Targeting the E3 Ub ligase Herc1 exacerbates Ara-C-induced apoptosis in AML cells. (A) MA cells were plated for 24 hours in triplicates into 6-well plates and treated with ±Ara-C (200 nM) or H2O (vehicle). The values are presented as means ± standard error of the mean (SEM) (n = 3). Significance was determined using a 2-way ANOVA analysis, followed by Holm-Šídák multiple comparisons test. ∗P ≤ .05. (B) Representative flow cytometry analysis of MA cells treated with ±Ara-C (200 nM) or H2O (vehicle) for 24 hours and stained with annexin V/DAPI. (C) Representation of the annexin V/DAPI analysis displayed in panel B for MA cells. The values are means ± SEM for n = 4 independent replicates. Significance was determined using a 1-way ANOVA, followed by Holm-Šídák multiple comparisons test. ∗∗P ≤ .005; ∗∗∗∗P ≤ .0001. (D) Representation of the annexin V/DAPI analysis displayed in panel B for HM cells. Values are means ± SEM for n = 3 independent replicates. Significance was determined using a 1-way ANOVA, followed by Holm-Šídák multiple comparisons test. ∗P ≤ .05. (E) Representation of the annexin V/DAPI analysis displayed in panel B for U937 cells. Values are means ± SEM for n = 4 independent replicates. Significance was determined using a 1-way ANOVA, followed by Holm-Šídák multiple comparisons test. ∗P ≤ .05. (F) Representative flow cytometry analysis of MA cells treated with Ara-C (200 nM) or H2O (vehicle) for 0 hours, 6 hours, and 12 hours and stained with propidium iodide or DAPI for cell cycle analysis. (G) Representation of the cell cycle analysis displayed in panel B for MA cells with ±Ara-C (200 nM) or water (Vehicle) for 0 hours and 6 hours (n = 3 independent experiments with 3 technical replicates) and for 12 hours (1 independent experiment, 3 technical replicates). Significance was determined using mixed-effects analysis followed by Holm-Šídák multiple comparisons test. ∗P ≤ .05; ∗∗P < .01; ∗∗∗∗P < .0001.
