Journal: Cells

Article Title: Organismal and Cellular Stress Responses upon Disruption of Mitochondrial Lonp1 Protease

doi: 10.3390/cells11081363

Figure Lengend Snippet: Effects of LonP1 disruption on mitochondrial function and motility in cancer cells. ( A ) MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays were performed in fibrosarcoma (HT1080) and metastatic melanoma (WM266-4) cell lines after LonP1 silencing (left) or 1 μΜ CDDO-Me treatment for 24 h (right). MTT assays were carried out at least three times, whereas significance was assessed by paired t -test (**** p < 0.0001). ( B ) Western blotting of ATP6 (25 kDa) upon LonP1 silencing (above) or 1 μM CDDO-Me treatment for 24 h (below) in WM266-4 and HT1080 cancer cells. β-ACTIN was used as a protein of reference. ( C ) Scratch-wound assays were carried out for 24 h using HT1080 and WM266-4 cancer cells under control conditions versus LonP1 silencing or treatment with 500 nM of CDDO-Me. Observations were made under an inverted microscope, and pictures were taken at 4× magnification. Experiments were repeated three times, while here, one representative experiment is shown.

Article Snippet: Western blots were performed with the primary antibodies: Anti-LONP1 (Sigma-Aldrich, St. Louis, MO, USA, 1:4000), Anti-ATF4 (Cell Signaling, Danvers, MA, USA, 1:1000), Anti-HSP70 (Cell Signaling, Danvers, MA, USA, 1:1000), Anti-PARP (Cell Signaling, Danvers, MA, USA, 1:500), Anti-ATP6 (Elabscience, Houston, TX, USA, 1:1000), Anti-β-ACTIN (Cell Signaling, Danvers, MA, USA, 1:1500), and the secondary antibodies Anti-Rabbit (Santa Cruz Biotech, Dallas, TX, USA, 1:5000) or Anti-Mouse (Millipore, Burlington, MA, USA, 1:2000).

Techniques: Western Blot, Inverted Microscopy