Figure 3
MTX downregulates the expression of Beclin-1, Atg12–Atg5 and LC3 in SCC-15 cells, but increases their levels in SCC-9 cells. (a) Total protein extracts were prepared from SCC-9 and SCC-15 cells after treatment with 10 μg/ml MTX for the indicated time intervals. The expression levels of Beclin-1 and Atg12–Atg5 conjugate were determined using western blotting. β-Actin was used as an internal control. (b) SCC-9 and SCC-15 cells were treated with 10 μg/ml MTX. Cytosolic protein extracts were prepared at various time points. LC3 protein expression and caspase-3 cleavage were assessed using western blotting. β-Actin was used as an internal control. (c) SCC-15 cells were treated with or without 10 μg/ml MTX in the presence or absence of E64d (30 μ M) and pepstatin A (15 μ M). After culturing for 12 or 24 h, cytosolic protein extracts were analyzed for LC3 protein expression using western blotting. β-Actin was used as an internal control
