Fig. 3: Inhibition of sulfatide synthesis impacted mitochondrial morphology.

a Caspase-3 activity in Co01 cells after treatment for 48 h with 2 μM Manidipine-2HCl and/or 5 nM A-1155463 in the presence or absence of 50 μM sulfatides as determined by flow cytometry. C: Control; M: Manidipine-2HCl 2 µM; A: A-1155463 5 nM; MA: Manidipine-2HCl 2 µM + A-1155463 5 nM; n = 4; b Percentage of PI positive Co01 induced by 2 μM Manidipine-2HCl and/or 5nM A-1155463 in the presence or absence of 50μM sulfatides after 48 h, determined by flow cytometry. C: Control; M: Manidipine-2HCl 2 µM; A: A-1155463 5 nM; MA: Manidipine-2HCl 2 µM + A-1155463 5 nM; n = 4; c Co01 treated with or with 2 μM Manidipine-2HCl for 24 h were used to determine the level of sulfatides in isolated heavy membrane fraction that primarily consists of mitochondria by lipidomics; n = 3. d Oxygen consumption rate (OCR) of Co01 treated with 2 μM Manidipine-2HCl with or without 50μM sulfatides were measured by seahorse mito stress assay. The basal, ATP production related and spare respiratory capacity were plotted as shown by the right lane. C: Control; M: Manidipine-2HCl 2 µM; MS: Manidipine-2HCl 2 µM+sulfatides 50 µM; n = 4. e Mitochondrial mass in Co01 was measured by 50 nM Mitotracker Green. Cells were treated with 2 μM Manidipine-2HCl with or without 50 μM sulfatides for 24 h before measuring by flow cytometry; n = 4. f The mitochondrial morphology was determined with mitochondria targeting DsRed (MitoDsRed) after treatment with and without 2 μM Manidipine-2HCl and/or 50 μM sulfatides (left two panels). Mitochondria were imaged with confocal microscope at 63× magnification. Right two panels depict the mitochondrial morphology after fixation and imaging by electron microscopy. Size bars are indicated and significance was calculated with Mann–Whitney test; ns: not significant, ∗p ≤ 0.05.