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cas12253-sup-0001-FigS1.tifimage/tif40KFig. S1. Peneciraistin C (Pe-C) induces caspase-independent non-apoptotic cell death in three lung cancer cell lines. A549, H446, and H661 cells were treated with indicated concentrations of Pe-C or 5-fluorouracil (5-FU) in the absence or presence of pan-caspase inhibitor (Q-vd-OPh, 100 μM) for 48 h. The percentage of cell death was quantified by Trypan blue staining.
cas12253-sup-0002-FigS2.tifimage/tif85KFig. S2. Peneciraistin C (Pe-C) does not induce caspase activation in A549 lung cancer cells. A549 cells were treated with indicated concentrations of Pe-C or 5-fluorouracil (5-FU) for 48 h. The activated caspase-3, caspase-9 and poly(ADP-ribose) polymerase (PARP) were measured by immunoblot analysis. cl, cleaved.
cas12253-sup-0003-FigS3.tifimage/tif53KFig. S3. Reactive oxygen species (ROS) production induced by peneciraistin C (Pe-C) plays an essential role in the case of apoptosis. A549 lung cancer cells were transfected with Atg5-targeted siRNA (si ATG5) and the control siRNA (si Control) for 48 h. (a) After transfection, A549 cells were treated with 3 μM Pe-C for the indicated time, then ROS generation was detected using chloromethyl-2′,7′-dichlorofluorescein diacetate (CM-H2-DCFDA, 10 μM) by a flow cytometer. (b) After transfection, A549 cells were treated with Pe-C (3 μM) in the absence (–) or presence of antioxidants N-acetyl-l-cysteine (NAC, 10 mM) or Mito-TEMPO (100 μM) for 12 h. CM-H2-DCFDA was added 30 min before end of treatment. The ROS levels were detected with a flow cytometer. (c) After transfection, A549 cells were treated with Pe-C (3 μM) in the absence (–) or presence of antioxidants NAC (10 mM) or Mito-TEMPO (100 μM) for 48 h. Cell viabilities were determined by MTT assay.

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