Rapamycin dosing schedules in A J Tsc2 mice A J Tsc2 mice were assigned to one of three different rapamycin treatment cohorts or an untreated control group. The rapamycin cohorts included the following schedules daily 4 weeks plus weekly 8 weeks, daily 4 weeks, weekly 12 weeks. All animals contain started treatment at nine months of age and were eutha nized twelve weeks later. Mice in Group 1 were treated with 8 mg kg rapamycin administered by intraperitoneal injection Monday through Friday for four weeks fol Inhibitors,Modulators,Libraries lowed by weekly doses of 8 mg kg rapamycin IP for eight weeks. Mice in Group 2 were treated with 8 mg kg rapamycin IP Monday through Friday for four weeks and received no drug treatment for the next 8 weeks. Mice in Group 3 were treated with weekly 8 mg kg rapamycin IP for twelve weeks.

Rapamycin powder was obtained from LC Laboratories and a 20 mg ml stock of rapamycin was made in ethanol. The stock solution was diluted to 1. 2 mg ml in vehicle for the 8 mg kg dose. Rapamycin treatments were administered within two hours of their prepara tion. All animals were checked daily, Inhibitors,Modulators,Libraries and general health and behavior were noted. All rapa mycin treated animals were weighed at 9 months, and again at the time of euthanasia at 12 months. All mice were euthanized at approximately twelve months of age according to institutional animal care guidelines. The severity of kidney disease was calculated using quantitative histopathology as described previously. Untreated A J Tsc2 mice from the 9 month and 12 month cohorts were weighed at the time of necropsy for comparison.

All experiments were done according to animal protocols approved by our institutional animal protocol review committee and were compliant with federal, local, and institutional guidelines on the care of experimental animals. Treatment of subcutaneous tumors with asparaginase, vincristine, sunitinib, bevacizumab, and rapamycin Inhibitors,Modulators,Libraries Nude mice were obtained from Charles River Laboratories, Inc. and injected subcuta neously on the dorsal flank with Inhibitors,Modulators,Libraries 2. 5 million NTC T2null cells. NTC T2null cells are mouse embryonic fibroblasts that have been described previously. A total of 80 CD 1 nude mice were divided into 10 randomly assigned groups untreated control group, single agent rapamycin, single agent Inhibitors,Modulators,Libraries asparaginase, combination asparaginase plus rapamycin, single agent vincristine, combination vincristine plus rapamycin, single agent sunitinib, combination sunitinib plus rapamycin, single agent bevacizumab, and combina tion bevacizumab plus rapamycin.

As soon as tumors became visible, they were measured Monday through Friday using calipers. Tumor volumes were calculated using the formula length width width 0. 5. All mice began treatment during when tumors reached a volume of 100 mm3. All mice were euthanized once tumors reached 3000 mm3 in accordance with institutional animal care guidelines.