Extended Data Fig. 2: Testing the impact of glutamine, glutaminolysis, AMPK pathway in resting NK cells and the role of glucose in activated NK cells.

(a) Sorted CD56^Dim NK cells were cultured in the indicated medium for 4 h. ECAR (a-b) and OCR (c-e) were measured. The results (Mean +SD) obtained from 3 independent donors are shown. ATP/ADP ratio (f) or ATP levels (g) in sorted CD56^Dim NK cells cultured in the indicated medium for 4 h was measured and is shown (Mean +SD, n = 3 or 4 independent donors). (h-l) Sorted CD56^Dim NK cells were cultured in the presence or absence of pyruvate for 4 h. (h) Absolute AMP concentration measured by mass spectrometry is shown (Mean +SD, n = 3 independent donors). The expression of AMPKα and p-AMPK (i-j) or p-ACC S79 (k-l) was measured by capillary WB. A representative image (i and k) or the result of the quantification (j and l) are shown (Mean +SD, n = 5 independent donors). For p-ACC quantification, sorted NK cells were treated for 1 h with MK8722, an activator of AMPK, to obtain a positive control (l). (m-n) In vitro activated NK cells were cultured in the indicated medium for 4 h. ECAR (m) and OCR (n) were measured. Left panels: One representative experiment (Mean +SD) out of three is shown. Right panels: individual points representing independent experiments are shown. (o) Sorted CD56^Dim NK cells were cultured in the indicated medium and the ATP level (Mean +SD, n = 6 independent donors) measured at the indicated time points are shown. One-way ANOVAs were performed with corrections for multiple testing in panels a, b, c, d, e, and f. Two-tailed T-test were performed comparing the indicated conditions in panel g, h, j, and l. Exact p-values are shown.

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