Rapamycin increases markers of autophagy without inducing cell toxicity.
A, porcine chondrocytes treated with no additives or 50 μm rapamycin for 12 h were assessed for injury by measuring LDH levels in medium and uptake and conversion of MTT into a colored formazan. Results are expressed as a percent of control (no rapamycin). There were no differences in cell injury markers or caspase-3 levels with rapamycin (n = 3, p > 0.05). B, porcine chondrocytes treated with no additives or 50 μm rapamycin for 4–12 h were lysed and lysates were used in Western blotting with antibodies to LC3 and Beclin. Increased levels of LC3-II occur with rapamycin treatment at 4, 6, and 8 h. Increased levels of Beclin were noted at 12 h. C, RNA was isolated from porcine chondrocytes incubated with and without rapamycin for 4–12 h. mRNA for LC3 and Beclin were measured with end-point PCR and showed no changes in Beclin mRNA levels and increases in LC3 at 8 h. D, chondrocytes were isolated and plated in iso-osmolar medium containing normal (5 mm) or high (25 mm) glucose. After 18 h, medium were removed, identical fresh medium was added, and ACVs were enumerated in conditioned medium for an additional 4–12 h.