Fig 1.
Characterization and validation of the novel NOX1 mouse monoclonal antibody.
(A, B) HEK293 cells were stably transfected with the pCMV-NOX1 plasmid or an empty vector and selected with G418. NOX1 overexpression was confirmed (A) at the mRNA level by RT-PCR (***p<0.001 vs. untransfected cells), and (B) at the protein level by Western blot analysis. (C, D) Transient knockdown of NOX1 expression in the colon cancer cell line LS513 with a scrambled control or a NOX1-specific siRNA. (C) A 4-fold decrease in NOX1 expression compared to parental (**p<0.01) and 6-fold decrease compared to cells transfected with scrambled siRNA (***p<0.001) was noted after 72 h at the mRNA level by RT-PCR. NOX1 mRNA level is given relative to β-actin. Data represent mean ± SD for at least 3 independent experiments. (D) Western blot analysis confirmed the NOX1 decrease at the protein level. (E) Immunodetection of NOX1 in HEK293-NOX1 and HEK293-vector control, LS513, and HT-29 cells by confocal microscopy. The cells were immunostained with NOX1 mouse mAb (green). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue), and mouse anti-IgG was used as a negative control. Digital images were taken at 63X magnification. NOX1 protein expression is qualitatively labeled as -, no expression; +, relatively low expression; ++, relatively higher expression; +++, highest expression. (F) To demonstrate a lack of cross-reactivity of the NOX1 antibody with other NOX isoforms, protein levels were measured by Western blot analysis for HEK293 cells stably transfected with the pCMV-NOX1 plasmid, the Myc-DDK-tagged-NOX2 plasmid, the pCMV-MycDDK-HsNOX4 plasmid, or an empty vector; UACC-257-vector and UACC-257-NOX5 stable overexpressing clones; and BxPC-3 cells with or without IL-4 stimulation (25 ng/ml for 24 h). The antibodies used to detect the NOX isoforms are listed in the Materials and Methods section. (G) RNA levels of the NOX isoforms were measured by RT-PCR. (H) NOX1 detection in a real-world system, LS513 colon cancer cells, that express both NOX1 and DUOX2 following 24-h incubation with IL-4 plus IL-17A, and treated with either a NOX1- or DUOX2-specific small interfering RNA (siRNA), or a nonspecific scrambled siRNA.
Fig 2.
Detection of NOX1 in human colon cancer cell lines.
(A) NOX1 expression was evaluated in a series of 30 human colon cancer cell lines obtained from the American Type Culture Collection. NOX1 mRNA level is given relative to β-actin. Data represent mean ± SD for at least 3 independent experiments. (B) NOX1 expression determined in parallel experiments for a panel of 7 colon cancer cell lines plus HEK293-NOX1 and HEK293-vector control at the protein level by Western blot analysis (2-min exposure).
Fig 3.
Functional expression of NOX1 isoforms in human colon cancer cell lines.
(A) RT-PCR and subsequent DNA gel analyses were performed to detect NOX1-L and NOX1-S/NOX1-Lv in a subset of 8 colon cancer cell lines plus HEK293-NOX1. (B) The subset of cell lines was evaluated for PMA-stimulated superoxide production by luminescence assay. (C-G) Basal and PMA-stimulated superoxide production were also measured in (C) HEK293-vector control, (D) HEK293-NOX1, (E) LS513, (F) HT-29, and (G) RKO cells using the luminol assay. Superoxide dismutase-polyethylene glycol (PEG-SOD, 200 U/ml) was used to confirm superoxide production, with measurements taken every 2 min for up to 120 min. (H) Mean rate of superoxide production at 60 min in nmol/h/106 cells. 2X106 cells per well were suspended in 200 μl of HBSS-HEPES containing 100 μM acetylated cytochrome c with or without 200 nM PMA and/or 200 U/ml PEG-SOD. The change in optical density at 550 nm was quantified using the kinetics of cytochrome c reduction. To eliminate superoxide-independent cytochrome c reduction, the absorbance value with PEG-SOD was subtracted from the absorbance value with cytochrome c alone for each cell line system.
Table 1.
Expression of NOX isoforms and accessory molecules in human colorectal cancer cell lines.
Fig 4.
Expression of NOX1 and RAS mutation status in colon cancer cell lines and human colorectal tumor specimens.
(A) NOX1 expression determined for a panel of 12 colon cancer cell lines plus HEK293-NOX1 at the mRNA level by qRT-PCR. NOX1 mRNA level is given relative to β-actin. Data represent mean ± SD for at least 3 independent experiments. Bars are color-coded according to KRAS mutation status. #, the RKO cell line is KRAS WT. (B) NOX1 expression was evaluated in a panel of 27 human colorectal cell lines at the mRNA level by quantitative real time PCR and is displayed relative to β-actin. Data represent mean ± SD for at least 3 independent experiments. (C, D) Relative NOX1 mRNA expression Z score in (C) 62 cell lines of the Cancer Cell Line Encyclopedia (CCLE) and (D) 623 colorectal adenocarcinoma specimens from The Cancer Genome Atlas (TCGA) stratified according to RAS mutation status. Each dot represents a cell line or tumor sample and the coloring corresponds to the RAS mutation status. (E) Mean NOX1 mRNA expression in 51 normal colorectal tissues from TCGA colorectal cohort was compared to expression in the 623 colorectal adenocarcinoma tumor samples from the same cohort shown in (D), divided into RAS WT (n = 384) and RAS mutant (n = 239) groups. Data are ± SEM. Statistical significance for RAS WT vs. RAS mutant subcategories by the Mann-Whitney test: *p<0.05, **p<0.01, ***p<0.001; n.s. denotes the absence of statistical significance.
Table 2.
RAS mutation status and NOX1 mRNA expression in 28 colon cell lines from the ATCC.
Fig 5.
Immunohistochemical detection of NOX1 protein in tumors and corresponding non-neoplastic tissue of the same origin.
Representative tumors/lesions and normal/non-neoplastic tissue pairs from a tissue microarray are shown. (A) Normal and abnormal colon, (B) normal and malignant small intestine, and (C) chronic atrophic gastritis and malignant gastric tissue. All images were taken at 10X digital magnification. The scale bar represents 400 μm.
Table 3.
NOX1 protein expression in human malignant and benign tissue.
Fig 6.
NOX1 expression and patient survival.
In the TCGA colorectal adenocarcinoma dataset, 373 patients had known NOX1 mRNA and overall survival information; 329 patients had known NOX1 mRNA and disease/progression-free survival. Kaplan-Meier curves displaying (A) overall survival and (B) disease/progression-free survival of these patients stratified into high and low NOX1 mRNA expressers. (A, B) The median NOX1 expression value was used as a cutoff, and no significant differences in overall survival or progression free survival were detected between high and low NOX1 expressers.