Extended Data Fig. 7: Peroxisomal β-oxidation and ROS levels regulate ATGL levels and lipolysis.
From: Peroxisomal β-oxidation acts as a sensor for intracellular fatty acids and regulates lipolysis
(a) 72 h after knocking down Cat and Acox1 in iBAs, ATGL lipase activity assays were conducted using the WCE (N = 4, F = 70.21). (b) IBAs were treated with 0.5 mM H2O2 for 9 h or 2 mM NAC for 24 h. ATGL lipase activity assays were conducted using the WCE (N = 4, F = 43.33). (c-g) IBAs were transfected with Nc and Pex2 siRNAs. Under this background, cells were treated with Cat siRNA, Acox1 siRNA, 100 μM C26:0 and C24:0, 0.5 mM H2O2 or 2 mM NAC, as indicated. IB was conducted to check protein levels by indicated antibodies (f, N = 3 in H2O2 treatment and 6 in control; N = 4 in c, d, e, g; F = 13.29 in Nc and 0.08457 in Pex2 siRNA in c; F = 119.5 in Nc and 0.06723 in Pex2 siRNA in d; F = 16.26 in Nc and 0.214 in Pex2 siRNA in e). (h) PEX2-FLAG and ATGL-FLAG were co-transfected in HEK293T cells. After 48 h, cells were treated by 0.5 mM H2O2 and harvested at indicated time points. IB was conducted by indicated antibodies. Repeated 3 times. (i) Wild type PEX2 and PEX2C1-7G (siRNA resistant) were expressed in HEK293T cell under the background of endogenous PEX2 depletion. After 48 h, cells were treated by 0.5 mM H2O2 and harvested at indicated time points. IB was conducted by indicated antibodies. Repeated 3 times. (j) IBAs were transfected with Cpt1b and Cpt2 siRNAs to inhibit mitochondrial fatty acid oxidation. After 72 h, IB was conducted to check protein levels by indicated antibodies (N = 4 in Cpt1b&Cpt2 and 8 in Nc siRNA, F = 0.125). (k) IBAs were treated with mitochondrial fatty acid oxidation inhibitor etomoxir (ETO) at indicated time points. IB was conducted to check protein levels by indicated antibodies (N = 3 in ETO treatment and 9 in control, F = 1.016). (l) HepG2 cells were transfected with CPT1A and CPT2 siRNAs to inhibit mitochondrial fatty acid oxidation. After 72 h, IB was conducted to check protein levels by indicated antibodies (N = 4 in CPT1A&CPT2 and 8 in Nc siRNA, F = 0.3906). (m) HepG2 cells were treated with mitochondrial fatty acid oxidation inhibitor ETO at indicated time points. IB was conducted to check protein levels by indicated antibodies (N = 3 in ETO treatment and 9 in control, F = 0.3629). (n) IBAs were treated with mitochondria targeted antioxidant MitoQ at indicated doses for 24 h. IB was conducted to check protein levels by indicated antibodies (N = 4, F = 0.7356). Results are shown as mean ± SEM and analyzed using Student’s two-sided t test (g) and ANOVA method with Dunnett correction for multiple comparisons between control and other groups (a-f, j-n).
