Article Figures & Data
Figures
- Figure 1.
A, Tsc-null mouse embryonic fibroblasts have a higher level of S6K phosphorylation than control mouse embryonic fibroblasts in serum-starved conditions. Cells were starved for 24 h (−). Cells were starved and then stimulated for 15 min with 20% fetal bovine serum (+). Phosphorylated and total levels of S6K were examined as described in Materials and Methods. Results are representative of three independent experiments. B, growth of Tsc1−/− and control mouse embryonic fibroblasts in serum-free medium (left); growth of Tsc2−/− and control mouse embryonic fibroblasts in serum-free medium (right). Twenty-four hours after seeding, cells were changed to medium without serum. Cell viability was assayed using an MTT assay. Points, means; bars, SD. C, Tsc2−/− mouse embryonic fibroblasts do not arrest in G0/G1 during serum starvation. Tsc2 mouse embryonic fibroblasts were collected at 60% confluency, whereas they were exponentially growing in normal medium or after serum starvation for 24 h. Cell cycle profile was then analyzed as described in Materials and Methods. Results are representative of three independent experiments. D, cell cycle profile of Tsc2−/− mouse embryonic fibroblasts were analyzed in exponentially growing conditions (48 h, 60% confluency) or in contact inhibited conditions (72 h, 100% confluent). Results are representative of three independent experiments.
- Figure 2.
A, FTIs inhibit the farnesylation of Rheb. pCDNA3mycHA-Rheb (WT and C181S) were transiently expressed in human embryonic kidney-293 cells. Cells were treated for 24 h with the indicated concentration of FTI starting 16 h after transfection. Rheb expression was detected by immunoblotting with anti-myc 9B11 antibody. Results shown are representative of three independent experiments. B, farnesylation is required for the activation of S6K by Rheb. pCMV5-hemagglutinin-Rheb (WT and C181S) and pCMV5-Flag-p70S6K were transiently coexpressed in COS-7 cells. Cells were pretreated with the indicated concentration of BMS-225975 for 24 h, and then starved in PBS for 30 min. Phosphorylated and total levels of S6K were examined as described in Materials and Methods. Rheb expression was confirmed by the use of anti-hemagglutinin antibody. C, Tsc1−/− mouse embryonic fibroblasts (left) and Tsc2−/− mouse embryonic fibroblasts (right) were treated with 1 or 2 μmol/L BMS-225975 or 10 nmol/L rapamycin in serum-free medium for 48 h. Phosphorylated and total levels of S6K or S6 were examined as described in Materials and Methods. Inhibition of farnesylation was confirmed by the mobility shift of HDJ-2 protein. Results are representative of three independent experiments. D, Tsc−/− mouse embryonic fibroblasts were plated in 96-well plates, and after 24 h, medium was changed to serum-free medium and the indicated drug (day 0). Cells were treated with 1 μmol/L BMS-214662 or BMS-225975, or 10 nmol/L rapamycin. Cell viability was then assayed by MTT assay at the indicated times. Tsc1−/− mouse embryonic fibroblasts (left) and Tsc2−/− mouse embryonic fibroblasts (right). Points, means; bars, SD.
- Figure 3.
FTIs can inhibit the anchorage-independent growth of Tsc−/− mouse embryonic fibroblasts. A, Tsc-null mouse embryonic fibroblasts are able to grow in soft agar. Mouse embryonic fibroblasts were cultured in soft agar for 2 wks at different densities, i.e., 3 × 103 cells per well (top) or 3 × 104 cells per well (bottom). Results are representative of three independent experiments. B, Tsc1−/− mouse embryonic fibroblasts (top) and Tsc2−/− mouse embryonic fibroblasts (bottom) were cultured for 2 wks in soft-agar in the presence of various inhibitors as indicated (0.25 μmol/L FTIs, 2.5 nmol/L rapamycin or 33 μmol/L LY249002). Results are representative of three independent experiments.
- Figure 4.
FTIs induced the rearrangement of actin cytoskeleton in Tsc2−/− mouse embryonic fibroblasts. Tsc2 mouse embryonic fibroblasts were treated with DMSO, FTIs (2 μmol/L) or rapamycin (10 nmol/L) for 72 h in normal growth medium. Actin filaments of Tsc2+/+ (left) and Tsc2−/− (right) mouse embryonic fibroblasts were visualized by TRITC-phalloidin as indicated in Materials and Methods. Results are representative of three independent experiments.
- Figure 5.
Tsc2−/− mouse embryonic fibroblasts overexpressing myc-tagged Rheb (M184L) are resistant to FTI-induced rearrangement of actin cytoskeleton. Overnight after transfection, cells were treated with or without FTIs (2 μmol/L) and actin filaments were visualized by TRITC-phalloidin as described in Materials and Methods (left). Rheb expressing cells detected by anti-myc antibody and FITC anti-mouse antibody are indicated with arrows (right). Results are representative of three independent experiments.
Volume 4, Issue 6
