Figure 3

The cytotoxicity of borrelidin is linked to the induction of the amino acid starvation response.

(a–f) HUVEC cells grown in full serum media were exposed to the indicated concentrations of BN (a–c) or BC194 (d–f) and standardized to 0.05% DMSO. Cropped images from western blots of cell extracts were analyzed using antibodies recognizing phospho-eIF2α and cleaved-caspase 3 with β-tubulin as a loading control. Full images of blots can be found in Supplementary Figures S2a and S2b. Quantification of phospho-eIF2α and cleaved-caspase 3 for BN (b,c) and BC194 (e,f) relative to β-tubulin; mean ± SEM, n ≥ 3, *p < 0.05 relative to 0 nM (one-way ANOVA, Tukey Test). The apparent drop in the levels of phospho-eif2α as BC194 increased to 1000 nM and the exclusion of 1000 nM BN-treated data (designated by #) are due to the fact that endothelial cells exposed to high macrolide concentrations were visually apoptotic, making estimations of total protein loaded difficult. As such, we believe that the variability observed in these data at higher concentrations was more related to the severe status of the cells rather than a change in the amino acid starvation response. (g,h) Western blot (g) quantification of eif2α amounts relative to β-tubulin for both BN- and BC194-treated cells (100 nM) in the presence of various threonine concentrations (0–10 mM); mean ± SEM, n ≥ 3, *p < 0.05 relative to 0 mM threonine (one-way ANOVA, Tukey Test), #p < 0.01 between compounds at same threonine concentration (two-way ANOVA, Sidak Test). Full images of blots can be found in Supplementary Figure S2c. See also Supplementary Figure S1 for cell cycle, proliferation effects and Supplementary Figure S3 for BC220 data.