Comparison of altered genes at different times after iAs exposure.

<p>Venn diagram depicting genes affected exclusively at 2(left circle), 12 hrs of exposure to 5 µM of iAs (right circle) and both.</p

Major genes altered by iAs exposure.

<p>Cellular functions performed by the proteins encoded for the ten most up-regulated (A) and down-regulated (B) genes after 2 and 12 hrs of exposure to 5 µM of iAs.</p

Response of Nrf2-regulated genes to iAs exposure.

<p>Evaluation of mRNA levels of the indicated Nrf2-target genes by RT-qPCR in CL-1 cell line after exposure to 5 µM of iAs in a time-response curve. A) <i>HMOX1</i> and <i>NQO1</i> expression after dose-response exposure at 12 hrs, B) <i>HMOX1</i> and <i>NQO1</i> time response at 5 µM of iAs. Data values are means ± SD and were normalized using <i>GADPH</i> as an endogenous gene and fold changes were calculated using the ΔΔCt method. Three independent cell cultures were used for each assay data point. **<i>p</i><0.001, *<i>p</i><0.01.</p

Validation of genes with altered expression after iAs exposure.

<p>Total RNA from CL-1 cell lines exposed to 5 µM of iAs for 2 and 12 h of exposure as indicated was used as template for RT-qPCR assays. Control: black bars, 2 hrs: grey bars and 12 hrs: white bars. A) Down-regulated genes, B) Up-regulated genes, C) Overexpressed genes in CL-45 cell lines treated with 5 µM of iAs for 12 hrs. Control: black bars, treated: white bars. D) Expression levels of <i>HMOX1</i> and <i>NQO1</i> genes in lymphoblastoid primary cultures with 5 µM of iAs for 12 hrs. Control: black bars, <i>HMOX1</i>: white bars and <i>NQO1</i>: grey bars. Data values are means ± SD and were normalized using <i>GADPH</i> as an endogenous gene. Fold change was related to untreated samples. Three independent cell cultures were used for each assay. **** <i>p</i><0.001, *** <i>p</i><0.005, ** <i>p</i><0.01, * <i>p</i><0.05.</p

Canonical pathways associated with iAs exposure.

<p>Canonical pathways associated with iAs exposure.</p

Transcripts altered by iAs exposure.

<p>Volcano plots of differential transcripts up- (black dots) and down-regulated (grey dots) after (A) 2 and (B) 12 hrs of exposure to 5 µM of iAs.</p

Schematic representation of the mitotic catastrophe and P73-dependent apoptosis mechanisms triggered by curcumin treatment in the K562 cells.

<p>Schematic representation of the mitotic catastrophe and P73-dependent apoptosis mechanisms triggered by curcumin treatment in the K562 cells.</p

Curcumin induces DNA fragmentation and cell death in K562 cells.

<p><b>A)</b> A fraction of the arrested K562 cells treated with 20 μM of curcumin showed DNA damage as indicated by the presence of TUNEL-positive cells and FACS analysis. <b>B)</b> The percentage of TUNEL-positive cells is represented by graphic bars, and <b>C)</b> production of a typical apoptotic DNA ladder is shown. <b>D)</b> Cell death was confirmed by death assays; FACS analysis, and <b>E)</b> the results are also shown as graphic bars. As positive controls of DNA fragmentation, we used cells treated with 100 nM nocodazole for 24 h. As a positive control of cell death, K562 cells were exposed to UV (40 mJ/cm²) for 2 min in a cross-linker GS Gene linker-UV chamber (Bio-Rad, Hercules CA, USA) and were recovered 24 h post-irradiation.</p

Mitotic spindles in K562 cells were altered by curcumin.

<p><b>A</b>) Expression levels of phosphorylsted-histone 3 in serine 10 (p-H3S10) were increased in K562 cells after curcumin treatment, as determined by western blot and <b>B</b>) FACS analysis. <b>C</b>) The relative numbers of p-H3S10-positive cells are presented as a histogram. K562 cells were stained with DAPI or immunostained using an antibody against α-tubulin (green fluorescence), and the cells with defects in the mitotic spindles were counted. <b>D</b>) The percentage of cells with monopolar (white bars), bipolar (black bars) and multipolar (gray bars) nuclei were determined in K562 cells. <b>E</b>) Representative fluorescence images of K562 cells bearing mitotic spindle alterations after 12 h, 18 h and 24 h of curcumin treatment are shown. <b>F</b>) Images of the most representative mitotic spindle alterations are shown. Actin was used as loading control.</p

Apoptosis is produced by activation of P73.

<p><b>A)</b> Impairment of the expression of the anti-apoptotic proteins BCL-2 and XIAP is shown. <b>B</b>) The decreased expression of the anti-apoptotic proteins was caspase independent, as shown by the use of Z-VAD-FMK pancaspase inhibitor. Samples were treated with 2 pulses of 40 μM of pan-caspase inhibitor, Z-VAD-FMK (R&D, Systems), to 12 and 15 h of curcumin treatment. K562 cells were harvested after 18 h, lysed and total protein extracts were obtained for western blot analysis, using specific antibodies against of Caspase-3, PARP, BCL-2 and XIAP. <b>C</b>) The diminution of the expression of the antiapoptotic proteins was consistent with decreases in the IκBα and <b>D</b>) activation and nuclear translocation of the P73 protein, as consequence of the <b>E)</b> diminished expression of the P73 promoter repressor protein C/EBPα; M = medium, D = DMSO.</p