Fig. 4

Induction of Bim expression is required for apoptosis following protein tyrosine kinase 6 (PTK6) downregulation. a MDA-MB-453 and UACC893R1 cells expressing control or PTK6 shRNA(s) (C9, 12 or 49) were lysed at two time points following shRNA lentiviral infection. Lysates were probed with indicated antibodies. b UACC893R1 cells expressing either empty or wild-type (WT) PTK6 cDNA vector were super-infected with control or PTK6 shRNA lentivirus that targets the 3′UTR of PTK6 (49). Cells were lysed 96 h after infection and lysates were probed with the indicated antibodies. c RNA from UACC893R1 or MDA-MB-453 expressing PTK6 shRNA (C9, 12, or 49) was extracted and levels of Bim transcript were assessed. d UACC893R1 cells expressing control or Bim shRNA (51) were superinfected with control or PTK6 shRNA lentivirus. Cells were harvested and lysates were probed with antibodies to cleaved poly ADP ribose polymerase (PARP) and Bim. All experiments were performed three times. Numbers below blots indicate quantification of band intensity performed using Image J, normalized to respective loading control bands. e UACC893R1 cells co-infected with the indicated viruses (EV, empty vector; PTK6 shRNA 49; Bim shRNA 51) were harvested 96 h after infection, stained with Annexin-V and propium iodide, and analyzed by flow cytometry. The percentage of Annexin-V positive cells is plotted. Right, the levels of Bim or PTK6 transcript were evaluated in parallel samples. Statistics were applied to results obtained with triplicate experiments; *P <0.05. P-values were determined by comparing control shRNA to PTK6 and/or Bim shRNA-treated samples: ^δPTK6; ^#Bim