Figure 3
From: Aloperine executes antitumor effects against multiple myeloma through dual apoptotic mechanisms
Aloperine activates caspase-8 via downregulation of cFLIP. (A) U266 and MM.1S cells were treated with 80 μM aloperine for the indicated durations. Protein expression levels of cFLIP, Noxa, Bimel, cIAP-1, cIAP-2, XIAP, survivin, Mcl-1, and β-actin were analyzed using Western blotting after treatment for 0, 12, 24, or 48 h. (B, C) U266 cells were transfected with FLIPS, FLIPL, or the corresponding empty vectors (empty vector FLIPL: FLIPL-EV; empty vector FLIPS: FLIPS-EV; FLIPL and FLIPS overexpression: FLIPL-OE and FLIPS-OE; WT: FLIPL, FLIPS). Expression of cFLIP and caspase-8 was verified by Western blotting in EV and cFLIP OE U266 cells without (B) or with (C) different doses of aloperine. (D) Cells were treated with 80 or 160 μM aloperine for 48 h, and apoptosis was determined via FACS analysis of DNA fragmentation of propidium iodide-stained nuclei. Background apoptosis: FLIPL-EV: 1.5%; FLIPS-EV: 1.4%; FLIPL: 0.9%; FLIPS: 1.3%. Data represent the mean ± SD of three independent experiments carried out in triplicate; a P < 0.05. GAPDH/β-actin was assessed by Western blotting. All proteins levels were quantified densitometrically and normalized to GAPDH/β-actin.