Figure 4. RhoJ Inhibits Drug-Induced Cell Cycle Arrest and Apoptosis.

A. RhoJ Depletion Synergizes with Chemotherapy to Inhibit Proliferation. SK-MEL-28 cells were transfected with 50nM siRNAs and incubated with 1µM cisplatin or vehicle for 48 hours. EdU incoporation was quantified. Representative images are contained in Figure S4D. B. RhoJ Depletion Synergizes with Cisplatin to Induce S phase arrest. C8161 melanoma cells expressing control or RhoJ shRNAs were incubated in the presence and absence of 15µM cisplatin for 24hrs and subjected to FACS analysis as described. Note the accumulation of cells in the peak between G1 (200) and G2 (400). C. RhoJ/Pak1 Modulates Cisplatin-induced p53 accumulation in p53 wild type cells. MNT-1 cells were transfected with the indicated siRNAs and incubated with 30µM cisplatin for 24hr. The accumulation of pSer20-p53 (Chk1 dependent phosphorylation site) and p53 was measured. D. RhoJ/Pak1 does not modulate Cisplatin-induced p53 accumulation in p53 mutant cells. SK-MEL-28 cells expressing the indicated shRNA (left panel) or transfected with the indicated siRNAs (right panel) were incubated with the indicated doses of cisplatin for 24 hours. As pSer20-p53 could not be detected in these cells (data not shown), the accumulation of p53 was measured. E. p53 Depletion Mitigates the Effects of RhoJ Depletion on Cisplatin induced PARP cleavage in p53 wild type cells. P53 wild type (C8161, MNT-1) and p53 mutant (SK-MEL-28) melanoma cells were incubated with the indicated siRNAs for 48 hours followed by 30 µM cisplatin for 24 hours. Relative accumulation of cleaved PARP was measured. F. p53 Depletion partially mitigates the effects of RhoJ Depletion on Cisplatin induced Apoptosis. MNT-1 melanoma cells were transfected with the indicated siRNAs for 48 hours. Subsequently cells were incubated with 30 mM cisplatin for 48 hours. Apoptosis was quantified by measuring the intensity of Annexin V staining.