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. 2022 Sep 2;11:e75908. doi: 10.7554/eLife.75908

Figure 2. AML cells can import the secreted acetate in co-culture to use it in TCA cycle and lipid biogenesis.

(A) Schematic of label distribution arising from [2-13C]acetate in TCA cycle intermediates. Black circles correspond to positions expected to be labelled. (B) 13C percentages of label incorporation in polar metabolites from labelled acetate in SKM-1 and MS-5 cells after two hours of incubation with [2-13C]acetate (4 mM) derived from 1H-13C-HSQC NMR spectra. Bars represent the mean of the 13C percentages and error bars represent the standard deviations for n=3 independent experiments. (C) 13C percentages on polar metabolites in SKM-1 and MS-5 cells in co-culture. Cells were co-cultured for 24 hr before the addition of extra 4 mM sodium [2-13C]acetate and culture for additional 30 min (upper panel) or 8 hr (lower panel). Bars represent the mean of the 13C percentages and error bars represent the standard deviations of n=3 independent experiments. 13C natural abundance is represented as a black bar at %13C=1.1. (D) 1H 1D NMR of lipids extracted from SKM-1 cells. Cells were co-cultured with MS-5 cells for 24 hr before the addition of extra 4 mM sodium [2-13C]acetate and culture for additional 48 hr. Lower panel represents overlay of spectra (n=3) from cells grown in 12C- and 13C—labelled acetate (black and red, respectively). Upper panel is the zoomed section of the spectra show 1H13C-methyl signal multiplets at 1.05ppm to 0.7pm as indicated by an arrow (the shift of the 1H13C methyl satellite signal is caused by the scalar JCH coupling of 125–128 Hz). (E) Cell viability measured by propidium iodide staining after culturing for 72 hr in glucose-free media containing 4 mM acetate or normal media, in the presence of DMSO, AraC 1 µM, and ACSS2i 20 uM. Bars represent the mean and error bars represent the standard deviations for n=3 independent experiments. A Tukey’s multiple comparison test was performed comparing each treatment and different medium conditions and p-values are represented by n.s. for not significant * for p-value <0.05, ** for p-value <0.01 and *** for p-value <0.001.

Figure 2—source data 1. Data and stats for panels included in Figure 2.

Figure 2.

Figure 2—figure supplement 1. Titration of acetate concentration in co-culture and acetate consumption by AML cells.

Figure 2—figure supplement 1.

(A) Linear regression of acetate concentrations and detected intensities in 1H-NMR spectra (Intensity = 1.38·106±1.6·104 x acetate concentration +2.27·105±3.3·104; R2=0.9987). The intensities detected in samples of co-culture were interpolated to obtain the estimate acetate concentration in co-culture (3.09 mM). Each point represents the mean of n=3 independent experiments and error bars represent standard deviation. (B) Extracellular acetate levels in SKM-1 cells cultured with 0.25 mM and 3 mM acetate medium after 0, 24, and 48 hrs. Each point represents the mean of n=3 independent experiments and error bars represent standard deviation. An unpaired Student’s t-test was applied for each condition (black brackets) and p-values are represented by * for p-value <0.05, ** for p-value <0.01 and *** for p-value <0.001.
Figure 2—figure supplement 1—source data 1. Raw values for acetate titration (A) and acetate consumption by SKM1 (B).
Figure 2—figure supplement 2. Label incorporation from [2-13C]acetate in SKM-1 and MS-5 cells cultured alone.

Figure 2—figure supplement 2.

(A) Example of metabolites assigned in a 1H-13C-HSQC spectrum from a polar extract from SKM-1 cells cultured with [2-13C]acetate for 2 hr. 13C percentages were calculated in 13 carbons from 11 different metabolites. The exact chemical shifts for each carbon are: acetate C2, 1.91 ppm (1H) – 26.1 ppm (13C); aspartate C2, 3.89 ppm (1H) – 55.0 ppm (13C); aspartate C3, 2.82 ppm (1H) – 39.4 ppm (13C); acetylcarnitine C9, 2.15 ppm (1H) – 23.4 ppm (13C); citrate C2, 2.52 ppm (1H) – 48.2 ppm (13C); fumarate C2, 6.51 ppm (1H) – 138.2 ppm (13C); glutamate C4, 2.34 ppm (1H) – 36.1 ppm (13C); glutamine C4, 2.44 ppm (1H) –33.7 ppm (13C); oxoglutarate C4, 2.43 ppm (1H) – 33.4 ppm (13C); glutathione C4, 2.55 ppm (1H) – 34.2 ppm (13C); malate C2, 4.28 ppm (1H) – 73.2 ppm (13C); malate C3, 2.68 ppm (1H) – 45.4 ppm (13C); and proline C4, 2 ppm (1H) – 26.6 ppm (13C). (B and C), 13C percentages on polar metabolites in Kasumi-1 (B) and HL-60 (C) cells in co-culture with MS-5 cells. Cells were co-cultured for 24 hours before the addition of extra 4 mM sodium [2-13C]acetate and incubated for 30 minutes or 8 hours. Bars represent the mean of the 13C percentages and error bars represent the standard deviation of n=3 independent experiments. 13C natural abundance is represented as a black bar at %13C=1.1.
Figure 2—figure supplement 2—source data 1. Raw values for different metabolites showing label incorporation from acetate in coculture 30 min incubation.
Figure 2—figure supplement 3. Acetate label incorporation analysis in lipids in AML cells in co-culture after 8 hr of labelling.

Figure 2—figure supplement 3.

1H 1D NMR of lipids extracted from SKM-1 (A), Kasumi-1 (B) and HL-60 (C) cell lines after being in co-culture with MS-5 cells for 24 hr and being labelled with 4 mM acetate for extra 8 hr (n=3). Lower panel represents overlay of spectra (3 batches) from cells grown in 12C- and 13C-labelled acetate for 8 hr, respectively (‘Black’ and ‘Red’). Upper panel is the zoomed section of the spectra (1.05ppm to 0.7ppm) for the lipids extracted from AML cells showing no 1H-13C3 peaks confirming no labelling for the methyl groups.
Figure 2—figure supplement 4. Acetate label incorporation analysis in lipids in AML cells in co-culture after 48 hr of labelling.

Figure 2—figure supplement 4.

1H 1D NMR of lipids extracted from Kasumi-1 (A) and HL-60 (B) cell lines after being in co-culture with MS-5 cells for 24 hr and being labelled with 4 mM acetate for extra 48 hr (n=3). Lower panel represents overlay of spectra (3 batches) from cells grown in 12C- and 13C-labelled acetate for 8 hr, respectively (‘Black’ and ‘Red’). Upper panel is the zoomed section of the spectra (1.05ppm to 0.7ppm) for the lipids extracted from AML cells showing clearly 1H-13C3peaks confirming labelling for the methyl groups.
Figure 2—figure supplement 5. Acetate label incorporation in TCA metabolites after 24 hr of ACSS2i treatment.

Figure 2—figure supplement 5.

13C percentages on polar metabolites in SKM-1 cells. Cells were co-cultured for 24 hr with 20 µM ACSS2i before the addition of extra 4 mM sodium [2-13C]acetate and incubated for 24 hr (n=1). Bars represent the 13C percentages. 13C natural abundance is represented as a black bar at %13C=1.1.
Figure 2—figure supplement 5—source data 1. Raw values of acetate labelling in SKM-1 cells +/-ACSS2 i.