Cav-1 and PTRF/Cavin are required for IGF-IR internalization and plasma membrane recovery.

<p>HaCat cells were transfected with siRNA for Cav-1 (Cav-1-siRNA), for PTRF/Cavin (PTRF/Cavin-siRNA), for Clathrin Heavy Chain (Clathrin HC-siRNA) and with scrambled control siRNA (Ctr-siRNA) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014157#s3" target="_blank">materials and methods</a>. (A) 72 hours after transfection, serum-starved cells were treated with IGF110 nM for the indicated times, trypsined, washed, blocked and incubated with a mouse PE-conjugated IGF-IR antibody. PE-conjugated IGF-IR labeled cells were analyzed by flow-cytometry to measure plasma membrane IGF-IR expression as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014157#s3" target="_blank">Materials and Methods</a>. (B) Ctr-siRNA, Cav-1-siRNA, PTRF/Cavin-siRNA and Clathrin HC-siRNA HaCat cells were serum-starved and subjected to a biotinylation based endocytic assay with NH-SS-biotin at 4°C (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014157#s3" target="_blank">Materials and Methods</a>). The cells were then warmed at 37°C with medium containing IGF110 nM to allow IGF-IR internalization. Glutathione was used to reduce the proteins not internalized from the plasma membrane. IGF-IR was immunoprecipitated with IGF-IR antibody and the internalized IGF-IR was revealed by Western Blot with a Streptavidin-HRP antibody. Data were quantified using NIH-Image and plotted in the graph. The amount of biotinylated internalized IGF-IR was expressed as a percentage of the amount of IGF-IR on the surface at 4°C which we set as 100%. (C) 72 hours from the transfection serum-starved cells were lysed and equal amount of Ctr-siRNA and Cav-1-siRNA or Ctr-siRNA and PTRF/Cavin-siRNA and Clathrin HC-siRNA cell lysates were separated on SDS–PAGE, transferred on nitrocellulose and blotted with an antibody directed against Cav-1, PTRF/Cavin, Clathrin HC, IGF-IR, Flotillin-2 and actin proteins. Data are expressed as the mean ± SD. Statistical analysis was performed using Student's <i>t</i> test. *p<0.05.</p

Expression of CavY14F mutant decreases IGF-IR internalization in IGF1 stimulated cells.

<p>(A) Hacat cells were transiently transfected with pEGFPN1 Cav-1-wt and pEGFN1 Cav-1Y14F plasmids. 48 hours after transfection serum starved HaCat cells were stimulated with IGF1 10 nM for 5 min, fixed in 4% formaldehyde, permeabilized with Methanol at −20°C, labeled with a rabbit anti-PTRF/Cavin (red) and a mouse anti-IGF-IR (blue) imaged by confocal immunofluorescence microscopy. Merged fields show co-localization (white) of Cav-1wt, PTRF/Cavin, IGF-IR (upper pannels) and Cav-1Y14F, PTRF/Cavin and IGF-IR (bottom panels). (B) 48 hours from the transfection, serum-starved cells were treated with IGF110 nM for the indicated times, trypsined, washed, blocked and incubated with a mouse PE-conjugated IGF-IR.antibody. PE-conjugated IGF-IR labeled cells were analyzed by flow-cytometry to measure plasma membrane IGF-IR expression as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014157#s3" target="_blank">Materials and Methods</a>. Data are expressed as the mean ± SD. Statistical analysis was performed using Student's <i>t</i> test. *p<0.05.</p

Cav-1 and PTRF/Cavin co-immunoprecipitation with IGF-IR.

<p>Serum starved HaCat cells were stimulated with IGF110 nM for the indicated times and then lysed. Equal amount of cell lysates were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies. The graphs represent quantification of co-immunoprecipitation experiments following densitometric analysis of bands and are expressed as fold of increase. Data shown are representative of three independent experiments and are expressed as the mean ± SD.</p

IGF1 does not Induces IGF-IR and Clathrin Heavy Chain co-localization.

<p>HaCat cells were treated with IGF1(10 nM) for 5 min, fixed in 4% formaldehyde, permeabilized with Methanol at −20°C, labeled with a rabbit anti-Clathrin Heavy Chain (red) and a mouse anti-IGF-IR (green), antibody and imaged by confocal immunofluorescence microscopy.</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 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

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

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 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