Extended Data Figure 5 : Confirmation of CPT1AKD using a second shRNA, additional effects of CPT1AKD, silencing another FAO-related gene in LECs, CPT1A overexpression does not induce the expression of LEC markers and CPT1AKD in VECs does not change the expression of blood endothelial cell markers.

From: The role of fatty acid β-oxidation in lymphangiogenesis

Extended Data Figure 5

Unless otherwise listed, CPT1AKD refers to knockdown 1 (KD1) of the 2 non-overlapping shRNAs tested. a, Representative immunoblot for CPT1A and β-ACTIN in LECs after silencing of CPT1A with two non-overlapping shRNAs (abbreviated as KD1 and KD2, respectively). b, Relative FAO flux in LECs at baseline and upon CPT1AKD versus VECs (n = 3). c, Relative [3H]thymidine incorporation in DNA in CPT1AKD LECs (using KD2) versus control LECs (n = 3). The same control samples were used from Fig. 3a. d, Total sprout length in CPT1AKD LEC spheroids (using KD2) versus control LEC spheroids (n = 3). The same control samples were used as Fig. 3c. e, Quantitation of the number of sprouts per spheroid in control LECs and upon CPT1AKD (using KD1 or KD2) (n = 3). f, Measurement of lactate dehydrogenase (LDH) in the cell culture media as a measure of cell death in control and CPT1AKD LECs (using KD1 or KD2) (n = 3). g, Relative FAO flux in pLECs at baseline and upon CPT1AKD versus VECs (n = 3). hj, Fold change in ITGA9 (h), CCL21 (i) and PROX1 (j) mRNA expression in pLECs at baseline and upon CPT1AKD versus VECs (n = 3). k, Fold change in VEGFR3 mRNA expression in pLECs at baseline and after treatment with etomoxir versus VECs (n = 3). l, Fold change in VLCAD mRNA expression in pLECs at baseline and upon VLCADKD (n = 3). m, Relative FAO flux in LECs at baseline and upon VLCADKD (n = 3). n, Fold change in VEGFR3 mRNA expression in pLECs at baseline and upon VLCADKD (n = 3). o, Relative FAO flux in VECs overexpressing CPT1A (CPT1AOE) versus control-transduced VECs (ctrl) (n = 3). pr, Fold change in CPT1A (p), PROX1 (q) and VEGFR3 (r) mRNA expression in CPT1AOE VECs versus ctrl VECs (n = 3). sv, Fold change in CPT1A (s), KDR (t), EFNB2 (u) and EPHB4 (v) mRNA expression in CPT1AKD VECs versus ctrl VECs (n = 3). Mean ± s.e.m. Statistical test: ANOVA and Bonferroni post-hoc test were used in multiple group comparisons. t-test was used for comparison of two groups. *P < 0.05; NS, not statistically significant.