Journal: Neuro-Oncology

Article Title: Dual blocking of mTor and PI3K elicits a prodifferentiation effect on glioblastoma stem-like cells

doi: 10.1093/neuonc/noq103

Figure Lengend Snippet: Inhibition of mTor reduces A172 sphere formation and the expression of NSC/progenitor markers. A172 cells were cultured in the stem/progenitor cell culture medium with EGF and bFGF in the absence or the presence of rapamycin for 3 days. The numbers of generated primary spheres were counted (A). The primary spheres were dissociated and cultured with EGF and bFGF in the absence of rapamycin for 3 days. The numbers of secondary spheres were counted (B). (C–E) Cells were cultured as described in (A). The expression of cd133 or gapdh mRNA was analyzed by RT-PCR (C). Cell lysates were subjected to immunoblot analysis with the indicated antibodies (D and E). (F–K) Cells were transfected with the indicated siRNA. Then, cells were reseeded in the stem/progenitor cell culture medium containing EGF and bFGF for assays to count primary sphere numbers (G and H), RT-PCR (J), and immunoblot (F and K) analysis after 3 days. The primary spheres were dissociated and cultured with EGF and bFGF for 3 days. The numbers of secondary spheres were counted (I). Results in (A), (B), (H), and (I) are presented as the mean ± SD of 3 independent experiments. *P < .05.

Article Snippet: Because A172 cells (1 × 10 7 ) from spheres cultured for 14 days did not form tumors after subcutaneous injection into 5-week-old male BALB/cAJcl− nu/nu mice (CLEA Japan, Inc.), we cultured A172 CSLCs by the monolayer culture method.

Techniques: Inhibition, Expressing, Cell Culture, Generated, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection

Journal: Neuro-Oncology

Article Title: Dual blocking of mTor and PI3K elicits a prodifferentiation effect on glioblastoma stem-like cells

doi: 10.1093/neuonc/noq103

Figure Lengend Snippet: Inhibition of PI3K reduces A172 sphere formation and the expression of NSC/progenitor markers. (A) A172 cells were cultured in the stem/progenitor cell culture medium with EGF and bFGF in the absence or the presence of LY294002 for 3 days. The numbers of generated primary spheres were counted. (B) The primary A172 spheres were dissociated and cultured in the stem/progenitor cell culture medium with EGF and bFGF in the absence of LY294002 for 3 days. The numbers of secondary spheres were counted. (C–E) Cells were cultured as described in (A). Cell lysates were subjected to immunoblot analysis with the indicated antibodies (D and E). The expression of cd133 or gapdh mRNA was analyzed by RT-PCR (C). Results in (A) and (B) are presented as the mean ± SD of 3 independent experiments. *P < .05.

Article Snippet: Because A172 cells (1 × 10 7 ) from spheres cultured for 14 days did not form tumors after subcutaneous injection into 5-week-old male BALB/cAJcl− nu/nu mice (CLEA Japan, Inc.), we cultured A172 CSLCs by the monolayer culture method.

Techniques: Inhibition, Expressing, Cell Culture, Generated, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Neuro-Oncology

Article Title: Dual blocking of mTor and PI3K elicits a prodifferentiation effect on glioblastoma stem-like cells

doi: 10.1093/neuonc/noq103

Figure Lengend Snippet: Effect of combination treatment with rapamycin and LY294002 on A172 cells. (A–E, G, and H) A172 cells were cultured in the stem/progenitor cell culture medium with EGF and bFGF in the absence or the presence of rapamycin (Rap) and/or LY294002 (LY) for 3 days. Cell lysates were subjected to immunoblot analysis with the indicated antibodies (A–C and H). The expression of cd133 or gapdh mRNA was analyzed by RT-PCR (G). Cells were cultured as described above and then observed under a phase-contrast microscope (magnification ×40) (D). The numbers of generated primary spheres were counted (E). The primary A172 spheres were dissociated and cultured with EGF and bFGF in the absence of the indicated inhibitors for 3 days. The numbers of secondary spheres were counted (F). Results in (E) and (F) are presented as the mean ± SD of 3 independent experiments.

Article Snippet: Because A172 cells (1 × 10 7 ) from spheres cultured for 14 days did not form tumors after subcutaneous injection into 5-week-old male BALB/cAJcl− nu/nu mice (CLEA Japan, Inc.), we cultured A172 CSLCs by the monolayer culture method.

Techniques: Cell Culture, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Microscopy, Generated

Journal: Neuro-Oncology

Article Title: Dual blocking of mTor and PI3K elicits a prodifferentiation effect on glioblastoma stem-like cells

doi: 10.1093/neuonc/noq103

Figure Lengend Snippet: Combination treatment with rapamycin and LY294002 inhibits self-renewal and elicits differentiation of A172 CSLCs. (A and B) A172 CSLCs were cultured in the stem/progenitor cell culture medium with EGF and bFGF for 14 days before inhibitor treatment. Then, A172 CSLCs were treated with the indicated inhibitors for 3 days, and observed under a phase-contrast microscope (magnification ×100). Arrows show neurite-like morphology (A). The numbers of spheres were counted (B) (rapamycin [Rap, 50 nM] and/or LY294002 [LY, 10 µM]). (C) Cells were cultured as described in (A) and plated on ornithine-coated coverslips with 10% serum for 6 h to facilitate cell adhesion to the dish. Cells were then immunolabeled with the indicated antibodies. (D) Results of quantitative analysis of 3 independent experiments for (C) are shown (Rap: rapamycin, 50 nM and/or LY: LY294002, 10 µM). Results in (B) are presented as the mean ± SD of 3 independent experiments. *P < .05. Data in (D) are the mean ± SD of values obtained from at least 200 cells in each of 3 independent experiments. *P < .05.

Article Snippet: Because A172 cells (1 × 10 7 ) from spheres cultured for 14 days did not form tumors after subcutaneous injection into 5-week-old male BALB/cAJcl− nu/nu mice (CLEA Japan, Inc.), we cultured A172 CSLCs by the monolayer culture method.

Techniques: Cell Culture, Microscopy, Immunolabeling

Journal: Neuro-Oncology

Article Title: Dual blocking of mTor and PI3K elicits a prodifferentiation effect on glioblastoma stem-like cells

doi: 10.1093/neuonc/noq103

Figure Lengend Snippet: NVP-BEZ235 inhibits A172 sphere formation and reduces the expression of NSC/progenitor markers. (A–C, E, and F) A172 cells were cultured in the stem/progenitor cell culture medium with EGF and bFGF in the absence or the presence of NVP-BEZ235 (BEZ) for 3 days. Cell lysates were subjected to immunoblot analysis with the indicated antibodies (A and F). The expression of cd133 or gapdh mRNA was analyzed by RT-PCR (E). Cells were cultured as described above and then observed under a phase-contrast microscope (magnification ×40) (B). The numbers of generated primary spheres were counted (C). The primary A172 spheres were dissociated and cultured with EGF and bFGF in the absence of the indicated inhibitors for 3 days. The numbers of secondary spheres were counted (D). Results in (C) and (D) are presented as the mean ± SD of 3 independent experiments.

Article Snippet: Because A172 cells (1 × 10 7 ) from spheres cultured for 14 days did not form tumors after subcutaneous injection into 5-week-old male BALB/cAJcl− nu/nu mice (CLEA Japan, Inc.), we cultured A172 CSLCs by the monolayer culture method.

Techniques: Expressing, Cell Culture, Western Blot, Reverse Transcription Polymerase Chain Reaction, Microscopy, Generated

Journal: Neuro-Oncology

Article Title: Dual blocking of mTor and PI3K elicits a prodifferentiation effect on glioblastoma stem-like cells

doi: 10.1093/neuonc/noq103

Figure Lengend Snippet: NVP-BEZ235 inhibits self-renewal and elicits differentiation of A172 CSLCs. (A) A172 CSLCs were cultured in the stem/progenitor cell culture medium with EGF and bFGF for 14 days before NVP-BEZ235 (BEZ) treatment. Then, A172 CSLCs were treated with BEZ for 3 days and observed under a phase-contrast microscope (left, magnification ×100). The numbers of generated primary spheres were counted (right). (B and C) Cells were cultured as described above and were plated on ornithine-coated coverslips with 10% serum for 6 h to facilitate cell adhesion to the dish. Cells were then immunolabeled with the indicated antibodies. Results in (A) are presented as the mean ± SD of 3 independent experiments. *P < .05.

Article Snippet: Because A172 cells (1 × 10 7 ) from spheres cultured for 14 days did not form tumors after subcutaneous injection into 5-week-old male BALB/cAJcl− nu/nu mice (CLEA Japan, Inc.), we cultured A172 CSLCs by the monolayer culture method.

Techniques: Cell Culture, Microscopy, Generated, Immunolabeling

Journal: Neuro-Oncology

Article Title: Dual blocking of mTor and PI3K elicits a prodifferentiation effect on glioblastoma stem-like cells

doi: 10.1093/neuonc/noq103

Figure Lengend Snippet: NVP-BEZ235–treated cells have reduced capacity of tumor formation in vivo. Monolayer-cultured A172 CSLCs were treated with NVP-BEZ235 (BEZ, 1 µM) for 3 days, and the levels of indicated proteins were analyzed by immunoblotting (A and B). (B) The numbers of generated spheres were counted (right upper). Representative images of spheres are shown (right lower). Results in the graph are presented as the mean ± SD of 3 independent experiments. (C) Monolayer-cultured A172 CSLCs (left) and SJ28P3 cells (right) treated with vehicle (DMSO) or NVP-BEZ235 for 3 days in vitro (1 × 105) were injected subcutaneously into BALB/c− nu/nu mice (5 mice per group), and the size of subcutaneous tumors was measured at the indicated time points. (D) Monolayer-cultured A172 CSLCs (left) and SJ28P3 cells (right) treated with vehicle (DMSO) or NVP-BEZ235 for 3 days in vitro (1 × 104) were injected intracranially into BALB/c− nu/nu mice (5 mice per group), and the survival of mice was evaluated by the Kaplan–Meier analysis (log-rank test: *P = .048 and **P = .049; upper). Alternatively, mice were sacrificed at 30 days after the intracranial injection, and the tissue sections were stained with hematoxylin and eosin (lower).

Article Snippet: Because A172 cells (1 × 10 7 ) from spheres cultured for 14 days did not form tumors after subcutaneous injection into 5-week-old male BALB/cAJcl− nu/nu mice (CLEA Japan, Inc.), we cultured A172 CSLCs by the monolayer culture method.

Techniques: In Vivo, Cell Culture, Western Blot, Generated, In Vitro, Injection, Staining