PKCδ overexpression is accompanied by DNA damage.

<p>NB cells, transiently transfected with empty vector (EV) or PKCδ plasmid, were treated with 1 mM BSO for 24 h. Where indicated, cells were pre-treated with 25 nM DPI for 30 min. DNA fragmentation (standard) and oxidation (fpg) were evaluated by comet test. The reported values derive from tail moment analyses.</p><p>**<i>p</i><0.01 vs EV CTR/fpg;</p><p>°°<i>p</i><0.01 vs PKCδ CTR standard;</p>§<p><i>p</i><0.05 vs EV + BSO;</p>§§<p><i>p</i><0.01 vs EV + BSO;</p>••<p><i>p</i><0.01 vs PKCδ CTR fpg;</p>◊◊<p><i>p</i><0.01 vs PKCδ+BSO.</p

Silencing of PKCδ prevents the genotoxic effects triggered by etoposide and BSO in SH-SY-5Y and in SK-N-BE-2C cells, respectively.

<p>NB cells, silenced for PKCδ, were treated with 0.07 µM etoposide (SH-SY-5Y) and 1 mM BSO (SK-N-BE-2C) for 24 h. DNA fragmentation (standard) and oxidation (fpg) were evaluated by comet test. The reported values derive from tail moment analyses.</p><p>**<i>p</i><0.01 vs NoT CTR fpg;</p>♦♦<p><i>p</i><0.01 vs NoT CTR standard;</p>§§<p><i>p</i><0.01 vs NoT + ETOPO/BSO standard,</p>##<p><i>p</i><0.01 vs NoT ETOPO/BSO fpg.</p

PKCδ overexpression increases ROS production in untreated and BSO-treated NB cells.

<p><b>A</b>, SH-SY-5Y, ACN, GI-MEN and SK-N-BE-2C cells were transfected with empty vector (EV) or PKCδ plasmid. 24 h after transfection, cells were treated with 1 mM BSO for 24 h. Where indicated, cells were pre-treated with 25 nM DPI for 30 min. ROS analysis was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014661#s2" target="_blank">Materials and Methods</a>. Histograms summarize quantitative data of means ± SD of five independent experiments. * p<0.01 vs. EV transfected cells; ° p<0.01 vs. PKCδ transfected cells;^§ p<0.01 vs. EV transfected cells +BSO; ^# p<0.01 vs PKCδ transfected cells +BSO. <b>B</b>, Immunoblot of SH-SY-5Y and SK-N-BE-2C protein extracts (10 µg/lane) was performed with anti-PKCδ (left panels) and anti-phosphoThr (505) PKCδ (right panels) antibodies. Lanes 1 show the protein extracts from untreated cells, lanes 2 from BSO-treated cells, lanes 3 from DPI-treated cells and lanes 4 from DPI+BSO-treated cells. Protein extracts from SH-SY-5Y cells transfected with PKCδ and exposed to 100 µM H₂O₂ (4 h) were used as positive control (lane 5). β-actin signals represent the immunoblot loading control. PKCδ activity was measured by phosphorylation of H1 histone, utilized as a substrate (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014661#s2" target="_blank">Materials and Methods</a>). The immunoblots shown are representative of five independent experiments.</p

PKCδ overexpression amplifies the genotoxic effects of etoposide.

<p>SH-SY-5Y cells, transiently transfected with empty vector (EV) or PKCδ plasmid, were treated with 0.07 µM etoposide or 1 mM BSO for 24 h. Where indicated, cells were pre-treated with rottlerin for 30 min. DNA fragmentation (standard) and oxidation (fpg) were evaluated by comet test. The reported values derive from tail moment analyses.</p><p>**<i>p</i><0.01 vs EV CTR/fpg;</p><p>°°<i>p</i><0.01 vs PKCδ CTR standard;</p>§§<p><i>p</i><0.01 vs EV + BSO/ETOPO;</p>##<p><i>p</i><0.01 vs EV ETOPO fpg;</p>••<p><i>p</i><0.01 vs PKCδ CTR fpg;</p>◊◊<p><i>p</i><0.01 vs PKCδ+BSO/ETOPO.</p

PKCδ subcellular localization and cell viability in NB cells treated with BSO or etoposide.

<p><b>A,</b> Confocal microscopy images of SH-SY-5Y cells transiently transfected with PKCδ and treated with etoposide (0.07 µM) or BSO (1 mM) for 1, 3 and 6 h. After treatment, cells were fixed, permeabilized and stained with anti-PKCδ antibody (green), propidium iodide (red) and MitoTraker (blue). <b>B</b>, Confocal microscopy images of SH-SY-5Y cells transfected with pEGFP-PKCδ/pEGFP-PKCδ-DN plasmid and treated with 1 mM BSO (3 h) or 0.07 µM etoposide (1 h). After treatment, cells were fixed and labelled as detailed above. <b>C,</b> Immunoblots of PKCδ–transfected SH-SY-5Y protein extracts (10 µg/lane) were performed with isoform specific anti-PKCδ and anti phospho-PKCδ (Thr505) antibody (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014661#s2" target="_blank">Materials and Methods</a>). <b>D</b>, Fluorescence microscopy images of PKCδ–transfected cells, treated as indicated and labelled with FITC-conjugated Annexin V/PI. The figures shown are representative of experiments performed in triplicate.</p

Overexpression of PKCδ sensitizes NB cells to BSO-induced apoptosis.

<p><b>A</b>, After transfections and treatments (performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014661#pone-0014661-g001" target="_blank">Fig. 1</a>), apoptosis was analyzed by fluorescence microscopy using Annexin-V/PI assay (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014661#s2" target="_blank">Materials and Methods</a>). Histograms summarize quantitative data of means ± SD of five independent experiments. * p<0.01 vs. EV transfected cells; ° p<0.01 vs. PKCδ transfected cells; ^§ p<0.01 vs. EV transfected cells +BSO; ^# p<0.01 vs PKCδ transfected cells +BSO. <b>B,</b> Caspase-3 activity was analyzed by Caspase-3 Cellular Activity Assay Kit (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014661#s2" target="_blank">Materials and Methods</a>) in SH-SY-5Y (left panel) and SK-N-BE-2C cells (right panel). The percentage of living cells was determined by MTT analyses. Histograms summarize quantitative data of means ± SD of three independent experiments. * p<0.05 vs. EV transfected cells; ** p<0.01 vs. EV transfected cells; ° p<0.05 vs. PKCδ transfected cells; °° p<0.01 vs. PKCδ transfected cells.</p

PKCδ silencing prevents apoptosis and ROS over-production triggered by etoposide and BSO.

<p>Down-regulation of endogenous PKCδ was obtained using the technique of RNA silencing, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014661#s2" target="_blank">Materials and Methods</a>. After 48 h transfection, SH-SY-5Y (left panels) and SK-N-BE-2C cells (right panels) were stimulated for 24 h with 0.07 µM etoposide or 1 mM BSO. <b>A</b>, Immunoblots of SH-SY-5Y and SK-N-BE-2C protein extracts (20 µg/lane) were performed with anti-PKCδ and anti-β actin antibodies. Lanes 1 show the protein extracts from untreated cells, lanes 2 from etoposide/BSO-treated cells, lanes 3 from untreated cells transfected with PKCδ siRNA, lanes 4 from cells transfected with PKCδ siRNA and then treated with etoposide/BSO, lanes 5 from cells transfected with siCONTROL RNA (NoT), lanes 6 from cells exposed to the agent used for silencing (INT, INTERFERin^TM) and lanes 7 from the positive control (as detailed above). The immunoblots shown are representative of five independent experiments. <b>B,</b> After silencing and treatments, apoptosis was analyzed by fluorescence microscopy using Annexin-V/PI assay (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014661#s2" target="_blank">Materials and Methods</a>). <b>C;</b> ROS analysis was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014661#s2" target="_blank">Materials and Methods</a>. Histograms summarize quantitative data of means ± SD of five independent experiments. * p<0.01 vs. untreated cells; ° p<0.01 vs. cells treated with etoposide/BSO.</p

data_sheet_1_microRNA-494 Favors HO-1 Expression in Neuroblastoma Cells Exposed to Oxidative Stress in a Bach1-Independent Way.docx

<p>Heme oxygenase 1 (HO-1) is crucially involved in cell adaptation to oxidative stress and has been demonstrated to play an important role in cancer progression and resistance to therapies. We recently highlighted that undifferentiated neuroblastoma (NB) cells are prone to counteract oxidative stress through the induction of HO-1. Conversely, differentiated NB cells were more sensitive to oxidative stress since HO-1 was scarcely upregulated. In this work, we investigated the role played by miR-494, which has been proved to be involved in cancer biology and in the modulation of oxidative stress, in the upregulation of HO-1. We showed that NB differentiation downregulates miR-494 level. In addition, endogenous miR-494 inhibition in undifferentiated cells impairs HO-1 induction in response to exposure to 500 µM H₂O₂, reducing the number of viable cells. The analysis of Bach1 expression did not reveal any significant modifications in any experimental conditions tested, proving that the impairment of HO-1 induction observed in cells treated with miR-494 inhibitor and exposed to H₂O₂ is independent from Bach1. Our results underline the role played by miR-494 in favoring HO-1 induction and cell adaptation to oxidative stress and contribute to the discovery of new potential pharmacological targets to improve anticancer therapies.</p

image_1_microRNA-494 Favors HO-1 Expression in Neuroblastoma Cells Exposed to Oxidative Stress in a Bach1-Independent Way.PDF

<p>Heme oxygenase 1 (HO-1) is crucially involved in cell adaptation to oxidative stress and has been demonstrated to play an important role in cancer progression and resistance to therapies. We recently highlighted that undifferentiated neuroblastoma (NB) cells are prone to counteract oxidative stress through the induction of HO-1. Conversely, differentiated NB cells were more sensitive to oxidative stress since HO-1 was scarcely upregulated. In this work, we investigated the role played by miR-494, which has been proved to be involved in cancer biology and in the modulation of oxidative stress, in the upregulation of HO-1. We showed that NB differentiation downregulates miR-494 level. In addition, endogenous miR-494 inhibition in undifferentiated cells impairs HO-1 induction in response to exposure to 500 µM H₂O₂, reducing the number of viable cells. The analysis of Bach1 expression did not reveal any significant modifications in any experimental conditions tested, proving that the impairment of HO-1 induction observed in cells treated with miR-494 inhibitor and exposed to H₂O₂ is independent from Bach1. Our results underline the role played by miR-494 in favoring HO-1 induction and cell adaptation to oxidative stress and contribute to the discovery of new potential pharmacological targets to improve anticancer therapies.</p

Gene expression in IS-treated M/2M.

<p>RT-PCR for (A) AhR, (B) AhRR, (C) Nrf2, (D) HO1, (E) IL-6 and (F) CCL2. Results were plotted as mRNA fold induction in IS-treated versus Control cells; * p <0.05; ** p <0.01; ***p<0,001 vs. control cells.</p