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. 2020 Jul 21;6:61. doi: 10.1038/s41420-020-00296-w

Fig. 5. MLN4924 exhibits p53-independent synergy with SN38.

Fig. 5

a Clonogenic assay in HCT116 p53+/+ and p53−/− cells co-treated with MLN4924 and SN38 for 24 h prior to media replenishment. Colonies were allowed to form for 7 days prior to fixing and staining with Crystal Violet. b Western blot analysis of p53, PARP, CUL3, and β-actin following 48 h treatment of HCT116 p53+/+ and p53−/− cells with 5 nM SN38 and 30 nM MLN4924, single agent, and in combination. c Annexin-V/PI flow cytometric analysis of HCT116 p53+/+ and p53−/− cell lines treated for 48 h with 5 nM SN38 and 30 nM MLN4924 alone or in combination. d Caspase-3/7 activity in HCT116 p53+/+ and p53−/− cells treated with 5 nM SN38, 30 nM MLN4924, or a combination of both compounds for 48 h. e Annexin-V/PI flow cytometric analysis of LoVo shScr and shp53 cell lines treated for 48 h with 5 nM SN38 and 30 nM MLN4924 alone or in combination. f Annexin-V/PI flow cytometric analysis of HCT116 BAX/BAK CRISPR-knockout and matched Empty Vector (EV) cell lines treated for 48 h with 5 nM SN38 and 30 nM MLN4924 alone or in combination. g Western blot analysis of PARP, BAX, BAK, and β-actin in the HCT116 BAX/BAK CRISPR-knockout and matched EV cell lines treated for 48 h with 5 nM SN38 and 30 nM MLN4924 alone or in combination. Two-way ANOVA with multiple comparisons test was used to compute p-values for mean ± SD of three independent experiments. Asterisks indicate statistically significant changes (*p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001; n.s denotes not significant).