Inhibition of autophagy with PI3K inhibitor 3-MA and lysosome inhibitor NH₄Cl suppress chLym-1induced apoptosis, antibody-dependent cellular cytotoxicity(ADCC) and complement-dependent cytotoxicity(CDC) in Raji cells.

A: 3-MA and NH₄Cl significantly inhibit apoptosis induced by chLym-1 in Raji cells. Raji cells were treated with 10 µg/ml of chLym-1 or/and autophagy inhibitors for 24 h, while Vehicles were treated with full medium. Thus, FCM was used to detect apoptotic rates of Raji cells. B: ChLym-1-induced ADCC is inhibited by PI3K inhibitor 3-MA. Raji cells, together with PBMCs (effect cells: target cells = 60∶1, 40∶1 and 20∶1), were treated with autophagy inhibitor 3-MA or/and chLym-1 for 5 h. Autophagy inhibitor 3-MA was added 12 h before chLym-1 treatment. Then, ADCC mediated by chLym-1 on Raji cells was measured in a 5 h LDH release assay. C: ChLym-1-induced ADCC is inhibited by Anti-ATG5 RNA inference. Raji cells (non-treated, transferred with control siRNA or transferred with Anti-ATG5 siRNA), together with PBMCs (effect cells: target cells = 60∶1, 40∶1 and 20∶1), were treated with chLym-1 for 5 h. ATG5 RNA-inference is performed 48 h before chLym-1 treatment. Then, ADCC mediated by chLym-1 on Raji cells was measured in a 5 h LDH release assay. D: ChLym-1 mediated CDC is blocked by PI3K inhibitor 3-MA or lysosome inhibitor NH₄Cl. Raji cells were treated with autophagy inhibitor 3-MA/NH₄Cl or/and chLym-1 for 1 h. Autophagy inhibitor 3-MA and NH₄Cl was added 24 h before chLym-1 treatment. Then, CDC mediated by chLym-1 on Raji cells was measured immediately by MTT assay.