Morphological features, expression of ICAM1 and intracellular ROS production in HPMC monolayers during in vitro induced senescence.

<p>A) Quantitative evaluation of senescence-related morphological features in HPMC monolayers from the peritoneal wash of the non-cancer patient (#002), from that of the colon cancer patient (#062) and from that of the gastric cancer patient (#219) after the first confluence (P2). The #062 HPMCs shows an higher percentage of multinucleated and multivacuolated cells respect to the other mesothelial cells (Kruskal-Wallis test: <i>*p</i> <0.05 vs #002 and #219). B) Quantitative evaluation of the senescence morphological features in #062 HPMC monolayer during in vitro induced senescence. The percentages of multinucleated and multivacuolated cells increases from passage 2 (P2) to passage 4 (P4) confirming the enhanced level of senescence of P4 (paired Student’s t test: *<i>p <</i>0.05). C) Quantitative evaluation of the percentage of ICAM1 positive cells in the HPMC monolayers. The HPMCs #062 from the colon cancer patient shows an higher percentage of ICAM1 positive cells respect to the other mesothelial monolayers (Kruskal-Wallis test: <i>*p</i> <0.01 vs #002 and <i>p</i> <0.05 vs #219). D) Immunofluorescence analysis of the percentage of ICAM1 positive cells in #062 HPMC monolayer during in vitro induced senescence. The quantitative analysis of the percentage of ICAM1 positive cells in P2 and P4 passages shows the increase in the percentage of positive cells from P2 to P4. Quantitative immunofluorescence analysis with anti-ICAM1 antibodies shows that both the number of ICAM1 positive cells, displaying a clear plasma membrane staining, and the fluorescence intensity of the signal are increased in P4 cultures respect to P2 HPMCs. The parallel phase contrast observations show that the ICAM1 positive cells are enlarged, multinucleated and vacuolated as expected for senescent cells (left panel, arrowheads). The cellular nuclei were stained with DAPI. Results in the first graph are expressed as mean values ± SE; paired Student’s t test: <i>*p<</i>0.001 vs P2. The quantitative evaluation of the fluorescence intensity of the ICAM1 signal was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057659#s2" target="_blank">materials and methods</a>: results in the second graph are expressed as mean values ± SE. paired Student’s t test: <i>*p</i> <0.01 vs P2. E) Optimization of the in vitro test for evaluation of ROS production in HPMCs from #062 patient. Treatment of HPMCs at P2 passage was performed with the pro-oxidant Cumene hydroperoxide (CUH) at different doses (100 and 200 µM) in presence or not of the anti-oxidant Vitamin E (15 µg/ml) before the addition of DCFH-DA. Increase of the fluorescence intensity is evident in cells treated with the oxidant in a dose-dependent manner, while a clear decrease in the signal is induced by the incubation with the anti-oxidant. F) Evaluation of ROS production in HPMCs #062 at P2 and P4 passages was performed with addition of DCFH-DA (2′,7′-dichlorofluorescein diacetate) and fluorescence detection by confocal microscopy as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057659#s2" target="_blank">materials and methods</a>. The increase in the fluorescence intensity signal of DCFH-DA in the late passage P4 compared with the earlier P2 confirm the enhancement of ROS generation induced by senescence of the mesothelial cells. Results are expressed as FUI mean values ± SE. Paired Student’s t test: <i>*p</i> <0.001 vs P2. G) Basal intracellular ROS generation in HPMCs #062 at P2 and P4 passages performed by cytofluorimetric assessment of DCFH-DA fluorescent signal. A representative flow cytometry histogram showed two distinct peaks of fluorescence intensity, with enhancement of DCFH-DA signal in HPMCs #062 P4 (purple histogram) respect to P2 (black histogram). The mean fluorescence intensity (MFI), calculated from three independent experiments, was higher in the late passage P4 compared with the earlier P2. Results are expressed as MFI relative values ± SE. Kruskal-Wallis test: *<i>p</i> <0.05.