Figure 3. GLS1 Regulates Glutamine-mediated ROS Scavenging in PHD2^KD Cells and Contributes to Cell Survival.

(A) Glutamine uptake rate in cultured control (ctr) and PHD2^KD cells (n=6).

(B) GLS1 and β-actin immunoblot (n=3).

(C-D) Fractional contribution of [U-¹³C]-glutamine to glutamate (C) and GSH (D) (n=3-6).

(E-F) GSH to GSSG levels (E) and total intracellular ROS levels (F) in control and PHD2^KD cells after genetic silencing of GLS1 (scrambled shRNA (shScr: -) or shGLS1 (+)) (n=6).

(G) Control, PHD2^KD and PHD2^KDGLS1^KD cell viability after treatment with 25 μM H₂O₂ (n=6). PHD2^KDGLS1^KD cells are PHD2^KD cells transduced with shGLS1; ctr and PHD2^KD cells are transduced with shScr.

(H) Rescue of cell viability of BPTES-treated PHD2^KD cells during oxidative stress (25 μM H₂O₂) with glutamate (Glu), but not with dimethyl α-ketoglutarate (DM α-KG), citrate (Cit), malate (Mal) or oxaloacetate (OAA) (n=9).

(I) TUNEL immunostaining of the scaffold center 3 days after implantation, and quantification (scale bar, 100 μm; n=4).

(J) Cell viability of control, PHD2^KD and hGLS1-overexpressing (hGLS1^OE) cells after treatment with 25 μM H₂O₂ (n=6).

(K) Fractional contribution of [U-¹³C]-glutamine to GSH (n=6).

(L) Ratio of GSH to GSSG (n=6).

(M) Total intracellular ROS levels with and without H₂O₂ treatment (n=6).

Data are means ± SEM. *p<0.05 vs. ctr, **p<0.01 vs. ctr, ***p<0.001 vs. ctr (Student’s t-test), #p<0.05 (ANOVA).