Figure 4

DMC activates paraptotic signals in malignant breast cancer cells. (a) MDA-MB 435S cells were pre-treated with CHX at the indicated concentrations and further treated with 20 μ M DMC for 24 h. Cell viability was assessed using calcein-AM and EthD-1. (b) MDA-MB 435S cells were pre-treated with or without 2 μM CHX and further treated with 20 μ M DMC for 16 h. Cellular morphologies after treatments were observed under a phase contrast microscope. Bar, 20 μm. (c) MDA-MB 435S cells were treated with 20 μ M DMC or 40 μ M curcumin for the indicated time points. Cell extracts were prepared for western blotting. Compared with control (untreated cells), the fold change of protein levels was determined. (d) MDA-MB 435S cells were pre-treated with the indicated specific inhibitors (PD98059, U0126, L-JNKI and SB203580) and further treated with 20 μ M DMC for 24 h. Cell viability was assessed using calcein-AM and EthD-1. (e) MDA-MB 435S cells treated with 20 μ M DMC or 40 μM curcumin for the indicated time points were subjected to western blotting of the indicated proteins. Western blotting of β-actin was served as a loading control. (f) MDA-MB 435S cells were pre-treated with the indicated concentrations of various antioxidants and further treated with 20 μ M DMC for 24 h. Cell viability was assessed using calcein-AM and EthD-1. (g) MDA-MB 435S cells were treated with 20 μ M DMC for the indicated time periods or treated with DMC at the indicated concentrations for 4 h and stained with MitoSOX-Red. Treated and stained cells were processed for FACS analysis. (h) MDA-MB 435S cells were treated with DMC or curcumin at the indicated concentrations for 4 h and stained with MitoSOX Red. Treated and stained cells were processed for FACS analysis. (i) MDA-MB 435S cells treated with 20 μ M DMC for the indicated time points were subjected to western blotting of the indicated proteins. Western blotting of β-actin was served as a loading control. Compared with control (untreated cells), the fold change of protein levels was determined (j) MDA-MB 435S cells were pre-treated with or without 2 μ M CHX and further treated with 20 μ M DMC for 24 h. Cell extracts were prepared for western blotting. Compared with control (untreated cells), the fold change of protein levels was determined