Fig. 5.

Slc4a11 reduces Gln-induced ROS and oxidative damage. (A) Effect of Gln on mitoROS. MCEC WT and KO cells in Complete Media were washed with assay media that contained DMEM (see Supplementary Table 2 for list of amino acids), 0.5% dialyzed-serum, 5.5 mM glucose, and ±0.5 mM Gln for 2–24 h. Cells were stained for 20 min with 2.5 μM MitoSOX 37 °C, followed by one wash with HBSS and analyzed by flow cytometry into MitoSOX⁺ or MitoSOX⁻ populations (see Supplementary Fig. 2B), n = 3, *p < 0.05 vs Gluc, same genotype. δ: p < 0.05 vs WT, same treatment at 24 h. (B) Effect of Gln on mitochondrial polarization. MCEC WT and KO were incubated in assay media ±0.5 mM Gln over 24 h. Cells were stained for 20 min with 100 nM TMRE 37 °C, followed by one wash with HBSS and analyzed by flow cytometry into TMRE⁺ or TMRE⁻ populations (see Supplementary Fig. 3), n = 3, *p < 0.05 vs Gluc, same genotype. δ: p < 0.05 vs WT, same treatment at 24 h. (C) Hyperpolarization MMP by Gln shown by Geometric mean of TMRE⁺ population, n = 3, *p < 0.05 vs Gluc, same genotype. δ: p < 0.05 vs WT, same treatment at 24 h. (D) Effect of Gln on Apoptosis. MCEC WT and KO were incubated in assay media ±0.5 mM glutamine over 24 h. Cells were stained with AnV-FITC for 5 min, followed by one wash with HBSS and analyzed by flow cytometry, n = 3, *p < 0.05 vs Gluc, same genotype. δ: p < 0.05 vs WT, same treatment at 24 h. (E) Rescue of KO cells by MitoQ. MCEC WT and KO were incubated in assay media +0.5 mM glutamine, and 1 μM MitoQ, or 10 mM N-Acetyl-cysteine (NAC) for 24 h, n = 3, *p < 0.05 ± drug. (F) MitoQ reduces Gln-dependent mitoROS, n = 3, *p < 0.05 ± MQ. (G) 1 μM MitoQ for 24 h increases %TMRE⁺ KO cells, n = 3, *p < 0.05 ± MQ. (H) [ATP] in WT and KO MCEC after 24 h n = 3, *p < 0.05 ± MQ.