CAL-101 induces autophagy. (A) INA-6 and LB MM cells were treated with 5μM CAL-101 for 6 hours. CAL-101 treatment induced LC3 accumulation in LB and INA-6 cells, evidenced by fluorescence microscopy or transmission electron microscopy. Autophagosome formation was defined by the accumulation of LC3; arrows indicate autophagosomes. (B) INA-6 cells were treated with 5μM CAL-101 or serum starvation for 6 hours with or without 100μM 3-MA, stained with 1 μg/mL acridine orange for 15 minutes, and analyzed by fluorescence microscopy. (C) LC3 and beclin-1 protein levels were determined by Western blotting using LC3 and beclin1 Abs of lysates from INA-6 cells treated with CAL-101, with or without 3-MA. GAPDH served as a loading control.