Fig. 4: Inhibition of autophagy undermines autophagic process in hematopoietic-related cells of AGM cells.

a Atg5 deficiency led to an increased percentage of RFP⁺GFP⁺ cells and decreased the percentage of RFP⁺GFP⁻ cells in the EC, pre-HSC I and pre-HSC II fractions. Error bars represent mean ± SEM. n = 7 biologically independent embryos, *p = 0.0155, **p < 0.01, ***p < 0.001, ****p < 0.0001. Colored circle = LC3^R/G, colored inverted square = LC3^R/G-KO. Yellow represents RFP⁺GFP⁺ signal, dark red represents RFP⁺GFP⁻ signal and black presents RFP⁻GFP⁻ signal. b The frequency of RFP⁺GFP⁺, RFP⁺GFP⁻ and RFP⁻GFP⁻ cells in the hematopoietic clusters from E11.5 LC3^R/G-KO AGM region. Error bars represent mean ± SEM. n = 7 biologically independent embryos, **p < 0.01, ****p < 0.0001. Hem cluster = hematopoietic clusters (CD31⁺cKit⁺). Yellow represents RFP⁺GFP⁺ signal, dark red represents RFP⁺GFP⁻ signal and black presents RFP⁻GFP⁻ signal. c Representative histograms showing the GFP fluorescence level in the EC, pre-HSC I, pre-HSC II and hematopoietic clusters. Red line = LC3^R/G group, blue line = LC3^R/G-KO group. d Comparison analysis of the geomean fluorescence intensity (GeoMFI) of GFP signals in the RFP⁺GFP⁺ EC, pre-HSC I, pre-HSC II and hematopoietic clusters of LC3^R/G and LC3^R/G-KO. Error bars represent mean ± SEM. n = 7 biologically independent embryos, *p = 0.0437, **p = 0.0013, ***p < 0.001. e The GeoMFI of RFP in the different fractions of EC, pre-HSC I, pre-HSC II and hematopoietic clusters of the LC3^R/G and LC3^R/G-KO. Error bars represent mean ± SEM. n = 7 biologically independent embryos. Circle = LC3^R/G, inverted square = LC3^R/G-KO. f The GeoMFI ratios of TMRE/MTG showing the reduction of mitochondrial activity in the EC, pre-HSC II, but not in the pre-HSC I and hematopoietic clusters of KO compared to control. Error bars represent mean ± SEM. n = 7 biologically independent embryos, *p < 0.05. Statistical significance was determined by one side Student’s t-test.