Propidium Iodide-Fluorescent Activated Cell Sorting (PI-FACS) representative graphs

Each sample from one minute radiofrequency (RF) treatment with and without gold nanoparticles (GNPs) at 67 μM/L. Panel A: Hep 3B human hepatocellular cancer cells control with DMEM; Panel B: Hep 3B GNPs; Panel C: Panc-1 human pancreatic cancer cells control with DMEM; Panel D: Panc-1 GNPs.<p><b>Copyright information:</b></p><p>Taken from "Intracellular gold nanoparticles enhance non-invasive radiofrequency thermal destruction of human gastrointestinal cancer cells"</p><p>http://www.jnanobiotechnology.com/content/6/1/2</p><p>Journal of Nanobiotechnology 2008;6():2-2.</p><p>Published online 30 Jan 2008</p><p>PMCID:PMC2276230.</p><p></p

Thermographic results of heating of solutions of gold nanoparticles (GNPs) exposed to external radiofrequency (RF) fields at different RF generator power outputs

Panel A: Graphic depiction of heating rate of deionized water with increasing concentrations of GNPs treated at 200 W of power. B. RF treatment at 400 W of power. C. RF treatment at 600 W of power. D. RF treatment at 800 W of power. Heating curves which conclude prior to 300 seconds are indicative of specimen boiling.<p><b>Copyright information:</b></p><p>Taken from "Intracellular gold nanoparticles enhance non-invasive radiofrequency thermal destruction of human gastrointestinal cancer cells"</p><p>http://www.jnanobiotechnology.com/content/6/1/2</p><p>Journal of Nanobiotechnology 2008;6():2-2.</p><p>Published online 30 Jan 2008</p><p>PMCID:PMC2276230.</p><p></p

Transmission electron microscopy of Panc-1 cells treated with 67 μM/L gold nanoparticles

Panel 1: 2 minutes of external radiofrequency (RF) field treatment. Note loss of nuclear stability and prominent vacuolization. Panel 2: No RF treatment. Nuclear integrity and normal appearing organelles.<p><b>Copyright information:</b></p><p>Taken from "Intracellular gold nanoparticles enhance non-invasive radiofrequency thermal destruction of human gastrointestinal cancer cells"</p><p>http://www.jnanobiotechnology.com/content/6/1/2</p><p>Journal of Nanobiotechnology 2008;6():2-2.</p><p>Published online 30 Jan 2008</p><p>PMCID:PMC2276230.</p><p></p

Additional file 1: Figure S1. of BMI1, a new target of CK2α

The spectrum obtained from MS analysis of Phospheptide of BMI1 (DFYAAHPSADAANGS#NEDRGEVADEDKR). Figure S2. Association between BMI1+ CK2α (categorized into “high BMI1 + high CK2α” vs “others”) and patient survival (PFS). Expression of CK2α and BMI1 in the ovarian cancer patient samples (N = 20) were determined by immunoblotting, quantified by densitometry analysis as described in the “methods section” and grouped as high BMI1/high CK2α expressers versus all others. While PFS was worse in the high BMI1 + high CK2α group, the result was not statistically significant (P = 0.4), possible due to small sample size. (DOCX 78 kb

Image1_Cystathionine β-Synthase Is Necessary for Axis Development in Vivo.PDF

<p>The cystathionine ß-synthase (CBS) is a critical enzyme in the transsulfuration pathway and is responsible for the synthesis of cystathionine from serine and homocysteine. Cystathionine is a precursor to amino acid cysteine. CBS is also responsible for generation of hydrogen sulfide (H₂S) from cysteine. Mutation in CBS enzyme causes homocysteine levels to rise, and gives rise to a condition called hyperhomocysteinuria. To date, numerous mouse knockout models for CBS enzyme has been generated, which show panoply of defects, reflecting the importance of this enzyme in development. In zebrafish, we and others have identified two orthologs of cbs, which we call cbsa and cbsb. Previous gene knockdown studies in zebrafish have reported a function for cbsb ortholog in maintaining ion homeostasis in developing embryos. However, its role in maintaining H₂S homeostasis in embryos is unknown. Here, we have performed RNA analysis in whole zebrafish embryos that showed a wide expression pattern for cbsa and cbsb primarily along the embryonic axis of the developing embryo. Loss-of-function analysis using a combination of approaches which include splice morpholinos and CRISPR/Cas9 genomic engineering show evidence that cbsb ortholog is responsible for anterior-posterior axis development, and cbsa function is redundant. Cbsb loss of function fish embryos show shortened and bent axis, along with less H₂S and more homocysteine, effects resulting from loss of Cbsb. Using a chemical biology approach, we rescued the axis defects with betaine, a compound known to reduce homocysteine levels in plasma, and GYY4137, a long term H₂S donor. These results collectively argue that cells along the axis of a developing embryo are sensitive to changes in homocysteine and H₂S levels, pathways that are controlled by Cbsb, and thus is essential for development.</p

Immunohistochemistry Analysis of Tumors from the PBS and ACG44 groups.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057522#pone-0057522-g004" target="_blank">Figure 4A and 4B</a> show representative images of H&E and Ki-67 stained tumor tissues, respectively, from the PBS treated group whereas <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057522#pone-0057522-g004" target="_blank">Figure 4C and 4D</a> show images of H&E and Ki-67 staining of tumor tissue from the ACG44 treated group. All images were taken with 20× magnification. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057522#pone-0057522-g004" target="_blank">Figure 4E</a> is quantification of the Ki-67 positive proliferative nuclei shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057522#pone-0057522-g004" target="_blank">Figures 4B and 4D</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057522#pone-0057522-g004" target="_blank">Figure 4F</a> is a tumor image of Ki-67 staining from the ACG44 treated group, taken at 100 X to show gold accumulation (black spots) at a high magnification.</p

Role of pre-incubation with serum, C225 and NBMPR on targeting efficacy of gold nanoconjugates and their <i>in vitro</i> biological function.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057522#pone-0057522-g002" target="_blank">Figure 2A</a> depicts the absorbance spectrums of GNP, AC4 and ACG44 before and after pre-incubation with serum either 15 minutes at room temperature or 2 hrs at 37°C. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057522#pone-0057522-g002" target="_blank">Figure 2B and 2D</a> are transmission electron microscopy images of the <i>in vitro</i> uptake of ACG44 and AIG44 in AsPC-1 cells, respectively. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057522#pone-0057522-g002" target="_blank">Figure 2C</a> describes the effect of pre-incubation with serum on the cellular uptake of the nanoconjugates into AsPC-1 cells analyzed for gold content utilizing INAA. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057522#pone-0057522-g002" target="_blank">Figure 2E</a> also depicts INAA analysis of cellular uptake of ACG44 and AIG44 in AsPC-1 cells, both with/without pre-incubation with C225 or NBMPR to demonstrate possible uptake mechanisms. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057522#pone-0057522-g002" target="_blank">Figure 2F</a> shows the anti-proliferative effect of ACG44, AIG44 and CG44 on AsPC-1 cells determined through ³H-thymidine incorporation.</p

Bmi-1 knockdown perturbs the GSH biosynthesis pathway.

<p>The top panel represents total cellular GSH measured (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017918#s4" target="_blank">Materials and methods</a>) in ovarian cancer cells transfected with scrambled control or Bmi-1 siRNA treated with or without cisplatin for 24 h. The bottom panel represents fold change in gene expression (normalized with beta actin and compared to scrambled control) as determined by quantitative RT-PCR of ovarian cancer cells transfected with scrambled control or Bmi-1 siRNA for 48 h.</p

Effect of Bmi-1 knockdown on orthotopic chemoresistant ovarian cancer growth.

<p>To assess the effects of siRNA therapy on tumor growth, treatment was initiated 1 wk after i.p. injection (1.0×10⁶ CP20) of tumor cells. Mice were divided into four groups (n = 10 mice per group): (a) control siRNA-DOPC (150 µg/kg i.p. twice weekly), (b) control siRNA-DOPC + cisplatin (160 µg/mouse i.p. weekly), (c) Bmi-1 siRNA-DOPC (150 µg/kg i.p. twice weekly), and (d) Bmi-1 siRNA-DOPC + cisplatin (doses same as individual treatments). Treatment was continued until 4 weeks after tumor inoculation before sacrifice. (A) Total RNA was isolated from a portion of the tumor tissues and subjected to RT-PCR using primers for Bmi-1 and beta actin. The comparative C_t method was used to calculate the relative abundance of mRNA compared with that of beta actin expression. The experiment was performed in triplicate and significance determined using two-sided Student's t test, P<0.05 was considered significant. (B) Mouse and tumor weights and (C) the number of tumor nodules for each group were compared using Student's t test (for comparisons of two groups). A two-tailed P≤0.05 was deemed statistically significant.</p

Bmi-1 knockdown augments engagement of the DDR pathway in cisplatin treated ovarian cancer cells.

<p>(A) Ovarian cancer cells transfected with scrambled control or Bmi-1 siRNA were treated with or without cisplatin for 48 h. Western blot was performed for phospho Chk-2, total Chk-2, phospho-H2AX, total H2AX and beta actin using respective antibodies. (B) Scrambled control or Bmi-1 siRNA transfected CP-70 cells were subjected to confocal microscopy using 53BP1 antibody (red) and DAPI (blue nuclear staining) to demonstrate nuclear foci formation.</p