Figure 3

CD157 knockdown increases AraC-induced cell death in U937 cells. (A) THP1 (black line), OCI-AML3 (dashed black line) or U937 (red line) cells were treated with increasing concentrations of AraC for 24 h to assess the sensitivity of each AML cell line to AraC. (B) The sensitivity of CD157-high (red line) versus CD157-low (blue line) U937 cells to AraC was compared by Presto Blue assays. Six replicates for each condition have been performed. Results are the mean ± SEM of four experiments. ****p < 0.0001, Student’s t-test. The EC₅₀ for U937 cells with high or low CD157 was calculated using the GraphPad software (see “Materials and methods”). (C) Representative dot plot (left) and histogram (right) of CD157-high and CD157-low U937 cells treated with AraC for 24 h, obtained by flow cytometry analysis after AnnexinV/PI staining. Histograms show the mean ± SEM of eight independent experiments. **p < 0.01, two-way ANOVA with Sidak’s multiple comparison test. (D) U937/CD157-high and U937/CD157-low were treated with AraC for 24 h, then whole cell lysate was subjected to Western blotting. Numbers below blots indicate fold change in the expression of each protein relative to untreated CD157-high cells, normalized to the corresponding β-actin and full length protein (PARP, Bax and Caspase-3). β-Actin was used as loading control. Left panels: the levels of Mcl-1, pGSK-3β, cBax, cPARP, and cCaspase-3 were determined by densitometry analysis and are the mean of three experiments run in parallel. Uncropped blot is provided in Supplementary Fig. S10. Data are expressed as the mean ± SEM of band densities of western blots from three separate experiments. *p < 0.05, **p < 0.01, ****p < 0.0001, ns = not significant, two-way ANOVA with Sidak’s multiple comparison test.