Autophagy in CLL cells. (A) Confocal fluorescence microscopy for CLL cells untreated or after 4-hour starvation in HBSS or 4-hour treatment with rapamycin (5μM) or thapsigargin (1μM). LC3 (red) shows autophagosomes. Lamp2 (green) shows lysosomes. 4,6-diamidino-2-phenylindole (blue) shows nuclei. Images were collected with 60× objective and 4× optical zoom. (B) Quantification of LC3 immunofluorescence intensity. Integrated intensity was averaged in 5 microscopic fields relative to number of cells per field and then averaged in 6 CLL patients' cells. Differences from vehicle-treated were significant (P < .0001). (C) Immunoblot for LC3B I and II in CLL cells (representative of 4 experiments). GAPDH was used as loading control.