Figure 2

CD157 modulates cellular stress responses in AML cell lines. (A) OCI-AML3 cells were cultured overnight in the absence of FCS, then the SY11B5 anti-CD157 mAb or mIgG (both at 10 µg/ml) were added for 24 h. Cell viability was measured by AnnexinV/PI staining and flow cytometry analysis. Histograms show the effect of nutrient deprivation on cell viability expressed as fold change of viable cells compared to cells maintained in standard culture conditions, and are the mean ± SEM of six independent experiments performed in quadruplicate. *p < 0.05, ***p < 0.001, ns = not significant, one-way ANOVA with Tukey’s multiple comparison test. (B) OCI-AML3 were cultured overnight in serum-free medium then, SY11B5 or RF3 mAbs to CD157 (10 µg/ml) were added for 24 h. Whole cell lysates were subjected to Western blotting and probed with the indicated antibodies. Blots were re-probed using antibodies against the total proteins. β-Actin was used as loading control. Numbers below blots indicate fold change in the expression of each protein relative to untreated control, normalized to the corresponding β-actin and total protein (mTOR and AKT). Uncropped images are provided in Supplementary Fig. S9 (C) CD157-high or low OCI-AML3, THP1 and U937 cells were cultured in standard conditions or in serum-free medium for 24 h, and then subjected to AnnexinV/PI staining and flow cytometry analysis. Results are expressed as fold change of viable cells normalized to CD157-high cells maintained in standard culture conditions and are the mean ± SEM of three experiments. *p < 0.05, **p < 0.01, one-way ANOVA with Tukey’s multiple comparison test. (D) CD157-high and CD157-low THP1 cells and (E) CD157-high and CD157-low U937 cells were maintained for 18 h in FCS-free culture medium, then were stimulated with FCS for 10 min or 2 h. Whole cell lysates were subjected to Western blotting and probed with the indicated antibodies. Blots were re-probed using antibodies against the total proteins. β-Actin was used as loading control. 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 total protein (AKT and GSK3β). Panels (D) and (E) show one Western blot representative of three independent experiments performed. Uncropped blot images are provided in Supplementary Fig. S9.