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

Figure 5.

Figure 5

CTX1 induced upregulation of Fas and FasL in U937 cells through p38 MAPK-mediated phosphorylation of c-Jun and ATF-2. U937 cells were directly treated with 500 nM CTX1 for 4 h, or pretreated with 10 μM SB202190 for 1 h and then incubated with 500 nM CTX1 for 4 h. (A) Detecting the expression of Fas and FasL using qRT-PCR. qRT-PCR was conducted according to the procedure described in the Materials and Methods section (mean ± SD, * p < 0.05). (B) CTX1 treatment elicited increase in transcriptional activity of Fas promoter and FasL promoter (mean ± SD, * p < 0.05). After transfection with indicated promoter constructs for 24 h, the transfected cells were treated with 500 nM CTX1 for 4 h and then harvested for measuring luciferase activity (mean ± SD, * p < 0.05). (C) Effect of SB202190 on the expression of Fas and FasL proteins in CTX1-treated cells (* p < 0.05, SB202190/CTX1-treated cells compared to CTX1-treated cells). (D) Effect of SB202190 on the expression of Fas and FasL mRNAs in CTX1-treated cells (mean ± SD, * p < 0.05). (E) Effect of CTX1 treatment on levels of p-c-Jun, p-c-Fos and p-ATF-2 (* p<0.05, CTX1-treated cells compared to untreated control cells). (F) Effect of SB202190 on CTX1-induced c-Jun and ATF-2 phosphorylation (* p < 0.05, SB202190/CTX1-treated cells compared to CTX1-treated cells). (G) Depletion of c-Jun or ATF-2 using siRNA inhibited the transcriptional activity of Fas and FasL promoter constructs in CTX1-treated U937 cells. U937 cells were co-transfected with Fas promoter/FasL promoter constructs and indicated siRNAs. The used concentration of control siRNA, c-Jun, and ATF-2 was 100 nM. After 24 h post-transfection, the cells were treated with 500 nM CTX1 for 4 h (mean ± SD, * p < 0.05; NS, not statistically significant). (H) Suppression of c-Jun or ATF-2 expression abrogated CTX1-induced Fas and FasL upregulation in U937 cells. U937 cells were transfected with 100 nM control siRNA, c-Jun siRNA or/and ATF-2 siRNA, respectively. After 24 h post-transfection, the cells were treated with 500 nM CTX1 for 4 h (* p < 0.05, CTX1-treated c-Jun siRNA-, ATF-2 siRNA-, or c-Jun siRNA/ATF-2 siRNA-transfected cells compared to CTX1-treated control siRNA-transfected cells).