Fig. 10.
(A and B) Simvastatin promotes apoptosis in TSC2-null tumors. Tumor tissues collected at days 0, 10, and 20 of the experiment were analyzed using the In Situ Cell Death Detection Kit (green); DAPI staining (blue) was performed to detect nuclei. (A) Representative images of tumors collected at day 20 of treatment were taken using an Eclipse TE2000-E microscope at ×200 magnification with appropriate filters. (B) Statistical analysis. The data (mean values ± SE; tumors from a minimum of five animals per treatment condition were analyzed) represent the percentage of apoptotic cells per total number of cells (taken as 100%). A minimum of 300 cells were analyzed per condition in each tumor. *, P < 0.001 for simvastatin on day 10 versus the control on day 10, for simvastatin on day 10 versus RAPA on day 10, for RAPA plus simvastatin on day 10 versus the control on day 10, for RAPA plus simvastatin on day 10 versus RAPA on day 10, for simvastatin on day 20 versus the control on day 20, for simvastatin on day 20 versus RAPA on day 20, for RAPA plus simvastatin on day 20 versus the control on day 20, and for RAPA plus simvastatin on day 20 versus RAPA on day 20 by ANOVA (Bonferroni-Dunn). (C and D) Simvastatin and rapamycin inhibit TSC2-null cell growth in vivo. Tumor tissues were collected on days 0, 10, and 20 of the experiment and subjected to immunohistochemical analysis with anti-Ki67 antibody (red). DAPI staining (blue) was performed to detect nuclei. (C) Representative images of tumors collected at day 20 of treatment were taken using an Eclipse E400 microscope at ×200 magnification with appropriate filters. (D) Statistical analysis. The data (mean values ± SE; tumors from a minimum of five animals per treatment condition were analyzed) represent the percentage of Ki67-positive cells per total number of cells (taken as 100%). A minimum of 300 cells were analyzed per condition in each tumor. *, P < 0.01 for RAPA on day 10 versus the control on day 10 and for RAPA on day 20 versus the control on day 20; **, P < 0.05 for simvastatin on day 20 versus the control on day 20; ***, P < 0.01 for RAPA plus simvastatin on day 10 versus RAPA on day 10, for RAPA plus simvastatin on day 10 versus simvastatin on day 10, for RAPA plus simvastatin on day 20 versus RAPA on day 20, and for RAPA plus simvastatin on day 20 versus simvastatin on day 20 by ANOVA (Bonferroni-Dunn).