1. Retinoic acid–induced cell cycle arrest of human myeloid cell lines is associated with sequential down-regulation of c-Myc and cyclin E and posttranscriptional up-regulation of p27Kip1
by Anna Dimberg, Fuad Bahram, Inger Karlberg, Lars-Gunnar Larsson, Kenneth Nilsson, and Fredrik Öberg
Blood
Volume 99(6):
March 15, 2002
©2002 by American Society of Hematology

2. U-937 cells arrest in the G0/G1phase of the cell cycle and up-regulate the differentiation marker CD11c in response to ATRA treatment.(A) The graph indicates the percentage of cells in the S, G2, and M phases during ATRA differentiation.
U-937 cells arrest in the G0/G1phase of the cell cycle and up-regulate the differentiation marker CD11c in response to ATRA treatment.(A) The graph indicates the percentage of cells in the S, G2, and M phases during ATRA differentiation. U-937 cells were grown in the presence of ATRA and collected at the indicated times, and the distribution of cells in different cell cycle phases was determined by flow cytometry of PI-stained nuclei. Mean ± SD (n = 4). (B) Induction of CD11c expression during ATRA-induced differentiation. Cells were induced by ATRA, collected at the indicated times, and analyzed by flow cytometry. Data are presented as CD11c mean fluorescence intensity (MFI) corrected for background binding by subtracting the MFI value for isotype-specific IgG2b control antibody. Mean ± SD (n = 3).
Anna Dimberg et al. Blood 2002;99:
©2002 by American Society of Hematology

3. Cyclins A, B, D3, and E are down-regulated during ATRA-induced differentiation of U-937 cells.(A) Cells were induced by ATRA and harvested at the indicated times, and total mRNA was prepared.
Cyclins A, B, D3, and E are down-regulated during ATRA-induced differentiation of U-937 cells.(A) Cells were induced by ATRA and harvested at the indicated times, and total mRNA was prepared. The regulation of cyclin mRNA during ATRA treatment was analyzed by RPA using the hCYC-1 multiprobe template. (B) The protein levels of cyclins A, B, D2, D3, and E during ATRA-induced differentiation were determined by Western blot analysis using whole cell lysates.
Anna Dimberg et al. Blood 2002;99:
©2002 by American Society of Hematology

4. ATRA-induced cell cycle arrest is associated with up-regulation of CKIs p21 and p27.(A) RPA analysis of different CKIs during ATRA-induced differentiation was done using the hCC-2 multitemplate probe and total mRNA from ATRA-treated cells.
ATRA-induced cell cycle arrest is associated with up-regulation of CKIs p21 and p27.(A) RPA analysis of different CKIs during ATRA-induced differentiation was done using the hCC-2 multitemplate probe and total mRNA from ATRA-treated cells. (B) ATRA-induced regulation of p27 protein levels was determined by Western blot analysis using whole cell lysates of ATRA-treated cells harvested at the indicated times. (C) To determine the turnover of p27 protein, cells were grown with ATRA or left untreated, cycloheximide (CHX) was added after 72 hours of induction, and cells were harvested at 0, 1, 2, 4, 6, and 8 hours after CHX treatment. Whole cell lysates were prepared and analyzed by Western blot using antibodies directed against p27 or actin.
Anna Dimberg et al. Blood 2002;99:
©2002 by American Society of Hematology

5. ATRA treatment of U-937 cells results in decreased CDK activity and a reduction in hyperphosphorylated pRb.(A) CDK2, 4, and 6 kinase activity was measured in an in vitro kinase assay using pRb as a substrate.
ATRA treatment of U-937 cells results in decreased CDK activity and a reduction in hyperphosphorylated pRb.(A) CDK2, 4, and 6 kinase activity was measured in an in vitro kinase assay using pRb as a substrate. Cells were grown in the presence or absence of ATRA for 72 hours, whole cell lysates were prepared, and the activities of CDK2, CDK4, and CDK6 were determined as described in “Materials and methods.” The graph indicates quantification of the bands correlated to control. Mean ± SD (n = 4). The asterisk indicates significant differences from the corresponding controls (P < .05). (B-D) The levels of total pRb or Ser780, Ser795, or Ser807/811 phosphorylated pRb (B), p130 (C), or p107 (D) during ATRA-induced differentiation were determined by Western blot of whole cell lysates prepared at the indicated times using specific antibodies.
Anna Dimberg et al. Blood 2002;99:
©2002 by American Society of Hematology

6. HL-60 and NB-4 cells respond to ATRA treatment by down-regulation of cyclin E and up-regulation of p27.Cells were induced by ATRA for 96 hours, whole cell lysates were prepared, and the protein levels of cyclin E and p27 were analyzed by Western blot.
HL-60 and NB-4 cells respond to ATRA treatment by down-regulation of cyclin E and up-regulation of p27.Cells were induced by ATRA for 96 hours, whole cell lysates were prepared, and the protein levels of cyclin E and p27 were analyzed by Western blot.
Anna Dimberg et al. Blood 2002;99:
©2002 by American Society of Hematology

7. Exogenous expression of Myc inhibits ATRA-induced cell cycle arrest and the associated down-regulation of cyclin E and up-regulation of p27.(A) The expression of c-Myc is down-regulated in response to ATRA treatment in U-937 cells.
Exogenous expression of Myc inhibits ATRA-induced cell cycle arrest and the associated down-regulation of cyclin E and up-regulation of p27.(A) The expression of c-Myc is down-regulated in response to ATRA treatment in U-937 cells. Cells were treated with ATRA for 0, 12, 24, 48, and 72 hours as indicated; whole cell lysates were prepared and subjected to immunoprecipitation using pan-Myc antibodies. The immunocomplexes were collected, separated by SDS-PAGE, and analyzed by Western blot using biotin-coupled pan-Myc antibodies and streptavidin-HRP. (B) U-937-myc-2 cells and the parental subline U-937-GTB were grown in the presence of ATRA, collected at the indicated times, and the distribution of cells in different cell cycle phases was determined by flow cytometry of PI-stained nuclei. Mean ± SD (n = 3). The asterisk indicates significant differences from the corresponding U-937-GTB controls. (C) The protein levels of cyclin E and p27 after 72 hours of ATRA treatment in U-937-GTB and U-937-myc-2 cells were analyzed by Western blot.
Anna Dimberg et al. Blood 2002;99:
©2002 by American Society of Hematology

8. Kinetics of the expression of cell cycle regulatory proteins and dephosphorylation of pRB, associated with ATRA-induced differentiation.(A) The temporal changes in protein expression of the indicated cell cycle regulators based on the quantification of West...
Kinetics of the expression of cell cycle regulatory proteins and dephosphorylation of pRB, associated with ATRA-induced differentiation.(A) The temporal changes in protein expression of the indicated cell cycle regulators based on the quantification of Western blot data from Figures 2B and 3B; cyclin A (▴), cyclin B (♦), cyclin E (●), p27 (*). (B) Kinetics of pRb dephosphorylation at specific sites based on the quantification of Western blot data from Figure 4B; Ser780 (♦), Ser795 (▴), Ser807/811 (●). Quantified data are displayed with the peak value for each series arbitrarily set to 100. For cycling cells the actual percentage of cells in the S+G2/M phases of the cell cycle is shown.
Anna Dimberg et al. Blood 2002;99:
©2002 by American Society of Hematology