Mitochondrial Oxidative Stress due to Complex I Dysfunction Promotes Fibroblast Activation and Melanoma Cell Invasiveness
Figure 5
Conditioned media from mutated fibroblasts promotes melanoma cells invasiveness; NAC treatment blocks HIF-1α stabilization and reverts the increase of melanoma cells invasiveness.
(a) Boyden cell invasion assay. Control and mutated fibroblasts were incubated in low glucose serum-free media in normoxic or hypoxic conditions (1% O2) for 24 hours. Media are then collected, and monolayers of A375 human melanoma cells were incubated in these conditioned media for 24 hours. 15103 A375 melanoma cells were seeded into the upper compartment of Boyden chambers. Cells were allowed to migrate through the filter toward the lower compartment filled with complete medium for 24 hours. Cell invasion was evaluated after Diff-Quick staining by counting cell in six randomly chosen fields. The results are representative of three experiments with similar results ± < 0.05 media from mutated fibroblasts versus media from control fibroblasts, *< media from mutated fibroblasts versus media from control fibroblasts
(b) Lysates of fibroblasts cultured in low glucose serum-free medium for 24 hours in hypoxic condition (1% O2) with or without 20 mM NAC were subjected to HIF-1α and α-SMA immunoblot analysis
(c) Fibroblasts were cultured as in (b) A375 melanoma cells were then incubated for 24 hours with media collected from fibroblasts. 3 A375 melanoma cells were seeded into the upper compartment of Boyden chambers coated with Matrigel. Cells were allowed to migrate through the filter toward the lower compartment filled with complete medium for 24 hours. Cell invasion was evaluated after Diff-Quick staining by counting cell in six randomly chosen fields. The results are representative of four experiments with similar results. * < 0.005 media from mutated fibroblasts versus media from control fibroblasts
