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. 2016 Nov 2;7(49):80716–80734. doi: 10.18632/oncotarget.13032

Figure 4. Ru1-induced apoptosis is partially caspase 3-dependent by triggering ROS-mediated mitochondrial dysfunction in A549 cells.

Figure 4

(A–D) A549 cells were incubated with Ru1 for 24 h with, or without, 1-h z-DEVD-fmk (50 μM) pretreatment. (A) The levels of cleaved caspase-3, PARP, cleaved-PARP and cleaved caspase-9 were assessed by western blot analysis. Below, quantification of the bands normalized by β-actin. (B) Cell viability was measured by MTT assay. (C and D) Annexin V/PI assay was performed by flow cytometry. (E–H) A549 cells were treated with Ru1 for 24 h with, or without, antioxidants (NAC = 10 mM and Tiron = 5 mM). (E) Relative fluorescence intensity was measured by microplate analyser. (F) Cell viability was assessed by MTT assay. (G) The percentage of apoptotic cells was measured by flow cytometry. (H) The levels of cytochrome c, cleaved-caspase 3, PARP, cleaved-PARP and cleaved-caspase 9 were assessed by western blot analysis with, or without, antioxidants (NAC = 10 mM). Below, quantification of the bands normalized by β-actin. (I) Cell viability was assessed by MTT assay after Ru1 treatment for 24 h with, or without, 1-h CsA (2 μM) pre-treatment. Results were represented as mean ± SD (*p < 0.05, ***p < 0.001).