Journal: PLoS ONE
Article Title: Endoplasmic Reticulum Stress Signaling Is Involved in Mitomycin C(MMC)-Induced Apoptosis in Human Fibroblasts via PERK Pathway
Figure Lengend Snippet: Increases in ROS induced by MMC triggers ER stress that can be blocked by antioxidants. (A) ROS levels were detected by using DCFH-DA and observed via fluorescence microscopy (200x magnification) 48 hours after MMC treatment. Nuclei are shown in blue and ROS staining in green. The fibroblasts were pretreated with 10 mM NAC, 10 mM GSH and 10 µM edaravone for 2 hours, respectively, then treated with MMC (0.4 mg/ml, 5 minutes) and incubated for 48 hours. (B) MDA generation was measured and is shown in the histogram. (C) The mean of DCF fluorescence intensity, which is indicative of ROS generation, was measured via flow cytometry and is depicted graphically as the relative fold increase of the control. (D) Cell viability and (E) apoptosis rates were examined 48 hours after MMC treatment using CCK-8 assays or Annexin V/propidium iodide double staining. (F) Western blot analysis of related proteins in fibroblasts 24 hours after MMC treatment. The expression of GRP-78, P-PERK, PERK, CHOP, BIM, cleaved caspase-3, PARP, cleaved PARP and β-actin (loading control) was analyzed. (G) The band intensities for GRP-78, P-PERK, PERK, CHOP, BIM, cleaved caspase-3, and cleaved PARP are shown as a histogram. The control group without MMC treatment was normalized to a value of 1.0-fold. The gel data results from the gels come from experiments performed in triplicate with similar results. The presented bar graphs shown in panels B, C, D and E are the average results from three different experiments. *P
Article Snippet: The cells were then resuspended in binding buffer at a concentration of 1×106 cells/ml and incubated with Annexin V-FITC and propidium iodide (BD Biosciences, USA) to achieved double staining, according to the manufacturer’s instructions.
Techniques: Fluorescence, Microscopy, Staining, Incubation, Multiple Displacement Amplification, Flow Cytometry, Cytometry, CCK-8 Assay, Double Staining, Western Blot, Expressing