Figure 7
Figure 7. p21 knock-down in Cre;Sclfl/fl restores aspects of megakaryopoiesis. (A) Western blot analysis showing reduced levels of P21 in NIH3T3 cells on transfection of p21 shRNA expression vectors: pS indicates pSuper vector; and L, lentiviral vector subsequently used to infect primary MKs. C indicates control shRNA (scrambled p21 shRNA sequence); p21sh, p21 shRNA; and actin, loading control. Efficacy of p21 shRNA sequences is observed with both vectors. (B) Colony assays. Bone marrow MkPs purified from Cre;Sclfl/fl homozygous mice were infected with p21 shRNA-expressing lentivirus or control vector. Day 2 GFP+ cells were plated in methylcellulose. MK colonies were scored at day 7. Data are the mean ± SD of 3 independent experiments performed in triplicate. *P = .01. (Right) Representative MK colonies. The photographs were taken using an Olympus BX60 microscope with a Qimaging camera. Openlab, Version 3 software (Improvision) was used for image acquisition, and images were exported into Adobe Photoshop, Version CS2 (Adobe Systems). Scale bars represent 20 μm. (C) Cre;Sclfl/fl MkPs were transduced with p21 shRNA-expressing lentivirus or control vector. Day 2 GFP+ cells were grown in TPO-containing medium and assayed for AChE activity 4 days later. Data are the mean ± SD of 3 independent experiments. *P = .08. (Right) Representative MKs. The photographs were taken using an Olympus BX60 microscope with a Qimaging camera. Openlab, Version 3 software (Improvision) was used for image acquisition, and images were exported into Adobe Photoshop, Version CS2 (Adobe Systems). Scale bars represent 40 μm. (D) Analysis of the ploidy of Cre;Sclfl/fl MKs transduced with p21 shRNA-expressing lentivirus or control vector on the GFP+CD41+ population. Peaks representing each ploidy class are labeled. One representative experiment of 3 is shown as well as the mean ploidy ± SD of the 3 independent experiments. P < .05. (E) Cell-cycle analysis. Day 2 transduced Cre;Sclfl/fl MkPs were sorted for GFP expression and expanded for 4 days. Cell-cycle phases were determined by analysis of BrdU incorporation and 7-amino-actinomycin D staining on CD41+ GFP+ gated cells. The percentage of cells in G0/G1, S, and G2M phases is shown. One representative experiment of 4 is shown. The mean (± SD) percentage of cells in S phase of the 4 experiments is shown in parentheses. P = .001. (F) Knockdown of P21 in mutant MkPs increases CDK2 phosphorylation on Thr 160 (P-Thr 160). Cre:Sclfl/fl MkPs expressing control shRNA or p21 shRNA sequences were lysed and subject to immunoblotting analyses using specific antibodies against CDK2 and P-Thr 160 CDK2. β-tubulin was used as a loading control. Increase in CDK2 phosphorylation in mutant sample (1.5- to 2-fold) was calculated after normalization for the amount of total CDK2.

p21 knock-down in Cre;Sclfl/fl restores aspects of megakaryopoiesis. (A) Western blot analysis showing reduced levels of P21 in NIH3T3 cells on transfection of p21 shRNA expression vectors: pS indicates pSuper vector; and L, lentiviral vector subsequently used to infect primary MKs. C indicates control shRNA (scrambled p21 shRNA sequence); p21sh, p21 shRNA; and actin, loading control. Efficacy of p21 shRNA sequences is observed with both vectors. (B) Colony assays. Bone marrow MkPs purified from Cre;Sclfl/fl homozygous mice were infected with p21 shRNA-expressing lentivirus or control vector. Day 2 GFP+ cells were plated in methylcellulose. MK colonies were scored at day 7. Data are the mean ± SD of 3 independent experiments performed in triplicate. *P = .01. (Right) Representative MK colonies. The photographs were taken using an Olympus BX60 microscope with a Qimaging camera. Openlab, Version 3 software (Improvision) was used for image acquisition, and images were exported into Adobe Photoshop, Version CS2 (Adobe Systems). Scale bars represent 20 μm. (C) Cre;Sclfl/fl MkPs were transduced with p21 shRNA-expressing lentivirus or control vector. Day 2 GFP+ cells were grown in TPO-containing medium and assayed for AChE activity 4 days later. Data are the mean ± SD of 3 independent experiments. *P = .08. (Right) Representative MKs. The photographs were taken using an Olympus BX60 microscope with a Qimaging camera. Openlab, Version 3 software (Improvision) was used for image acquisition, and images were exported into Adobe Photoshop, Version CS2 (Adobe Systems). Scale bars represent 40 μm. (D) Analysis of the ploidy of Cre;Sclfl/fl MKs transduced with p21 shRNA-expressing lentivirus or control vector on the GFP+CD41+ population. Peaks representing each ploidy class are labeled. One representative experiment of 3 is shown as well as the mean ploidy ± SD of the 3 independent experiments. P < .05. (E) Cell-cycle analysis. Day 2 transduced Cre;Sclfl/fl MkPs were sorted for GFP expression and expanded for 4 days. Cell-cycle phases were determined by analysis of BrdU incorporation and 7-amino-actinomycin D staining on CD41+ GFP+ gated cells. The percentage of cells in G0/G1, S, and G2M phases is shown. One representative experiment of 4 is shown. The mean (± SD) percentage of cells in S phase of the 4 experiments is shown in parentheses. P = .001. (F) Knockdown of P21 in mutant MkPs increases CDK2 phosphorylation on Thr 160 (P-Thr 160). Cre:Sclfl/fl MkPs expressing control shRNA or p21 shRNA sequences were lysed and subject to immunoblotting analyses using specific antibodies against CDK2 and P-Thr 160 CDK2. β-tubulin was used as a loading control. Increase in CDK2 phosphorylation in mutant sample (1.5- to 2-fold) was calculated after normalization for the amount of total CDK2.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal