Fig 1: E2F transcription factor 1 (E2F1) regulates QSOX2 expression directly. (A) Chord diagram of all enriched hallmark gene sets in a GSEA. Black font: FDR < 0.05; Gray font: FDR = 0.05; Blue curve: negative enrichment; Red curve: positive enrichment. (B) WB analysis of NCI-H3255 and NCI-H1975 cells transfected with shRNA-control or shRNA-E2F1 (48 h) and evaluated with the indicated antibodies. (C) NCI-H3255 and NCI-H1975 cells transfected with the indicated plasmids and subjected to real-time PCR analysis for E2F1 and QSOX2 expression. *P < 0.05, **P < 0.01. (D) Schematic diagram of the putative bonding sites for E2F1 in the QSOX2 promoter region. BS1: binding site 1; BS2: binding site 2. (E) Chip-qPCR analysis of E2F1 binding to the promotor of QSOX2 in 293T cells after treatment with siRNA-NC or siRNAs-E2F1. Purified rabbit IgG was used as a negative control for the background enrichment signal; *P < 0.01; **P < 0.001. (F) Co-transfection of the QSOX2 promoter and siRNAs-E2F1 into 293T cells in triplicate. Relative QSOX2 promoter activities were measured at 48 h after transfection by a dual-luciferase assay; *P < 0.05, **P < 0.01.
Fig 2: Downregulation of QSOX2 expression represses the proliferation, cell cycle and survival of NSCLC cells. (A) DEG and KEGG pathway analyses of TCGA and GEO datasets. The bioinformatic data analysis workflow consisted of three publicly available datasets. (B) KEGG enrichment bubble chart. Negative log base 10 FDR values from the pathway enrichment analysis are plotted. Dark blue: FDR < 0.05; light blue: FDR = 0.05. (C,D) NSCLC cells transfected with the indicated plasmids and subjected to EdU assays. Quantitative analysis was performed using ImageJ software. The bar graph shows the mean ± SD; n = 3, *P < 0.05, **P < 0.01. (E,F) NSCLC cells transfected with the indicated plasmids for 48 h and subjected to cell cycle assays. The percentage of cells in each phase of the cell cycle was quantified in an accumulated histogram. *P < 0.05. (G,H) NSCLC cells transfected with the indicated plasmids and subjected to apoptosis assays. Quantitative analysis was performed using FlowJo software. The bar graph shows the mean ± SD; n = 3, *P < 0.05, **P < 0.01.
Fig 3: Quiescin sulfhydryl oxidase 2 (QSOX2) can be used as a circulating marker to monitor tumor growth. (A) Serum samples were collected from 22 patients in cohort 1 and 22 healthy persons and subjected to ELISA analysis for QSOX2 expression; *P < 0.05. (B) ROC curve analyses of QSOX2 signature to discriminate NSCLC patients from healthy persons. (C) NCI-H3255 and NCI-H1975 cells were treated with cisplatin (10 µg/ml) for 24 h, NCI-H1299 cells were treated with a graded dose of cisplatin (5/10/20 µg/ml) for 24 h, and all cells were harvested for WB analysis with an anti-QSOX2 antibody. (D) Concentrated cell culture medium supernatant from panel (C) was harvested and subjected to ELISA analysis for QSOX2 expression; *P < 0.05. (E) Representative QSOX2-stained sections from 0.9% NaCl- or cisplatin (5 mg/kg)-treated xenografts are shown. The yellow arrow represents QSOX2. (F) The boxplot compares the QSOX2 level in the serum of tumor-bearing mice treated with or without cisplatin; *P < 0.05. (G) The xenografts from were harvested for Western blot (WB) analysis with the anti-QSOX2 antibody. (H) The expression levels of QSOX2 were compared before and after 2 cycles of cisplatin-based chemotherapy. Then, sera were harvested from 17 NSCLC patient and subjected to ELISA and WB analysis for QSOX2 expression, *P < 0.05 and **P < 0.01.
Fig 4: Quiescin sulfhydryl oxidase 2 (QSOX2) is periodically expressed during the cell cycle. (A) Bioinformatic data analysis of GSE52100. Expression heatmap of QSOX2 and cyclin genes in two cell cycle courses after double thymidine block release. The purple bars indicate S phase, and the black arrows indicate mitosis. (B) Correlation analysis between QSOX2 and cyclin gene expression; red: positive correlation; blue: negative correlation. (C) 293T cells synchronized with a double thymidine block, released and evaluated at several time points. The cell cycle profiles were assessed by flow cytometry after propidium iodide (PI) staining of DNA. (D) The expression of QSOX2 and cyclin genes in the cell lysates from panel (C) assessed by Western blot analysis. (E) Protein–protein interaction network (PPI) analysis of QSOX2 assessed by STRING (http://string-db.org/) (F) WB analysis of NCl-H3255 and NCl-H1975 cells transfected with siRNA-control or siRNA-QSOX2 (48 h) and evaluated with the indicated antibodies. (G) Immunoblotting of ß-catenin in the nuclear extracts of NCl-H3255 and NCl-H1975 cells was carried out after QSOX2 silencing, and GAPDH and Histone 3 were used as a loading controls, respectively.
Fig 5: Quiescin sulfhydryl oxidase 2 (QSOX2) is highly expressed in NSCLC and correlates with prognosis. (A) Twenty-two pairs of resected frozen tissue samples were subjected to real-time PCR analysis for QSOX2 expression (cohort 1). (B) IHC detection of QSOX2 expression in representative NSCLC samples and matched adjacent normal lung tissue samples (n = 34, cohort 2). The areas of carcinoma and adjacent tissues are indicated. The red arrow head represents QSOX2. (C) The expression of QSOX2 according to the IHC score. Statistical significance was analyzed using the Wilcoxon matched-pairs signed-rank test. (D) Kaplan-Meier analysis of the overall survival of 92 NSCLC patients (data from TMA cohort 3). (E) IHC results revealed that the expression of QSOX2 was positively correlated with TNM stage and lymph node metastasis status in NSCLC. (F) Violin plot comparing the expression of QSOX2 between NSCLC and non-tumor tissue samples from the TCGA dataset. (G) Kaplan-Meier survival curves plotted by R Studio (survival package) according to the different expression levels (normal, slightly elevated, moderately elevated, and significantly elevated) of QSOX2 in stage III–IV NSCLC samples.
Supplier Page from Abcam for Anti-QSOX2 antibody