption by shRNA for a short time rapidly induced a dramatic decrease of c-Myc protein level in GSCs. (C) IB analysis of USP13, c-Myc, GFAP, and SOX2 in the GSCs transduced with Flag-USP13 expression or vector control through lentiviral infection in two GSC populations derived from T4121 and T387 xenografts. Forced expression of USP13 increased c-Myc protein levels in GSCs. (A–C) Mass is shown in kilodaltons. (D–F) The effect of USP13 knockdown on GSC tumorsphere formation. Disrupting USP13 by shUSP13-50 or shUSP13-52 impaired tumorsphere formation of GSCs (T387). (D) Representative images of GSC tumorspheres are shown. (E and F) Quantification showing that USP13 knockdown significantly reduced GSC tumorsphere number (E) and size (F). n = 4. (G and H) Growth curves of GSCs expressing shUSP13 or shNT control. GSCs derived from T387 xenograft (G) or T4121 xenograft (H) were transduced with shUSP13 (sh50 and sh52) or shNT and then measured for cell growth over a time course (day 0 to day 8). Disrupting USP13 significantly inhibited the growth of GSCs. n = 6. (I and J) Annexin V–FITC staining to detect apoptosis in GSCs expressing shUSP13 or shNT. GSCs (T387) were transduced with shUSP13 or shNT through lentiviral infection for 48 h, stained with annexin V-FITC and PI, and then analyzed by flow cytometry. (I) Representative FACS data are shown. The upper right dots represent late apoptotic cells (positive for both annexin V and PI). (J) Quantifications showing that disrupting USP13 significantly increased apoptosis of GSCs. n = 3. (K) RT-PCR analysis of expression of c-Myc downstream targets including cyclin B (CCNB), prohibitin (PHB), and DNA mismatch repair protein (MSH2) in the GSCs expressing shUSP13 (sh50 or sh52) or shNT. GSCs (T387) were transduced with shUSP13 shNT for 48 h through lentiviral infection, and the RNA samples were isolated for RT-PCR analysis with specific primers for CCNB, PHB, and MSH2. Disruption of USP13 also reduced expressions of c-Myc downstream targets in GSCs. n = 3. Data are mean ± SD. **, P < 0.01; ***, P < 0.001; shNT versus shUSP13. Student’s t test was used to assess the significance. Data are from three independent experiments.

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