Figure 5. CDK6 and CDC25A regulate S... | Rockefeller University Press

Figure 5.

$Figure 5. CDK6 and CDC25A regulate S phase in hESCs. (A) Quantitative RT-PCR analysis for the expression of CDK6 and CDC25A in H1, H9, and hES-NCL1 cell lines 42 h after the transfection of CDK6 and CDC25A siRNA. The data represent the mean ± SEM (error bars) from three independent experiments (one in each cell line). The value for the control siRNA was set to 1, and all other values were calculated with respect to this. (B) Down-regulation of CDK6 by flow cytometry 42 h after the transfection of CDK6 siRNAs in hESCs (a representative example from the H9 line is shown). (C) Down-regulation of CDC25A by Western blotting 42 h after the transfection of CDC25A siRNAs (a representative example from the H9 line is shown). Molecular masses are indicated in kilodaltons. (D) Reduction in CDK6 kinase activity upon knockdown of CDK6. The value for the control siRNA was set to 100%, and all other values were calculated with respect to this. (E) Reduction in CDC25A phosphatase activity upon knockdown of CDC25A. The value for the control siRNA was set to 100%, and all other values were calculated with respect to this. (D and E) The data represent the mean ± SEM from three experiments performed in the H9 cell line. (F) Flow cytometry images showing movement of cells through the cell cycle after transfection of CDK6 siRNAs and synchronization by nocodazole for 18 h assessed by propidium iodide staining. (G) Chart representation of the fraction of cells in S phase over time after transfection of CDK6 siRNA and synchronization by nocodazole for 18 h assessed by propidium iodide staining. (H) Flow cytometry images showing retention of cells in G1 phase of the cell cycle after transfection of CDC25A siRNA and NANOG siRNA and synchronization by nocodazole for 18 h assessed by propidium iodide staining. (F–H) The figures represent an example of at least two independent experiments performed in the H9 subline.$

CDK6 and CDC25A regulate S phase in hESCs. (A) Quantitative RT-PCR analysis for the expression of CDK6 and CDC25A in H1, H9, and hES-NCL1 cell lines 42 h after the transfection of CDK6 and CDC25A siRNA. The data represent the mean ± SEM (error bars) from three independent experiments (one in each cell line). The value for the control siRNA was set to 1, and all other values were calculated with respect to this. (B) Down-regulation of CDK6 by flow cytometry 42 h after the transfection of CDK6 siRNAs in hESCs (a representative example from the H9 line is shown). (C) Down-regulation of CDC25A by Western blotting 42 h after the transfection of CDC25A siRNAs (a representative example from the H9 line is shown). Molecular masses are indicated in kilodaltons. (D) Reduction in CDK6 kinase activity upon knockdown of CDK6. The value for the control siRNA was set to 100%, and all other values were calculated with respect to this. (E) Reduction in CDC25A phosphatase activity upon knockdown of CDC25A. The value for the control siRNA was set to 100%, and all other values were calculated with respect to this. (D and E) The data represent the mean ± SEM from three experiments performed in the H9 cell line. (F) Flow cytometry images showing movement of cells through the cell cycle after transfection of CDK6 siRNAs and synchronization by nocodazole for 18 h assessed by propidium iodide staining. (G) Chart representation of the fraction of cells in S phase over time after transfection of CDK6 siRNA and synchronization by nocodazole for 18 h assessed by propidium iodide staining. (H) Flow cytometry images showing retention of cells in G1 phase of the cell cycle after transfection of CDC25A siRNA and NANOG siRNA and synchronization by nocodazole for 18 h assessed by propidium iodide staining. (F–H) The figures represent an example of at least two independent experiments performed in the H9 subline.

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