Fig. 3

EIF4A inhibition and araC have contrasting effects on mitochondrial fitness and bioenergetic profile. AraC and CR-1-31-B synergize in MOLM-14 cells: a MOLM-14 cells were treated with CR-1-31-B (0-5 nM) in combination with araC (0-500 nM) for 24 h. Viability data was obtained using CellTiter-Glo and synergy between the drugs was assessed using the SynergyFinder application. CR-1-31-B hampers protein synthesis in the presence or the absence of araC: b (left) MOLM-14 cells treated with (i) vehicle, (ii) 250 nM araC (24 h), (iii) 10 nM CR-1-31-B (the last 1 h of treatment) and (iv) 250 nM araC (24 h) + 10 nM CR-1-31-B (the last 1 h of treatment). Sub-polysome, light-, and heavy-polysome fractions were obtained by ultracentrifugation using 5–50% sucrose gradients. During fractionation, UV absorbance at 254 nm (Abs 254 nm) was continuously monitored to obtain absorbance tracings. Position of 40S and 60S ribosomal subunits, monosome (80S) and polysomes are indicated. (right) The areas under the curve for the monosome (80S) and polysomes was used to calculate the polysome/80S ratio. AraC affects mitochondrial membrane potential in an eIF4A-dependent manner: c MOLM-14 cells were treated with vehicle, 2.5 nM CR-1-31-B, 250 nM araC, or their combination for 48 h. Mitochondrial membrane potential (Mitochondrial MP) and d mitochondrial superoxide (mtROS) content were quantified by flow cytometry using TMRE and mitoSOX, respectively; arbitrary units (a.u.). CR-1-31-B induces a shift towards a lower bioenergetic state: e MOLM-14 cells were treated with vehicle, 250 nM araC (48 h) and/or 2.5 nM CR-1-31-B (last 24 h of the 48 h incubation). The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) obtained using the Seahorse XF technology was used to calculate the ATP production rates by mitochondrial respiration (y-axis) and glycolysis (x-axis). Data related to Fig. 3e are shown in Supp. Fig. 2b-e. EIF4A inhibition and araC affect metabolite levels in MOLM-14 cells. f MOLM-14 cells were treated with vehicle, 250 nM araC (48 h) and/or 10 nM CR-1-31-B (treatment for the last 24 h of the total 48 h incubation with araC). Targeted analysis of metabolite content was done by GC/MS. The heatmap represents intracellular metabolites content relative to vehicle treated cells. Comparisons were assessed by two-way ANOVA followed by Šídák’s multiple comparisons test, *p < 0.05. Footnote. With the exception of targeted metabolite analysis (n = 2), the bars or circles represent the mean values of at least 3 independent experiments plus/minus the standard error of the mean (SEM) for c-d or the standard deviation for e. Comparisons between groups were assessed by One-way ANOVA followed by paired t-test, *p < 0.05 and ns indicates a p > 0.05. All functional analysis performed by flow cytometry were done for viable DAPI negative cells