HEK-293 cells stably expressing WT or mutant D2HGDH were plated on poly-D-lysine-coated glass coverslips in 24-well tissue culture plates. After 24h, full-media containing 250nM of the cell-permeable Mito Tracker probe (Invitrogen, #M22426) was added, and incubated for 20 minutes. Next, the cells were rinsed in PBS and fixed in 4% paraformaldehyde for 15 minutes, followed by permeabilization and blocking with 5% horse serum, 0.2% Triton X-100 in PBS. Upon removal of the blocking solution, the primary antibody (anti-D2HGDH, Proteintech, #13895-1-AP) was added at 1:100 in 0.3% Triton X-100/PBS solution, and incubated for 1 hour at room temperature. After three washes with PBS, the Cy3-labeled secondary antibody (1:200) (Jackson ImmunoResearch,# 711-166-152) was added for 1h at room temperature in the dark, followed by three washes in PBS. Coverslips were mounted on glass slides using Vectashield (Vector Laboratories) for imaging. Images were acquired by laser scanning confocal microscope (LSCM) with Olympus FV1000 imaging system,33 (link). The UPLANAPO 60x oil objective (1.42NA) was used for all analyses, and an additional electronic zoom of 3 was applied. Excitation and emission signals were respectively 488 and 500±35nm for GFP (expressed from a bicistronic construct), 534 and 560–660nm for Cy3 (D2HGDH), 644 and 665 nm for Cy5 (MitoTracker).