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. 2021 Aug 30;297(4):101149. doi: 10.1016/j.jbc.2021.101149

Figure 5.

Figure 5

FGFR–ERK signaling regulates CPT1A expression and FAO through PPARα.A and B, Western blot analysis (A) and densitometric quantification (n = 4 biological replicates from three independent experiments) (B) of CPT1A proteins in HDLECs treated with vehicle, the FGFR inhibitor ASP5878, or a combination of ASP5878 and the PPARα antagonist GW6471. α-Tubulin served as a loading control. C and D, Western blot analysis (C) and densitometric quantification (n = 3 replicates from two independent experiments) (D) of CPT1A proteins in HDLECs treated with nontargeting (control) siRNA, FGFR1 siRNA, or both FGFR1 siRNA and the PPARα inhibitor GW6471. α-Tubulin was examined as a loading control. E and F, quantitative PCR (qPCR) analysis (n = 3 technical replicates; representative of two independent experiments) (E) and Western blotting (representative of two independent experiments) (F) of CPT1A expression in HDLECs transfected nontargeting (control) siRNA, FGFR1 siRNA, or a combination of FGFR1 siRNA and PPARα siRNA. β-actin was examined as a loading control. G, 9,10-3H-palmitic acid–based measurement of FAO flux in HDLECs treated with vehicle, the FGFR inhibitor ASP5878, or a combination of ASP5878 and the PPARα antagonist GW6471 (n = 4 biological replicates; representative of two independent experiments). H, 9,10-3H-palmitic acid–based measurement of FAO flux in HDLECs treated with nontargeting (control) siRNA, FGFR1 siRNA, or both FGFR1 siRNA and the PPARα inhibitor GW6471 (n = 4 biological replicates). I, Western blot analysis of CPT1A proteins in HDLECs treated with vehicle, the FGFR inhibitor ASP5878, or both ASP5878 and the PPARγ antagonist GW9662 (representative of three independent experiments). α-Tubulin served as a loading control. J, qPCR analysis of PPARA mRNA (encoding PPARα) in HDLECs transfected with nontargeting (control) or FGFR1 siRNA (n = 3 independent experiments). K, qPCR analysis of PPARA mRNA in HDLECs showing that lentivirus-mediated expression of RAF1S259A-normalized FGFR1 knockdown-induced upregulation of PPARA mRNA (n = 3 independent experiments). Data represent mean ± SD; p values were calculated by unpaired t test (J) or one-way ANOVA with Sidak's multiple comparisons test (B, D, E, G, H, and K). CPT1A, carnitine palmitoyltransferase 1A; ERK, extracellular signal–regulated protein kinase; FAO, fatty acid β-oxidation; FGFR, fibroblast growth factor receptor; HDLEC, human dermal lymphatic endothelial cell; PPARα, peroxisome proliferator–activated receptor alpha.