Fig. 3. The accumulation of MDC is inhibited by inhibitors of the autophagic pathway. CHO cells were incubated in -MEM medium (control cells) or in EBSS medium (starved cells) for two hours in the absence or the presence of 10 mM 3-methyladenine (3-MA), 200 nM wortmannin (WM) or 2 µg/ml cycloheximide (CXM). In the case of 3-MA and CXM, cells were pre-incubated for 3 hours (3-MA) or 5 hours (CXM) with the corresponding inhibitor before the induction of autophagy. (A) Following the incubation period indicated above, cells were incubated with 0.05 mM MDC for 10 minutes at 37°C and then washed four times with PBS pH 7.4. Cells were immediately analyzed by fluorescence microscopy as indicated in Fig. 1. (B) Cells treated as indicated above were incubated with 0.05 mM MDC for 10 minutes at 37°C, washed four times with PBS pH 7.4, and then lysed in 10 mM Tris-HCl, pH 8 containing 0.1% Triton X-100. Intracellular MDC was measured by fluorescence photometry as indicated in Experimental Procedures. The data represent the mean±s.e.m. of two to four independent experiments.