Figure 5.
Actin- and CDC42-dependent autophagy in O5AΔ/Δ and human MSPCs. Mouse samples: (A) IP injection of 5-FU in the following genotypes: CTRL: O5A+/+ n = 5, 5Afl/fl n = 7 and O5AΔ/Δ n = 10. Analysis of cytoskeleton-associated proteins and autophagy in compact bone-derived MSPCs, isolated 8 days after 5-FU-treatment and cultured until passage 4. (B) Fluorescent microscopy images of F-actin (Phalloidin/green) and DAPI (blue) staining. The graph shows the results of stress fiber formation in pooled MSPCs of 3 independent experiments. (C) Fluorescent microscopy images of CDC42-GTP in MSPCs illustrated as relief image (Adobe Photoshop: v21.1.1/filter relief). Graphs showing the total pixel intensity of CDC42-GTP (left panel) and the cell edge (right panel) as measured by ImageJ software. (D) Confocal images of CDC42-GTP (green), LC3 (red), and DAPI (blue) in MSPCs. Colocalization was measured by ImageJ software (plugin colocalization) and visualized in white. The graph shows the measurement of colocalization in pooled MSPCs. (E) Fluorescent microscopy images of F-actin (Phalloidin/green) and LC3 (red) in MSPCs. Colocalization was measured by ImageJ software (plug-in: colocalization) and visualized in white. Human samples: (F) Fluorescent microscopy images of cultured human MSPCs (P2) from healthy young donors and SDS patients. Fluorescent staining of WNT5A (green, left), F-actin (green, middle), CDC42-GTP (red, middle), DAPI staining (blue, left and middle), and colocalization of LAMP1 (green, right) and LC3 (red, right) measured by ImageJ software. (G) Graphs showing the protein content of WNT5A (left) and CDC42-GTP (right). (H) Evaluation of the orientation of F-actin stress fibers stained with phalloidin. Cells showing intermediate orientation (for instance, at the cell edge only) or no orientation were taken together (see supplemental Methods). (I) LAMP1 staining (left) and colocalization pixels of LAMP1 and LC3 (right) in MSPCs (P2) from healthy young individuals, n = 7, healthy aged individuals, n = 7, samples from hypocellular BM patients (P_1-3; see supplemental Table 2 for details), n = 3, SDS patients (P_7-9; see supplemental Table 2 for details), n = 3 and post-allo-HSCT patients (P_4-6; see supplemental Table 2 for details), n = 3. Scale bars 10 µm. *P < .05 (nonparametric Mann-Whitney test: B-E,G-I). The results represent 2 to 3 independent experiments. Data are represented as mean ± SEM. Symbol legends shown in Figure 5A and underneath Figure 5H-I.

Actin- and CDC42-dependent autophagy in O5AΔ/Δ and human MSPCs. Mouse samples: (A) IP injection of 5-FU in the following genotypes: CTRL: O5A+/+ n = 5, 5Afl/fl n = 7 and O5AΔ/Δ n = 10. Analysis of cytoskeleton-associated proteins and autophagy in compact bone-derived MSPCs, isolated 8 days after 5-FU-treatment and cultured until passage 4. (B) Fluorescent microscopy images of F-actin (Phalloidin/green) and DAPI (blue) staining. The graph shows the results of stress fiber formation in pooled MSPCs of 3 independent experiments. (C) Fluorescent microscopy images of CDC42-GTP in MSPCs illustrated as relief image (Adobe Photoshop: v21.1.1/filter relief). Graphs showing the total pixel intensity of CDC42-GTP (left panel) and the cell edge (right panel) as measured by ImageJ software. (D) Confocal images of CDC42-GTP (green), LC3 (red), and DAPI (blue) in MSPCs. Colocalization was measured by ImageJ software (plugin colocalization) and visualized in white. The graph shows the measurement of colocalization in pooled MSPCs. (E) Fluorescent microscopy images of F-actin (Phalloidin/green) and LC3 (red) in MSPCs. Colocalization was measured by ImageJ software (plug-in: colocalization) and visualized in white. Human samples: (F) Fluorescent microscopy images of cultured human MSPCs (P2) from healthy young donors and SDS patients. Fluorescent staining of WNT5A (green, left), F-actin (green, middle), CDC42-GTP (red, middle), DAPI staining (blue, left and middle), and colocalization of LAMP1 (green, right) and LC3 (red, right) measured by ImageJ software. (G) Graphs showing the protein content of WNT5A (left) and CDC42-GTP (right). (H) Evaluation of the orientation of F-actin stress fibers stained with phalloidin. Cells showing intermediate orientation (for instance, at the cell edge only) or no orientation were taken together (see supplemental Methods). (I) LAMP1 staining (left) and colocalization pixels of LAMP1 and LC3 (right) in MSPCs (P2) from healthy young individuals, n = 7, healthy aged individuals, n = 7, samples from hypocellular BM patients (P_1-3; see supplemental Table 2 for details), n = 3, SDS patients (P_7-9; see supplemental Table 2 for details), n = 3 and post-allo-HSCT patients (P_4-6; see supplemental Table 2 for details), n = 3. Scale bars 10 µm. *P < .05 (nonparametric Mann-Whitney test: B-E,G-I). The results represent 2 to 3 independent experiments. Data are represented as mean ± SEM. Symbol legends shown in Figure 5A and underneath Figure 5H-I.

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