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. 2020 Aug 15;10(22):10290–10308. doi: 10.7150/thno.46728

Figure 2.

Figure 2

Celastrol activates pro-apoptotic ER stress and mitochondrial pathways via increasing ROS. (A) Rapid activation of the ER stress pathway by Celastrol. SGC-7901 cells were exposed to 3 µM Celastrol for indicated times. Lysates were probed for p-eIF2α, p-PERK, ATF4, and CHOP. eIF2α and GAPDH served as controls. (B) Effect of NAC on Celastrol-induced ER stress. SGC-7901 and BGC-823 Cells were pretreated 5 mM NAC for 1 h where indicated and exposed to Celastrol for 3 h (ATF-4, p-eIF2α, eIF2α, p-PERK) or 8 h (CHOP). Lysates were processed to Western blot assay. (C) Electron microscopy images of SGC-7901 cells exposed to 3 µM Celastrol for 8 h. Arrows pointing to ER [images shown are 10000 magnification in lower panel and 20000 magnification in upper panels]. (D) Mitochondrial membrane potential (Δψm) was detected by JC-1 dye. SGC-7901 cells were pretreated with 5 mM NAC for 1 h before exposure to 3 µM Celastrol for 12 h [scale bar = 40 µm]. (E, F) Western blot analysis of p-JNK/JNK (E) and Bax/Bcl-2 (F) in SGC-7901 cells exposed to Celastrol at indicated concentrations for 12 h. NAC pretreatment was carried out at 5 mM for 1 h. GAPDH was used as loading control. (G) NAC inhibits Celastrol-induced caspase-9 activation. SGC-7901 cells were exposed to Celastrol at indicated concentrations for 20 h, with or without 1 h pretreatment with 5 mM NAC. The caspase-9 activity was measured using a substrate kit (n = 3; **P<0.01, ***P<0.001 compared to Control; ##P<0.01 compared to Cela-3).