Fig. 5: Melanoma derived H₂O₂ induces MSC PGC1α expression.

a MSCs cultured with A375 in direct contact, in a transwell dish (TW), with A375 conditioned media filtered through a 0.22 µm filter (FM), or nutrient depleted media [54]. RNA extracted and PGC1α expression analysed by RT-PCR. b MSC were treated with 10 and 100 µM of H₂O₂ for 6 h. RNA extracted and PGC1α expression analysed by RT-PCR. c A375 were cultured for 1 and 4 h in fresh media. Media was collected and analysed for H₂O₂ using Amplex Red assay. d MSCs cultured with A375 in TW in the presence of 5 mM NAC for 24 h. RNA was extracted from MSC and PGC1α expression analysed by RT-PCR. All conditions except NuD induced PGC1a upregulation (a), suggesting that secreted factors were responsible for PGC1α upregulation in MSC. Others have shown that ROS can induce PGC1α in various cell types, therefore MSCs were cultured in the presence of H₂O_2. Both 10 and 100 µM induced PGC1α mRNA expression in MSCs. To determine if melanoma can produce H₂O₂ we use the Amplex Red assay. Melanoma produced detectable levels of H₂O₂ at 1 and 4 h post incubation. Finally, to determine if melanoma derived H₂O₂ is responsible for induced PGC1α mRNA expression in MSCs, H₂O₂ was quenched with the ROS scavenger N-acetyl-cysteine (NAC). PGC1α mRNA induced expression was inhibited in A375 cocultures in the presence of NAC. These data show that melanoma derived H₂O₂ induces MSC PGC1α expression.