Induction of p65 nuclear translocation and NF-κB activation by metallic nickel particles.

<p>JB6 cells, stably transfected with NF-κB luciferase reporter plasmid, were seeded onto 24-well plate and incubated overnight. Cells were treated with/without metallic nickel nano- or fine particles for 24 h. (A) Effect of nickel particles on nuclear translocation of the p65 subunit of NF-κB as measured by indirect immunofluorescence analysis using an anti-p65 primary antibody. The bright greenish areas represent nuclei that stained with anti-p65 antibody. After treatments, cells were fixed, permeabilized, blocked, and stained with NF-κB monoclonal p65 antibody for 3 h. Alexa Fluor 488 goat anti-rabbit secondary antibody was added for 1 h. NF-κB is 100% expression in all cell cytoplasm, metallic nickel nano- or fine particles may induce NF-κB translocation from cytoplasm into the cell nucleus. (B) The NF-κB activity was measured by the luciferase activity assay. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092418#s3" target="_blank">Results</a> are mean and standard error of four assay wells. The experiment was repeated two times. * <i>P</i><0.05 versus control; † <i>P</i><0.05 versus fine particles. TPA (20 ng/ml) was set as a positive control.</p

Effects of metallic nickel particles on colony formation of JB6 P⁺ cells in soft agar assay.

<p>Metallic nickel nano- or fine particle-treated or untreated JB6 P⁺ cells (1×10⁴) were incubated on soft agar medium for 21 days. TPA (20 ng/ml) was set as a positive control. (A) Images of colony formation of JB6 P⁺ cells in soft agar assay. (a) Control. (b) TPA (20 ng/ml). (c) Metallic nickel fine particles. (d) Metallic nickel nanoparticles. (B) Number of colonies/10⁴ cells in soft agar assay. The cell colonies were scored by a computerized image analyzer. * <i>P</i><0.05 versus untreated control.</p

Induction of c-Jun nuclear translocation and AP-1 activation by metallic nickel particles.

<p>JB6 cells, stably transfected with AP-1 luciferase reporter plasmid, were seeded onto 24-well plate and incubated overnight. Cells were treated with/without metallic nickel nano- or fine particles for 24 h. (A) Effect of metallic nickel particles on nuclear translocation of the c-Jun subunit of AP-1 as measured by indirect immunofluorescence analysis using an anti-c-Jun primary antibody. The bright greenish areas represent nuclei that stained with anti-c-Jun antibody. After treatments, cells were fixed, permeabilized, blocked, and stained with monoclonal c-Jun antibody for 3 h. Alexa Fluor 488 goat anti-rabbit secondary antibody was added for 1 h. AP-1 is 100% expression in all cell nuclei, metallic nickel nano- or fine particles may increase the expression (staining density) in the cell nucleus. (B) The AP-1 activity was measured by the luciferase activity assay. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092418#s3" target="_blank">Results</a> are means and standard errors of four assay wells. The experiment was repeated two times. * <i>P</i><0.05 versus control; † <i>P</i><0.05 versus fine particles. TPA (20 ng/ml) was set as a positive control.</p

A summative diagram of the potential pathways involved in nickel nanoparticles induced apoptosis and carcinogenesis in JB6 cell line.

<p>A summative diagram of the potential pathways involved in nickel nanoparticles induced apoptosis and carcinogenesis in JB6 cell line.</p

Effects of metallic nickel particles on p53 transcription activity and protein levels.

<p>JB6 cells, stably transfected with p53 luciferase reporter plasmid, were seeded onto a 24-well plate and incubated overnight. Cells were treated with/without metallic nickel nano- or fine particles for 24 h. The p53 transcription activity and protein levels were measured by the luciferase activity assay (A) and western-blot (B), respectively. Metallic nickel nanoparticles caused a greater decrease of p53 transcription activity and protein levels than fine particles. * <i>P</i><0.05 <i>versus</i> control. TPA (20 ng/ml) or UVB (4 kJ/m²) were set as a positive control.</p

Effects of metallic nickel particles on protein expressions of R-Ras, c-myc, c-Jun, p65, p50 and HIF-1alpha.

<p>JB6 cells were treated with 20 µg/cm² metallic nickel nano- or fine particles for 1, 3, 6 and 8 h. Western blot results show that both metallic nickel nano- and fine particles induced up-regulation of protein expressions of R-Ras, c-myc, c-Jun, p65, and p50 in a time-dependent manner. Metallic nickel nanoparticles elicited a higher stimulation on these protein expressions compared to fine particles. Nanoparticles induced a significant HIF-1alpha up-regulation after 6 and 8 h treatment.</p

Cell viability after cells were treated with Ni NPs alone or Ni NPs + EGCG.

<p>Note: *<i>P</i><0.05, Ni NPs alone compared with Ni NPs + EGCG; ++ <i>P</i><0.01, +++ <i>P</i><0.001, compared with control—(without any treatment); ## <i>P</i><0.01, ### <i>P</i><0.001, compared with control + 10 μM EGCG; error bars, SE. Abbreviations: Ni NPs, nickel nanoparticles; EGCG, epigallocatechin-3-gallate.</p

Comparison of apoptotic cell number^§ after cells were treated with Ni NPs alone or Ni NPs + EGCG.

<p>Notes: ^§including early and late-stage apoptotic cells; *** <i>P</i><0.001, Ni NPs alone compared with Ni NPs + EGCG; +++ <i>P</i><0.001, compared with control—(without any treatment); # <i>P</i><0.05, ### <i>P</i><0.001, compared with control + 10 μM EGCG; error bars, SE. Abbreviations: Ni NPs, nickel nanoparticles; EGCG, epigallocatechin-3-gallate.</p

Luciferase activity of AP-1 and NF-κB after JB6 cells were treated with Ni NPs alone or Ni NPs + EGCG.

<p>Notes: *<i>P</i><0.05, Ni NPs alone compared with Ni NPs + EGCG; + <i>P</i><0.05, ++ <i>P</i><0.01, +++ <i>P</i><0.001, compared with control—(without any treatment); # <i>P</i><0.01, ## <i>P</i><0.01, ### <i>P</i><0.001, compared with control + 10 μM EGCG. 20 nM TPA was set as a positive control. Abbreviations: Ni NPs, nickel nanoparticles; EGCG, epigallocatechin-3-gallate; TPA, phorbol ester.</p

Morphological changes after cells were treated with Ni NPs alone or Ni NPs + EGCG.

<p>Note: Magnification of the light microscope was at 20×. Abbreviations: Ni NPs, nickel nanoparticles; EGCG, epigallocatechin-3-gallate.</p