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. 2021 Aug 12;10(8):2073. doi: 10.3390/cells10082073

Figure 2.

Figure 2

CTX1 induced apoptosis of U937 cells. Without specific indication, U937 cells were treated with 500 nM CTX1 for 4 h. (A) Flow cytometry analyses of CTX1-treated U937 cells using annexin V-FITC/propidium iodide double staining. (Left) Untreated control cells. (Right) CTX1-treated U937 cells. (B) Western blot analyses showing degradation of procaspases and PARP in CTX1-treated cells (* p < 0.05, CTX1-treated cells compared to untreated control cells). (C) Viability of CTX1-treated cells was rescued by pretreatment with caspase inhibitors. U937 cells were pretreated with 10 μM Z-VAD-FMK (pan-caspase inhibitor), Z-DEVD-FMK (caspase-3 inhibitor), or Z-IETD-FMK (caspase-8 inhibitor) for 1 h, and then incubated with 500 nM CTX1 for 4 h. Cell viability was determined by MTT. The values represent averages of three independent experiments with triplicate measurement (mean ± SD, * p < 0.05). (D) Dissipation of mitochondrial membrane potential (ΔΨm) in CTX1-treated cells. The loss of ΔΨm was analyzed by flow cytometry. (E) Western blot analyses showing the production of t-Bid and the expression of BCL2 family proteins in CTX1-treated cells (* p < 0.05, CTX1-treated cells compared to untreated control cells). (F) Transfection of FADD siRNA abrogated CTX1-induced degradation of procaspase-8/-3 and production of t-Bid. U937 cells were transfected with 100 nM control siRNA or FADD siRNA, respectively. After 24 h post-transfection, the cells were treated with 500 nM CTX1 for 4 h (* p < 0.05, CTX1-treated FADD siRNA-transfected cells compared to CTX1-treated control siRNA-transfected cells). (G) FADD depletion rescued the viability of CTX1-treated cells (mean ± SD, * p < 0.05). (H) FADD depletion attenuated CTX1-induced ΔΨm loss (mean ± SD, * p < 0.05).