Ssage was lost at the 10th culture passage. We hypothesize that this signal loss may be 15900046 Epigenetics related to the cellsenescence and modification of oxidative stress, processes intrinsically linked to melanogenesis [47]. The physical parameters of the measured ESR signal strongly suggested its connection with melanin [17,20,48]. Eu- and pheomelanogenesis occur under low- and high- GSH levels conditions, Epigenetic Reader Domain respectively [49,50], and changes of GSH levels and increase of oxidative cellular stress are typically observed in melanoma [51]. The presence/absence of ESR signal in different cellular systems may thus depend on factors including senescence level, oxidative stress level, melanogenesis and eu/pheomelanin proportion. In human primary melanocytes (1.26106 cells) showing a dark 
cellular pellet, no ESR signal was recorded (Fig. 1B); on the contrary, melanoma cells SKMEL-28 and SKMEL-110 (1.06106 cells) show an intense ESR signal (Fig. 1A).Melanoma Diagnosis via Electron Spin ResonanceFigure 4. ESR signal within melanoma subgroups. A) Each subgroup was classified according to tumour thickness (High or Low Breslow’s depth). Bars report the ESR mean value of each subgroup with SEM; * indicates p#0.05. B) ANOVA analysis with Bonferroni Multiple Comparison Test, within the group containing nevi, melanomas “Low Breslow’s depth” (,1 mm) and melanomas “High Breslow’s depth” ( 1 mm); * indicates p,0.01 of melanomas “High Breslow” vs nevi and vs melanomas “Low Breslow”). C) ANOVA analysis with Bonferroni Multiple Comparison Test of the eu/ pheomelanin ratio (a/b), of nevi, melanomas “Low Breslow’s depth” (,1 mm) and melanomas “High Breslow’s depth” ( 1 mm) groups; * indicates p,0.01 of melanomas “High Breslow” vs nevi and vs melanomas “Low Breslow”). doi:10.1371/journal.pone.0048849.gInterestingly, previous data [52] report a 
clear ESR signal in melanocytes, in apparent contrast with our findings; however it should 15755315 be noted that such signal was recorded on 1006106 cells, i.e. a number of cells almost hundred times higher as compared to our experimental conditions, indicating that detection of an ESR signal in dark melanocytes strongly depends on the number of cells investigated. It is well known that melanocytes visual pigmentation consistently correlates with eumelanin but not always with pheomelanin levels [53] and that some melanoma cell lines with high pheomelanin content has not visible pigmentation [54]. Consistently with all such considerations, it is reasonable to speculate that ESR analysis may detect qualitative alteration of the eu/ pheomelanin proportion, likely independent form the visual pigmentation and absolute eumelanin content.The same ESR signal was specifically found in freshly drawn mouse melanoma-tissues as well as in paraffin-embedded primary human melanoma-tissues (Fig. 2) while it was absent in several mouse non-melanoma tissues (Fig. 2B). This signal appeared to be stable for at least 14 days upon freezing, suggesting that the conditions of preservation of the mouse tumours allowed successful measurements repeated in time. ESR signal recorded in human paraffin-embedded nevi was markedly lower than ESR signal recorded in human melanoma sections (Fig. 2C), further supporting the hypothesis that, under our experimental conditions, endogenous ESR signal may be related to qualitative melanin differences occurring particularly in melanoma. [10,55?8]. The fine analysis of ESR spectra may reveal the specific proportion of eume.Ssage was lost at the 10th culture passage. We hypothesize that this signal loss may be 15900046 related to the cellsenescence and modification of oxidative stress, processes intrinsically linked to melanogenesis [47]. The physical parameters of the measured ESR signal strongly suggested its connection with melanin [17,20,48]. Eu- and pheomelanogenesis occur under low- and high- GSH levels conditions, respectively [49,50], and changes of GSH levels and increase of oxidative cellular stress are typically observed in melanoma [51]. The presence/absence of ESR signal in different cellular systems may thus depend on factors including senescence level, oxidative stress level, melanogenesis and eu/pheomelanin proportion. In human primary melanocytes (1.26106 cells) showing a dark cellular pellet, no ESR signal was recorded (Fig. 1B); on the contrary, melanoma cells SKMEL-28 and SKMEL-110 (1.06106 cells) show an intense ESR signal (Fig. 1A).Melanoma Diagnosis via Electron Spin ResonanceFigure 4. ESR signal within melanoma subgroups. A) Each subgroup was classified according to tumour thickness (High or Low Breslow’s depth). Bars report the ESR mean value of each subgroup with SEM; * indicates p#0.05. B) ANOVA analysis with Bonferroni Multiple Comparison Test, within the group containing nevi, melanomas “Low Breslow’s depth” (,1 mm) and melanomas “High Breslow’s depth” ( 1 mm); * indicates p,0.01 of melanomas “High Breslow” vs nevi and vs melanomas “Low Breslow”). C) ANOVA analysis with Bonferroni Multiple Comparison Test of the eu/ pheomelanin ratio (a/b), of nevi, melanomas “Low Breslow’s depth” (,1 mm) and melanomas “High Breslow’s depth” ( 1 mm) groups; * indicates p,0.01 of melanomas “High Breslow” vs nevi and vs melanomas “Low Breslow”). doi:10.1371/journal.pone.0048849.gInterestingly, previous data [52] report a clear ESR signal in melanocytes, in apparent contrast with our findings; however it should 15755315 be noted that such signal was recorded on 1006106 cells, i.e. a number of cells almost hundred times higher as compared to our experimental conditions, indicating that detection of an ESR signal in dark melanocytes strongly depends on the number of cells investigated. It is well known that melanocytes visual pigmentation consistently correlates with eumelanin but not always with pheomelanin levels [53] and that some melanoma cell lines with high pheomelanin content has not visible pigmentation [54]. Consistently with all such considerations, it is reasonable to speculate that ESR analysis may detect qualitative alteration of the eu/ pheomelanin proportion, likely independent form the visual pigmentation and absolute eumelanin content.The same ESR signal was specifically found in freshly drawn mouse melanoma-tissues as well as in paraffin-embedded primary human melanoma-tissues (Fig. 2) while it was absent in several mouse non-melanoma tissues (Fig. 2B). This signal appeared to be stable for at least 14 days upon freezing, suggesting that the conditions of preservation of the mouse tumours allowed successful measurements repeated in time. ESR signal recorded in human paraffin-embedded nevi was markedly lower than ESR signal recorded in human melanoma sections (Fig. 2C), further supporting the hypothesis that, under our experimental conditions, endogenous ESR signal may be related to qualitative melanin differences occurring particularly in melanoma. [10,55?8]. The fine analysis of ESR spectra may reveal the specific proportion of eume.