Figure 4.
MK-specific deletion of Pdk1 results in defective actin polymerization with disrupted F-actin assembly, impaired spreading, as well as reduced podosome number and morphology upon adhesion on collagen. (A) Representative confocal images (maximum projection) of BM-derived Pdk1fl/fl and Pdk1−/− MKs adherent to collagen (50 μg/mL) and stained for F-actin and α-tubulin displaying a disrupted F-actin organization in the cytoskeleton of Pdk1−/− MKs with impaired spreading and a significant reduction of podosome density. Phalloidin-Atto647N staining was used for F-actin (red); α-tubulin-A488 staining was used for α-tubulin (green), and DAPI was used for nuclei staining (blue). White arrows indicate podosomes. Scale bar equals 40 μm (overview) or 10 μm (detail). (B) Arithmetic means ± SD (left; n = 36 to 59, Wilcoxon-Mann-Whitney test) of F-actin expression (reflected by MFI of Phalloidin-Atto647N) in Pdk1fl/fl and Pdk1−/− MKs upon adhesion to collagen for 3 hours.Arithmetic means ± SD (right; n = 40 to 48, Wilcoxon-Mann-Whitney test) of α-tubulin expression (reflected by MFI of anti–α-tubulin-Alexa488 antibody) in Pdk1fl/fl and Pdk1−/− MKs. (C) Arithmetic means ± SD (n = 74 to 86, unpaired 2-tailed Student t test) of Pdk1fl/fl and Pdk1−/− MK spreading area on collagen. (D) Means ± SD (n = 38 to 57, Wilcoxon-Mann-Whitney test) of podosome number in BM-derived MKs of Pdk1fl/fl and Pdk1−/− mice. (E) Representative confocal images of BM-derived Pdk1fl/fl and Pdk1−/− MKs adherent to collagen (50 μg/mL) and stained for polymerized F-actin using LifeAct-TagGFP2 (red). DAPI (blue) was used for nuclei staining. Scale bar equals 20 μm. (F) Arithmetic means ± SEM (n = 42 to 51, unpaired 2-tailed Student t test) of Pdk1fl/fl and Pdk1−/− MK spreading area on collagen. (G) Means ± SEM (n = 42 to 51, Wilcoxon-Mann-Whitney test) of podosome number in MKs derived from BM of Pdk1fl/fl and Pdk1−/− mice adherent on collagen (related to panel E). (H) Arithmetic means ± SEM (n = 20 to 23, Wilcoxon-Mann-Whitney test) of F-actin expression (reflected by MFI of LifeAct-TagGFP2) in Pdk1fl/fl and Pdk1−/− MKs upon adhesion to collagen for 3 hours. **P < .01 indicates statistically significant difference.

MK-specific deletion of Pdk1 results in defective actin polymerization with disrupted F-actin assembly, impaired spreading, as well as reduced podosome number and morphology upon adhesion on collagen. (A) Representative confocal images (maximum projection) of BM-derived Pdk1fl/fl and Pdk1−/− MKs adherent to collagen (50 μg/mL) and stained for F-actin and α-tubulin displaying a disrupted F-actin organization in the cytoskeleton of Pdk1−/− MKs with impaired spreading and a significant reduction of podosome density. Phalloidin-Atto647N staining was used for F-actin (red); α-tubulin-A488 staining was used for α-tubulin (green), and DAPI was used for nuclei staining (blue). White arrows indicate podosomes. Scale bar equals 40 μm (overview) or 10 μm (detail). (B) Arithmetic means ± SD (left; n = 36 to 59, Wilcoxon-Mann-Whitney test) of F-actin expression (reflected by MFI of Phalloidin-Atto647N) in Pdk1fl/fl and Pdk1−/− MKs upon adhesion to collagen for 3 hours.Arithmetic means ± SD (right; n = 40 to 48, Wilcoxon-Mann-Whitney test) of α-tubulin expression (reflected by MFI of anti–α-tubulin-Alexa488 antibody) in Pdk1fl/fl and Pdk1−/− MKs. (C) Arithmetic means ± SD (n = 74 to 86, unpaired 2-tailed Student t test) of Pdk1fl/fl and Pdk1−/− MK spreading area on collagen. (D) Means ± SD (n = 38 to 57, Wilcoxon-Mann-Whitney test) of podosome number in BM-derived MKs of Pdk1fl/fl and Pdk1−/− mice. (E) Representative confocal images of BM-derived Pdk1fl/fl and Pdk1−/− MKs adherent to collagen (50 μg/mL) and stained for polymerized F-actin using LifeAct-TagGFP2 (red). DAPI (blue) was used for nuclei staining. Scale bar equals 20 μm. (F) Arithmetic means ± SEM (n = 42 to 51, unpaired 2-tailed Student t test) of Pdk1fl/fl and Pdk1−/− MK spreading area on collagen. (G) Means ± SEM (n = 42 to 51, Wilcoxon-Mann-Whitney test) of podosome number in MKs derived from BM of Pdk1fl/fl and Pdk1−/− mice adherent on collagen (related to panel E). (H) Arithmetic means ± SEM (n = 20 to 23, Wilcoxon-Mann-Whitney test) of F-actin expression (reflected by MFI of LifeAct-TagGFP2) in Pdk1fl/fl and Pdk1−/− MKs upon adhesion to collagen for 3 hours. **P < .01 indicates statistically significant difference.

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