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

Bmi-1 knockdown increases cisplatin-mediated ROS production in ovarian cancer cells.

<p>Ovarian cancer cells transfected with scrambled control or Bmi-1 siRNA were treated with or without cisplatin for 24 h. Subsequently the cells were incubated with 5 µM carboxy-H2DCFDA in fresh HBSS for 30 min at 37°C. The cells were harvested with trypsin and fluorescence of the labeled cells was measured at an excitation wavelength of 485 nm and emission wavelength of 530 nm by using Fluorolog 3 (Jobin-Yvon Horiba). Ratio of mean fluorescence intensity (MFI) with respect to the untreated scrambled control is represented.</p

Effect of Bmi-1 knockdown on apoptotic markers.

<p>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 caspase-8, caspase-9 and PARP using respective antibodies.</p

Assessment of NRP-2 expression in human gastric cancer tissues and cell lines.

<p>(A) Immunohistochemical staining for NRP-2 expression in representative tissue sections (20X) of normal human gastric mucosa and gastric cancer specimens (B) Immunoblot analysis of NRP-2 expression in six human GI cancer cell lines. Vinculin served as an internal loading control. (C) Generation of stable CNDT 2.5 cell lines with NRP-2 knockdown. Immunoblot analysis of NRP-1 and -2 expression in CNDT 2.5 cells transfected with shcntr or shNRP-2 plasmids (shNRP-2 clones; C6 and C10). Vinculin served as a loading control. (D) MTT assay results. Growth rates were no different between the control cells and NRP-2 knockdown clones. Bars indicate SEM. (E) Top: Mean number of cells that migrated in a Boyden chamber assay. Bottom: Representative images (10X) of migration assays. (F) Top: Mean number of cells that invaded in BioCoat Matrigel invasion chamber assay. Bottom: Representative images (10X) of invasion assays.</p

Effect of NRP-2 knockdown on expression of known metastatic genes.

<p>(<i>A</i>) Autoradiographic image of the array membrane showing differential expression of metastatic genes in shcntr (<i>left</i>) and shNRP-2 (<i>right</i>) cells. Circled spots indicate the position of the S100A4 gene. (B) Validation of S100A4 protein expression level in cells by immunoblot analysis. Actin served as a loading control. (C) Reduction of the steady-state level of β-catenin by NRP-2 knockdown. β-catenin expression in shcntr and shNRP-2 cells was determined by immunoblotting. Vinculin served as a loading control. (D) Verification of reduced β-catenin expression in shNRP-2 cells by immunofluorescence staining. shcntr and shNRP-2 CNDT 2.5 cells growing in chamber slides were fixed and immunostained with anti-β-catenin antibody (red). Nuclei were counterstained with DAPI (blue). (E) Validation of β-catenin reduction in a second NRP-2 knockdown gastric cancer cell line. NCI-N87 gastric cancer cells were infected with lentivirus containing shcntr or shNRP-2 constructs. Immunoblot analysis showed reduction in β-catenin expression in NCI-N87 NRP-2 knockdown cells. Actin served as a loading control. (F) β-catenin level in the cytoplasmic (Cyto) and nuclear (Nuc) fractions of shcntr and shNRP-2 cells. HSP90 (cytoplasmic) and LaminB (nuclear) served as fractionation controls.</p

Effect of NRP-2 on β-catenin expression and function.

<p>(A) Assessment of TCF reporter activity using the β-catenin-responsive TOPflash or mutant FOPflash reporters. Luciferase activities were measured after transient transfection of the reporter plasmids into shcntr and shNRP-2 CNDT 2.5 cells (B) Increased proteasome-mediated degradation of β-catenin in shNRP-2 cells. shcntr and shNRP-2 CNDT 2.5 cells were treated with 30 µM MG132 or equal volume of DMSO (a solvent for MG132) for 2 hours. Cell lysates were analyzed by immunoblotting using anti-β-catenin antibody (‘φ’, no treatment; ‘Ub’, ubiquitinated β-catenin). (C) Immunoblot analysis showing restoration of β-catenin level in the cytoplasmic and nuclear fractions after treatment with 30 µM MG132 for 2 hours. (D) Decreased levels of phosphorylated GSK3β in NRP-2 knockdown cells. Immunoblot analysis of phosphorylated GSK3β (at Ser-9) and GSK3β. (E) Restoration of β-catenin level after blocking GSK3β activity in NRP-2 knockdown cells. shcntr or shNRP-2 CNDT 2.5 knockdown cells were treated with increasing concentrations of LiCl for 24 hours and harvested for immunoblot analysis to detect β-catenin.</p

Hypothetical schema for NRP-2-mediated activation of β-catenin signaling in gastrointestinal cancer cells.

<p>Left: In a GI cancer cell, increased inactive GSK3β inhibits destruction complex function, leading to stabilization of β-catenin that is free to translocate to the nucleus and activate transcription of target genes. Right: In a GI cancer cell with NRP-2 knockdown, the active destruction complex phosphorylates β-catenin and targets it for proteasome-mediated degradation, resulting in decreased activation of downstream target genes.</p

Effect of NRP-2 knockdown on chemosensitivity of CNDT 2.5 cells to 5FU treatment and on expression and activation of apoptotic mediators.

<p>(A) Annexin V assay on shcntr and shNRP-2 CNDT 2.5 cells after treatment without or with 5FU for 48 hours. (B) Western blot analysis of apoptotic markers in cell extracts from shcntr and shNRP-2 CNDT 2.5 cells treated without or with 5FU. Vinculin and actin served as loading controls.</p