Notably, with the exception of some early time points in some JunB knock-down L-540 cells, in particular JunB#1 shRNA, apoptosis was not a factor contributing to the reduced growth rate of cells (Supplementary Fig.?S3). Open in a separate window Figure 3 c-Jun/JunB knock-down results in a similar cell cycle alteration within cHL cell lines. example, has been described as a JunBCspecific target19,30, whereas both c-Jun and JunB have been suggested to promote transcription32. Similarly, while AP-1 activity appears to be required for cHL proliferation, it is unclear whether this phenotype can be directly attributed to c-Jun and/or JunB. Leventaki and and ideals were obtained by carrying out ANOVA with Tukeys test comparing settings to c-Jun/JunB shRNACexpressing cells with the exception that a two-tailed test was performed in (F). comparing control to JunB shRNACexpressing cells. ns; not significant, *value is the knock-down compared to control 002 and the second is compared to control 216. Stable knock-down of c-Jun or JunB in cHL cell lines resulted in a prolonged G0/G1 We next examined whether the decreased growth rate in the knock-down cell lines was due to a proliferation defect. BrdU and 7-AAD double staining experiments revealed that knocking down c-Jun or JunB expression in all cell lines resulted in a decreased percentage of cells in S phase and a concomitant increase in the percentage in G0/G1 (Fig.?3ACF; Supplementary Figs?S1 and S2), although this did not always reach statistical significance and the changes in JunB knock-down KM-H2 cells were modest (Fig.?3F). Notably, with the exception of some early time points in some JunB knock-down L-540 cells, in particular JunB#1 shRNA, apoptosis was not a factor contributing to the reduced growth rate of cells (Supplementary Fig.?S3). Open in a separate window Physique 3 c-Jun/JunB knock-down results in a similar cell cycle alteration within cHL cell lines. The percentage of cells at each stage of the cell cycle was measured by BrdU/7-AAD double staining of L-540 (A,B), L-428 (C,D) or KM-H2 (E,F) cells expressing control, c-Jun, or JunB shRNAs. The results represent the average and standard deviation of at least four impartial experiments from two individual infections. (G,H) Representative circulation cytometry plots and summary of Ki-67 expression within the G0/G1 populace of L-540 cells expressing the indicated shRNAs. The summaries represent the average and standard deviation of five impartial experiments from at least two individual infections. Notice: the control shRNA data in (E,F) is the same because c-Jun and JunB knock-down cells were examined together in the same experiments. values were obtained by performing ANOVA with Tukeys test comparing the c-Jun/JunB knock-down cells with control shRNACexpressing cells. A two-tailed test was performed in (F). comparing control and JunB shRNACexpressing cells. In (A,B), the first value is the knock-down compared to control 002 and the second is compared to control 216. We used the percentages of cells in each stage of the cell cycle (Fig.?3) and doubling occasions estimated from your growth curves in Fig.?2 to gain an appreciation of the time cells spent in each stage of the cell cycle38 (Table?1). We excluded the JunB shRNACexpressing L-540 cells because of the apoptosis observed in these cells early in the growth curve experiments. The most consistent difference observed was a statistically significant prolonged G0/G1 which was increased ~20C80% in the c-Jun/JunB knock-down cell lines (Table?1). Notably, the results were consistent when knock-down cells were compared to either control shRNA-expressing cells. Table 1 c-Jun or JunB knock-down in cHL cell lines is usually associated with a prolonged G0/G1. values were obtained by performing ANOVA with Tukeys test comparing the c-Jun/JunB knock-down cells with control shRNACexpressing cells. When two values are.Patrick Paszkowski for critically reading the manuscript. functions in cHL. For example, has been described as a JunBCspecific target19,30, whereas both c-Jun and JunB have been suggested to promote transcription32. Similarly, while AP-1 activity appears to be required for cHL proliferation, it is unclear whether this phenotype can be directly attributed to c-Jun and/or JunB. Leventaki and and values were obtained by performing ANOVA with Tukeys test comparing controls to c-Jun/JunB shRNACexpressing cells with the exception that a two-tailed test was performed in (F). comparing control to JunB shRNACexpressing cells. ns; not significant, *value is the knock-down compared to control 002 and the second is compared to control 216. Stable knock-down of c-Jun or JunB in cHL cell lines resulted in a prolonged G0/G1 We next examined whether the decreased growth rate in the knock-down cell lines was due to a proliferation defect. BrdU and 7-AAD double staining experiments revealed that knocking down c-Jun or JunB expression in all cell lines resulted in a decreased percentage of cells in S phase and a concomitant increase in the percentage in G0/G1 (Fig.?3ACF; Supplementary Figs?S1 and S2), although this did not always reach statistical significance and the changes in JunB knock-down KM-H2 cells were modest (Fig.?3F). Notably, with the exception of some early time points in some JunB knock-down L-540 cells, in particular JunB#1 shRNA, apoptosis was not a factor contributing to the reduced growth rate of cells (Supplementary Fig.?S3). Open in a separate window Physique 3 c-Jun/JunB knock-down results in a similar cell cycle Rabbit polyclonal to c-Kit alteration within cHL cell lines. The percentage of cells at each stage of the cell cycle was measured by BrdU/7-AAD double staining of L-540 (A,B), L-428 (C,D) or KM-H2 (E,F) cells expressing control, c-Jun, or JunB shRNAs. The results represent the average and standard deviation of at least four impartial experiments from two individual infections. (G,H) Representative circulation cytometry plots and summary of Ki-67 expression within the G0/G1 populace of L-540 cells expressing the indicated shRNAs. The summaries represent the average and standard deviation of five impartial experiments from at least two individual infections. Notice: the control shRNA data in (E,F) is the same because c-Jun and JunB knock-down cells were examined together in the same experiments. values were obtained by performing ANOVA with Tukeys test comparing the c-Jun/JunB knock-down cells with control shRNACexpressing cells. A (±)-BAY-1251152 two-tailed test was performed in (F). comparing control and JunB shRNACexpressing cells. In (A,B), the first value is the knock-down compared to control 002 and the second is compared to control 216. We used the percentages of cells in each stage of the cell cycle (Fig.?3) and doubling occasions estimated from your growth curves in Fig.?2 to (±)-BAY-1251152 gain an appreciation of the time cells spent in each stage of the cell cycle38 (Table?1). We excluded the JunB shRNACexpressing L-540 cells because of the apoptosis observed in these cells early in the growth curve experiments. The most consistent difference observed was (±)-BAY-1251152 a statistically significant prolonged G0/G1 which was increased ~20C80% in the c-Jun/JunB knock-down cell lines (Table?1). Notably, the results were consistent when knock-down cells were compared to either control shRNA-expressing cells. Table 1 c-Jun or JunB knock-down in cHL cell lines is usually associated with a prolonged G0/G1. values were obtained by performing ANOVA with Tukeys test comparing the c-Jun/JunB knock-down cells with control shRNACexpressing.