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. 2021 Feb 15;10(2):153. doi: 10.3390/biology10020153

Figure 5.

Figure 5

Figure 5

Effect of NAC and BSO on BZ- and CFZ-induced apoptosis and ER stress in B16-F1 cells. (A,B) Cells were pretreated with 5 mM NAC for 1 h (A) or 100 µM BSO (B) for 24 h and treated with a vehicle, 100 nM BZ or 100–200 nM CFZ for 24 h in 2% FBS/DMEM. Cell lysates were analyzed by Western blotting with antibodies against cleaved caspase 3, CHOP, and β-actin. The band densities were normalized against β-actin, and the fold changes compared to that in vehicle-treated control are indicated below each band. (C,D) Cells were pretreated with either NAC (C) or BSO (D) and then treated with vehicle, 50 nM BZ, or 100 nM CFZ in 2% FBS/DMEM. Ethanol-fixed cells were stained with PI and the gated sub-G1 fraction was determined by flow cytometry and is plotted as means ± standard deviations. (C,D) Cells were pretreated with NAC (C) or BSO (D), and then treated with vehicle, 5–20 nM BZ, or CFZ for 2 days in the culture medium. The viability of NAC- or BSO-pretreated cells was determined by MTT assay, and the percent cell viabilities are plotted as means ± standard deviations (n = 12 for total replicates). Significant difference between groups was determined by one-way ANOVA followed by Dunnett’s T3 post hoc tests. *** p < 0.001 compared with the vehicle-treated control cells. # p < 0.05, ## p < 0.01 and ### p < 0.001 compared with BZ or CFZ treatment alone without NAC or BSO pretreatment.