Figure 4
Aspa knock-down and elevated intracellular NAA levels reduce the brown phenotype in an additive manner.
(a–f) iBACs with stable knock-down of Aspa or control cells were treated with 10 mM NAA throughout seven days of differentiation (n = 3). Normal differentiated cells served as a control. Student’s t-test (n = 3) compared to ntc: *p < 0.05; **p < 0.01; ***p > 0.001. (a) Metabolites were extracted by boiling cell lysates in ethanol after addition of internal standard mix and analysed with HPLC/HRMS. (b) mRNA levels of NAA pathway genes, mitochondrial acetyl-coA synthetase 1 (Acss1), cytoplasmic Acss2, ATP-citrate lyase (Acly) and adipose triglyceride lipase (Atgl). (c) Free fatty acid release into cell culture medium. (d) mRNA levels of brown marker genes. (e,f) Aspa and Ucp1 protein levels measured by immunoblot. Beta-actin serves as loading control.
