a. Western blot analysis of lysates prepared from HeLa, CAL51, MCF7, A549, NCI-H226, PC3, DU145 and MCF10A cells. An antibody recognising WEE1 was used with β-tubulin as a loading control. WEE1 expression is significantly increased in HeLa and CAL51 cells compared to MCF7, A549, NCI-H226, PC3, DU145 and MCF10A cells. b. Left panel: Cell viability assay in cells transfected with WEE1 ONTARGETplus SMARTpool, or ONTARGETplus siControl. WEE1 silencing was selectively lethal to WEE1 overexpressing HeLa and CAL51 cells. Error bars represent the SEM from triplicate transfections. Right panel: Western blot analysis of lysates prepared from CAL51 cells transfected with WEE1 ONTARGETplus SMARTpool or ONTARGETplus siControl. An antibody recognising WEE1 was used with β-tubulin as a loading control. WEE1 ONTARGETplus SMARTpool significantly reduced WEE1 protein expression compared to siControl transfected cells. c. Cell viability assay in cells treated with WEE1 inhibitor. WEE1 inhibition was selectively lethal to WEE1 overexpressing HeLa and CAL51 cells. Error bars represent the SEM from triplicate cell treatments. d. Left hand panel: Western blot analysis of lysates prepared from cells treated with 5 µM WEE1 inhibitor for 0, 6, 24 and 48 hours. An antibody recognising PARP was used with β-tubulin as a loading control. After 24 hours WEE1 inhibition induced PARP cleavage (Clvd PARP) in WEE1 overexpressing HeLa and CAL51 cells but did not induce PARP cleavage in MCF7 and NCI-H226 cells which express WEE1 at normal levels. Right hand panel: Caspase 3,7 activity in cells treated with 5 µM WEE1 inhibitor for 24 hours. WEE1 inhibition induced caspase 3,7 activation in WEE1 overexpressing HeLa and CAL51 cells but did not induce caspase 3,7 activation in MCF7 and NCI-H226 cells which express WEE1 at normal levels. Error bars represent the SEM from triplicate cell treatments. e. Left hand panel: Western blot analysis of lysates prepared from cells transfected with WEE1 ONTARGETplus SMARTpool or ONTARGETplus siControl. An antibody recognising PARP was used with β-tubulin as a loading control. Silencing of WEE1 induced PARP cleavage in WEE1 overexpressing HeLa and CAL51 cells but did not induce PARP cleavage in MCF7 and NCI-H226 cells which express WEE1 at normal levels. Right hand panel: Caspase 3,7 activity in cells transfected with WEE1 ONTARGETplus SMARTpool or ONTARGETplus siControl. Silencing of WEE1 induced caspase 3,7 activation in WEE1 overexpressing HeLa and CAL51 cells but did not induce caspase 3,7 activation in MCF7 and NCI-H226 cells which express WEE1 at normal levels. Error bars represent the SEM from triplicate transfections.