Specificity and role of autophagy in CLL cells. (A) Confocal fluorescence microscopy of CLL cells incubated 4 hours with or without F-ara-A (5μM), chlorambucil (20μM), CAL-101 (1μM), or rituximab (10 μg/mL). 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, using Olympus Fluoview 1000 Laser Scanning Confocal microscope. (B) Quantification of LC3 fluorescence in CLL cells from (A; n = 6). LC3 fluorescence increases for F-ara-A and CAL-101 treatments were significant (P < .0001). (C) Confocal fluorescence microscopy of CLL cells treated 4 hours with agent alone versus agent + chloroquine (CQ, 0.5μM). (D) Quantification of correlation index for LC3 and Lamp2 in CLL cells (n = 5) treated with rapamycin (5μM), CAL-101 (1μM), thapsigargin (1μM), F-ara-A (5μM), with or without CQ (0.5μM). Correlations of LC3 and Lamp2 were significant (rapamycin, P = .0004; thapsigargin, P = .0012; CAL-101, P = .0002; F-ara-A, P = .0001). (E) Viability at 24 hours and 48 hours, shown as percent of annexin (ann) negative and PI-negative cells (n = 8) by flow cytometry. Cytotoxicity of thapsigargin was significantly enhanced by CQ addition (P = .0008). (F) Viability at 24 hours in CLL cells (n = 5) untransfected or transfected with scrambled siRNA or ATG5/7 siRNA and treated with F-ara-A (5μM) or thapsigargin (1μM). Thapsigargin cytotoxicity significantly increased in ATG5/7 siRNA samples (P = .0047).