Fig. 6.

RSK is essential for melanoma cell proliferation and tumour development in mice. (A) A375 and Colo829 cells stably expressing control vector or shRNA against RSK1/2 were grown in culture medium containing 5% FBS. The relative number of viable cells was measured during four days using an MTS assay. The data are normalized to control vector (Ctl). (B) As in (A), except that after 72 h of culture, the relative number of viable cells was measured by cell counts. (C) Graph showing the relative number of apoptotic cells after 48 h of culture in serum-growing conditions, as measured by FACS analysis of Annexin V binding. (D) As in (A), except that A375 cells were grown in the presence of indicated inhibitors. (E) As in (C), except that A375 cells were treated with the indicated inhibitor for 24 h prior to FACS analysis. As positive control, cells were treated with 0.5 μM doxorubicin (DOX). (F) and (G) A375 and Colo829 cells stably expressing a control shRNA or shRNAs against RSK1/2 were injected subcutaneously into the flanks of athymic mice. Mice were monitored for tumour development and graphs represent the growth rate of subcutaneous tumours. Values represent the average volume +/− SEM of 3 to 5 tumours (3 to 5 mice). Insets: Endogenous RSK1, RSK2 and β-actin protein levels in injected cells were monitored by immunoblotting.