Fig 2: FLVCR1a null endothelial cells accumulate heme and show reduced viability in vivoa Electron micrographs showing the nuclear morphology of a control (left) and an Flvcr1a null EC (right). The main apoptotic features as chromatin condensation and nuclear fragmentation are not observed in Flvcr1a null ECs. b Electron micrographs showing the single-membrane vacuoles (black arrowheads) in Flvcr1a null ECs from E11.5 forelimb transverse sections. c Optical imaging on CD31+ cells isolated from E13.5 control and eKO embryos. Black arrows indicate intracellular vacuoles. d, e qRT-PCR analysis of Flvcr1a d and Flvcr1b e in CD31-positive and -negative cell fractions from E13.5 embryos. Values represent mean ± SEM; n = 5; ***p < 0.001. f, g Flow cytometer analysis of CD31+ cells from E11.5 f and E14.5 g embryos after staining with Annexin V. The percentage of Annexin V/CD31 double-positive cells is reported. *p < 0.05, **p < 0.01. h Western blot analysis of full-length (35 kDa) and cleaved (17–19 kDa) caspase-3. The active form of caspase-3 was not detected in CD31+ cells from E13.5 eKO embryos. As positive control HUVEC cells treated for 3 h with 5 μM STS were used. i Western blot analysis of the autophagy protein LC3. The ratio between LC3-II (14 kDa) and LC3-I (17 kDa) does not change in CD31+ Flvcr1a null ECs. j Quantification of normalized LC3-II/I ratio. Values are expressed as fold increase. k Intracellular heme levels in CD31+ cells isolated from E13.5 embryos. Values for each sample are expressed as the ratio between the fluorescence of CD31+ cells and the total fluorescence (CD31+/-). *p < 0.05. l qRT-PCR analysis of Ho-1 in CD31+ cells from E13.5 control and eKO embryos. Values represent mean ± SEM; n = 5. eKO, Flvcr1a endothelial knockout; EC endothelial cell; N nucleus; STS Staurosporine