Fig. 4. The effect of combined Beclin-1 and rapamycin treatment on autophagy induction and cell growth in Ras-NIH 3T3/Mdr cells. (A) The cells were transiently transfected for 48 h with either a vector expressing full-length Beclin-1 or an empty vector. Ras-NIH 3T3 cells treated with paclitaxel were used as a positive control for apoptosis. Analysis of caspase-3 activity was performed as described in Fig. 2. (B) To quantify the incidence of autophagy, Beclin1-trans-fetced Ras-NIH 3T3/Mdr cells treated with or without rapamycin (10 nM) for 24 h were stained with MDC and visualized by fluorescence microscopy. The autophagic index was calculated as the percentage of MDC-labeled cells out of 200 cells from each treatment group. (C) Beclin1-transfected cells were treated with rapamycin as indicated. Electrophoretic mobility change of LC3 from nonautophagic (LC3-I) form to autophagic membrane recruited (LC3-II) form was determined by immunoblotting. (D) The cells were transiently transfected with either a vector expressing mCherry-Atg5 fusion proteins or an empty vector, together with GFP-LC3 and allowed to express for 24 h. Cells were incubated in the presence or absence of rapamycin (10 nM) for 24 h. To quantify autophagic cells, we counted the number of autophagic cells among 200 GFP-positive cells. (E) Beclin-1-transfetced Ras-NIH 3T3/Mdr cells were incubated in the presence or absence of rapamycin in 96-well plates for 3 days. Cell growth was measured by the WST assay. The viability of cells treated with vehicle alone was regarded as 100%. (F) To identify the effect of rapamycin and Beclin1 when used as single agents or in combination on Ras-NIH 3T3/Mdr cells clonogenic survival, Beclin-1-transfected Ras-NIH 3T3/Mdr cells were incubated in the absence and presence of rapamycin for 7 days. The cells were fixed with methanol and stained with 5% Giemsa solution, and colonies of more than 20 cells were scored. Values represent the mean ± SD of quadruplicate determinants from one of three representative experiments. **P < 0.01; *P < 0.05 as determined by the Dunnett’s t-test.