PFMA-3 very high dose rate altered SK-Mel28 cell cycle distribution more severely than XRT and finally induced the appearance of sub-G1 peak.

<p>Flow cytometric analysis of cell cycle distribution in SK-Mel28 cell line treated with <b>a)</b> 2 Gy <b>b)</b> 4 Gy <b>c)</b> 8 Gy PFMA-3 and XRT for different times. The histograms were representative of three separate experiments. ctrl, control cells. <b>d)</b> Western blot analysis documenting cyclin-B1 levels modulation in PFMA-3 and XRT irradiated cells. Antibody to b-actin served as a loading control. ctrl, control cells.</p

PFMA-3 causes a DNA-DSB more severe with respect to XRT in SK-Mel28.

<p>Induction of DSB was assessed through detection of phosphorylation of H2A.X at Ser139 (green) by immunofluorescence and microscopy analysis. <b>a)</b> DSB after 2 Gy treatment. <b>b)</b> DSB after 4 Gy treatment. <b>c)</b> DSB after 8 Gy treatment. ctrl, control cells. <b>d)</b> Western blot analysis documenting phospho-p53 (Ser51) levels modulation in PFMA-3 and XRT irradiated samples. Antibody to b-actin served as a loading control. ctrl, control cells.</p

PFMA-3 induced glutathione and lipid peroxidation production greater than XRT.

<p><b>a)</b> Visualization of the SK-Mel28 living cells 3 hrs after irradiation with PFMA-3 or XRT (at 2 and 4 Gy) and of those non irradiated (ctrl) loaded by the fluorescent dyes monochloromobimane (mBCl) and boron-dipyrromethene (BODIPY) for a qualitative GSH and lipid peroxidation detection, respectively. Epifluorescence images at 100 × of magnification. <b>b)</b> Representative FcMeOH/SECM images in constant height mode of non irradiated (ctrl) and irradiated SK-Mel28 cells at 4 Gy by PFMA-3 and XRT. The regeneration currents recorded were reported as a percentage increase/decrease of the signal in respect to the one recorded at the same tip-dish distance but far away from cells (I_T/I_Tdish). <b>c)</b> Histogram reported the values obtained by computing the mean (± SEM) regeneration current recorded on 4 different cells selected from the scanning electrochemical microscopy images. Asterisks indicated statistically significant differences (*P <0.05).</p

PFMA-3 affected SK-Mel28 migration abilities more than XRT.

<p><b>a)</b> Cell migration behaviour was evaluated during performance of a wound-healing assay after treatment with 2 Gy, 4 Gy and 8 Gy PFMA-3 and XRT. Data were representative of three independent experiments performed in quadruplicate. ctrl, control cells. Asterisks indicated statistically significant differences (*P <0.05, **P <0.005, ***P <0.0005). <b>b)</b> Western blot analysis documenting E-cadherin levels modulation in PFMA-3 and XRT irradiated samples. b-actin loading control was not shown. ctrl, control cells.</p

PFMA-3 causes radiation-induced clonogenic cell death greater than XRT.

<p><b>(a)</b> Colony forming unit assay on SK-Mel28 and A375 cells irradiated with PFMA-3 and XRT at 2, 4 and 8 Gy. Data are representative of three independent experiments performed in triplicate and SD is not shown being less than 10%. Black (SK-Mel28) and red (A375) asterisks indicate statistically significant differences (*P<0.05, **P <0.005). <b>(b)</b> Radiobiological parameters of SK-Mel28 and A375 cell lines for PFMA-3 and XRT 2, 4, 8 Gy irradiation. SF2, SF4 and SF8 survival fraction at 2, 4 and 8 Gy respectively. D50, 50% survival dose. The D50 values for XRT device were used as standard reference for the RBE evaluations. The MLQ curve has been built from parameters suggested for SK-Mel28 and for low energy X-ray sources with a correction for the exposure time [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199312#pone.0199312.ref037" target="_blank">37</a>].</p

PFMA-3 affected A375 migration abilities more than XRT.

<p>Cell migration behaviour was evaluated during performance of a wound-healing assay after treatment with 2 Gy, 4 Gy and 8 Gy PFMA-3 and XRT. Data were representative of three independent experiments performed in quadruplicate. ctrl, control cells. Asterisks indicated statistically significant differences (*P <0.05, **P <0.005, ***P <0.0005).</p