Fig. 5 |. LCL161 disrupts binding of BIRC2 to H3 and induces cell death.
a, Representative images of the PLA (red) between BIRC2 and H3 in SW480 cells treated with or without 10 μM LCL161 for 1 h. Nuclei are stained with DAPI (blue). Scale bars, 10 μm. The experiment was performed independently three times. b, Quantification of the PLA signal with at least 75 cells analyzed per condition. Statistical analysis is described in Statistics and reproducibility. Two-sided Wilcoxon tests. P < 2.2 × 10−16. c, Western blot analysis of peptide pulldowns of GST-tagged BIRC2BIR3 with the H3 peptide (amino acids 1–22) in the presence of increasing concentrations of LCL161. The experiment was performed independently three times. d, Western blot analysis of BIRC2 in SW480 cells treated with or without 10 μM LCL161 for 1 h. β-actin was used as loading control. The experiment was performed independently three times. e, Western blot analysis of BIRC2 in human macrophages obtained by ex vivo differentiation of normal blood monocytes and stimulated or nonstimulated with IFNγ for 24 h in the presence or absence of 10 μM LCL161. β-actin was used as loading control. The experiment was performed independently three times. f,g, RT–qPCR analysis of IL6 (f) and RANTES (g) mRNA expression levels in human macrophages stimulated or nonstimulated with IFNγ for 24 h in the presence or absence of 10 μM LCL161. Data represent mean ± s.e.m. of at least three independent experiments. Paired two-sided t-test. h,i, MTT assay showing survival of mouse MEF (h) or human glioma LN18 cells (i) incubated for 24 h in medium containing the indicated concentrations of LCL161 and/or TNF. Data represent mean ± s.e.m. from three independent biological replicates.