Figure 2. Dissociation of mitochondria–ER contacts diminishes mitochondrial respiration but not glycolysis.

(A) Left, representative PLA images of EM CD8⁺ T cells activated for 12 h with α-CD3/α-CD28 (3-28) mAb as in Figure 1A and concomitantly treated with DMSO (top) or nocodazole (10 μM, bottom). Cells were stained as in Figure 1D. Right, representative summary bar graph of PLA from 1 of 3 donors treated with DMSO (n = 28 cells) or nocodazole (n = 23 cells).

(B) Left, representative mitochondrial perturbation assay of EM CD8⁺ T cells activated as in Figure 1A or activated in the additional presence of nocodazole (10 μM). Right, bar graphs of basal respiration and glycolysis (n = 4 donors).

(C) Left, representative PLA images of Jurkat T cells cultured in the presence of DMSO (top) or nocodazole (10 μM, bottom) for 12 h. Right, bar graph showing the number of detected mitochondria–ER junctions per cell in DMSO (n = 17) and nocodazole (n = 17) treated cells. Representative of n = 2 independent experiments.

(D) Left, representative mitochondrial perturbation assay of Jurkat T cells cultured as in (C). Right, bar graphs of basal respiration and glycolysis of control and nocodazole treated Jurkat T cells (n = 3 independent experiments).

(E) Immunoblot of cellular fractions and total cell lysates (T) from Jurkat T cells. Cells were separated into fractions containing mitochondria–ER junctions (MEJ), pure mitochondria (PM), and remaining supernatant from MEJ (containing the majority of ER, Golgi, and cytoplasm – EGC). Blots were probed with antibodies targeting ACC, VDAC, Cox iv, Grp75, PACS2, KDEL motifs, and actin.

(F) Left, immunoblot analysis of lysates from non-transfected (non), scrambled siRNA (sc) transfected, and Grp75 siRNA (siRNA) transfected Jurkat T cells. Cells were cultured for 72 h after transfection. Blots were probed with Grp75 and actin antibodies. Right, quantification of Grp75 abundance in control and Grp75 siRNA transfected lysates normalized to actin (representative of n = 3 independent experiments).

(G) PLA images (left) and quantification of mitochondria–ER contact abundance (right) of control (sc, n = 32) and Grp75 siRNA (n = 63) transfected cells (n = 2 independent experiments).

(H) Left, representative mitochondrial perturbation assay of Jurkat T cells cultured for 72 h after transfection with scrambled (sc) and Grp75 targeted siRNA. Right, bar graphs of basal respiration and glycolysis from control (sc) and Grp75 siRNA transfected Jurkat T cells (n = 3 independent experiments).

Data are presented as mean±SEM. Two-tailed unpaired (A,C,G), and paired (B,D,H) Student’s t tests were used to compare groups. * P < 0.05, ** P < 0.01, *** P < 0.001 **** P < 0.0001, ns = not significant. See also Figure S2.