Fig. 1: Role of endothelial PCK2 in vessel sprouting.

a qRT-PCR analysis of PCK1 and PCK2 mRNA levels in ECs (HUVEC) (n = 4) and HepG2 cells (n = 4; used as positive control); nd, not detectable. b Representative immunoblot and densitometric quantification of PCK2 protein level in ECs in glucose-deprived (0 mM) versus control (24 h 5.5 mM glucose) conditions for the indicated time points (n = 4). GAPDH was used as a loading control. c Quantification of cell death (LDH release assay) in control and PCK2^KD1 ECs in 5.5 versus 0 mM glucose (n = 5). d Quantification of TUNEL⁺ cells in control and PCK2^KD1 ECs in 5.5 versus 0 mM glucose (n = 8). e ³H-thymidine incorporation into DNA in control and PCK2^KD1 ECs in 5.5 versus 0 mM glucose (n = 7); dpm, disintegrations per minute. f Number of sprouts and average sprout length per spheroid in mitomycin C (MitoC)-treated control and PCK2^KD1 ECs in 5.5 versus 0 mM glucose (n = 6). g Scratch wound closure (proxy of EC migration) in MitoC-treated control and PCK2^KD1 ECs in 5.5 versus 0 mM glucose (n = 6). h Representative immunofluorescence images of CD31-stained neovessels (gray) in corneal flat-mounts from mice after surgical implantation in the eye of bFGF pellets containing a negative control or Pck2-targeting siRNA and corresponding quantification of CD31⁺ vessel area as percentage of total cornea area (n = 13–14). Scale bars are 300 µm. Data are mean ± s.e.m. Statistics: ANOVA (c–g), two-tailed t-test with Welch’s correction (b, h); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns not significant.