Figure 2
From: Distinct outcomes of CRL–Nedd8 pathway inhibition reveal cancer cell plasticity
Distinct outcomes of MLN treatment. (a) Effect of MLN on VCaP cell cycle analyzed by flow cytometry (profiles are shown on the top). DNA content was measured with 7-AAD. The percentage of dead cells was estimated by counting cells with <2N DNA content. (b) Effect of MLN on cellular DNA synthesis measured by EdU incorporation. The percentage of EdU-positive cells is shown as a boxplot diagram (see also Supplementary Figure S4). (c) Immunofluorescence analysis of DNA breaks with γ-H2AX-specific antibody (γ-H2AX, green) and Hoechst dye (DNA, blue). Scale bar, 5 μm. Diagrams show percentage of nuclei with an indicated number of γ-H2AX foci at 0 nM MLN (n=100), 50 nM MLN (n=125), and 500 nM MLN (n=115). (d) Effect of MLN on selected cellular markers. Western blotting was performed with protein-specific antibodies. The protein level was normalized first to GAPDH, then to vehicle control. (e) Suppression of drug cytotoxicity by 50 nM MLN. Cells were treated for 5 days with indicated drugs with (blue) or without (gray) 50 nM MLN in 10% ChSM or 10% StdM (asterisk). Data are presented as boxplot diagrams with P-values indicated at top. For drug description see Supplementary Table S5.
