Defective autophagic vesicle delivery in MSPCs from O5A^Δ/Δ mice. (A) Experimental design for IP injection of 5-FU in the following genotypes: CTRL: O5A^+/+, n = 5, 5A^fl/fl, n = 7 and O5A^Δ/Δ, n = 10. Analysis of autophagy relevant mechanisms in compact bone-derived MSPCs 8 days after in vivo treatment. All experiments were performed either with sorted ([B], o/n culture) or compact bone-derived MSPCs ([C-F], passage 4). (B) Fluorescent staining of LC3 (red) and LAMP1 (green) in sorted primary MSPCs from the BM of 5-FU-treated mice, cultured overnight on 0.1%-gelatin-coated coverslips. (C) Fluorescent microscopy images of LC3 (red/left), SQSTM1 (=p62; red/right) and DAPI (blue) staining in compact bone-derived MSPCs (p4). The left graph shows the perinuclear distribution of LC3 measured with ImageJ software. SQSTM1 foci were counted with ImageJ software (graph, right). (D) Confocal images of perinuclear LAMP1 (green) and DAPI (blue) staining. The graphs show the perinuclear distribution of LAMP1 and the diameter of the lysosomes designated as feret's diameter measured with ImageJ software. (E) Confocal images of perinuclear colocalization of LAMP1 (green) and LC3 (red) measured by ImageJ software. Colocalization was measured by ImageJ software (PLUGIN: colocalization) and visualized in white. (F) Representative flow cytometry histogram of basal autophagy and quantification of Cyto-ID dye levels (DMFI: Cyto-ID dye level chloroquine treated-Cyto-ID dye level WITHOUT treatment). Data were measured by ImageJ software. Scale bars, 10 µm. *P < .05 (nonparametric Mann-Whitney test: B-F). The results of each panel represent 2 to 3 independent experiments. Each dot represents the measurement of 1 cell. Data are represented as mean ± SEM. Symbol legends are shown in Figure 3A,F.