Western blot analyses of total Seliciclib price cell lysates allowed detection of a PC PLC isoform with an apparent molecular weight of 66 kDa, which is in agreement with previous studies by our group and other groups on a number of different mammalian systems. Densitometric analyses con firmed that the MDA MB 231 cells expressed the high est PC PLC content, and the factor of increase was 6. 0 1. 6 in comparison with the non tumoral counterpart. All BC cells showed a higher PC PLC protein expression in comparison with MCF 10A cells, but the factors of increase were lower in SKBr3 and MCF 7 than in MDA MB 231 cells. As shown in Fig ure 1c, Amplex Red assays on total lysates from cells harvested at early confluence also showed a 6. 3 1.

2 fold increase in the PC PLC activity in MDA MB 231 cells Inhibitors,Modulators,Libraries in comparison with the non tumoral counterpart, whereas the factors of increase were lower for the other BC cells. By contrast, the PLD activity was not significantly differ ent among BC and non tumoral cells. Altogether, these results showed that the highest PC PLC upregulation occurred in the poorly differentiated MDA MB 231 cells. Cell proliferation arrest in MDA MB 231 cells exposed to D609 The absolute PC PLC activity of untreated MDA MB 231 cells increased in the log phase of growth from 0. 2 to 0. 4 pmol ug protein per minute between 24 and 72 hours and decreased thereafter. Cell exposure to D609 inhibited the PC Inhibitors,Modulators,Libraries PLC activity by 60% at 24 to 48 hours and by 80% at 72 hours. Continuous exposure of MDA MB 231 cells to this dose of D609 induced a long standing cell proliferation arrest Inhibitors,Modulators,Libraries up to at least 144 hours.

Similar anti proliferative effects were found for D609 treated SKBr3 and MCF 7 cells. The D609 induced inhibition of cancer cell growth was not due to general Inhibitors,Modulators,Libraries cytotoxicity, because the number of dead cells was practically maintained at the same levels in BC and in their control cultures. The difference in the percentage of dead cells in untreated compared with Inhibitors,Modulators,Libraries treated BC cell cul tures was therefore due to D609 induced inhibition of cell proliferation rather than to an increase in cell mor tality. Moreover, measurement of the percentages of Annexin V positive cells showed that, at this reference dose, D609 did not exert any substantial apoptotic effect on any of the investigated BC cells. A massive loss of cell viability was instead detected in MDA MB 231 cell cultures exposed to much higher D609 doses, as shown in panels a and b of Additional file 3. In cells treated for 48 hours, the percentage of dead cells increased from 12. 5% 4. 5% at the dose of 188 uM to 69. 3% 14. 1% at 500 uM and 88. 9% 8. 1% at 750 uM, compared with 5. 1% 2. 7% in control cells. Similar differential levels were detected at 72 hours.