Figure 4
shRNA–mediated knock-down of JunB in ALK+ ALCL cell lines results in reduced proliferation. Western blots showing JunB levels (A–C) and growth curves (D–F) of the indicated cell lines expressing either control or JunB shRNA. Growth curves represent five experiments from three infections (D), five experiments from five infections (E), and three experiments from one infection (F). Note: JunB#6 and JunB#1 shRNAs overlap in their seed sequence and are not distinct. BrdU/7-AAD double staining of Karpas 299 (G) or SUP-M2 (H) cells expressing control or JunB shRNA. Results represent the average and standard deviation of three independent experiments from three infections for Karpas 299 cells, and eight independent experiments from six infections for SUP-M2 cells. (I) Bar graph showing the percentage of cells in S phase (% BrdU positive cells) observed when control shRNA or JunB shRNA–expressing Karpas 299 cells were transfected with plasmids expressing either EGFP-P2A or EGFP-P2A-FLAG-JunB. The results represent the average and standard deviation of four independent experiments. (J) Approximate doubling times and time spent in each stage of the cell cycle was determined for Karpas 299 and SUP-M2 cells expressing control or JunB shRNA. (K) Representative flow cytometry data and summary of Ki-67 expression within the G0/G1 population of SUP-M2 cells expressing control or JunB shRNA. The summary represents the average and standard deviation of three independent experiments from two separate infections. P values were obtained by performing independent, two-tailed t tests comparing the JunB knock-down to control shRNA–expressing cells (D–F and K) or between JunB knock-down cells with or without JunB cDNA (I). ANOVA with Tukey’s post hoc test was performed in (I). *P < 0.05, **P < 0.01, ***P < 0.001. Molecular mass markers (in kDa) are indicated to the left of western blots.
