Figure 3
Knock-down of Aspa elevates intracellular NAA and changes ultrastructural features.
(a–h) Aspa knock-down in iBACs. Undifferentiated iBACs were incubated with supernatant containing lentiviral Aspa shRNA (siAspa) or non-targeting control (ntc) followed by antibiotic selection. Subsequently stably silenced cells were differentiated for seven days and harvested. Student’s t-test (n = 3) compared to ntc: *p < 0.05; **p < 0.01. (a) Aspa mRNA levels with qPCR and immunoblot for Aspa protein levels. (b) Aspa activity assay was measured via fluorometric detection of aspartate. (c) Intracellular NAA concentrations measured using LC-MS/MS. (d) Oil red O staining of mature iBACs to visualize neutral lipids. (e) Basal oxygen consumption measured with a Clark electrode. (f) Electron micrographs of differentiated iBACs. Scale bar is 1 μm. (g) Diameters of more than 800 lipid droplets were analyzed from all siAspa and ntc electron micrographs. (h) Mitochondria were counted from 99 electron micrographs derived from three replicates and related to the area.
