Fig 2: NADPH oxidase 1 (NOX1) promotes metastasis and reactive oxygen species (ROS) production in cervical squamous cell carcinoma (CSCC) cells and may serve as a prognostic marker in CSCC patients. (A,B) qRT-PCR and Western blot were employed to evaluate the knockdown efficacy of NOX1 in SiHa and MS751 (n = 3). (C,D) qRT-PCR and Western blot were employed to evaluate the overexpression efficacy of NOX1 in SiHa and HCC94 (n = 3). (E,F) Cell migration assay and Matrigel invasion assay were employed to investigate the effects of NOX1 knockdown cells (SiHa and MS751) (scale bar: 200 µm). (G,H) Cell migration assay and Matrigel invasion assay were employed to investigate the effects of NOX1-overexpressing cells (SiHa and HCC94) (scale bar: 200 µm). (I) ROS level was reduced in NOX1 knockdown cells (SiHa and MS751). (J) ROS level was increased in NOX1-overexpressing cells (SiHa and HCC94) (n = 3). (K) Representative images of the immunohistochemical (IHC) staining of NOX1 in human CSCC tissues and adjacent non-tumor tissues (scale bar: 200 µm). (L) Dot plots to show the IHC score of DDX19A expression using 76 pairs of CSCC tissues and adjacent non-tumor tissues tumor microarray (TMA) tissue sections (p < 0.001). (M) Correlation between lymph node metastasis and DDX19A expression in CSCC patients. Chi-square test was used. (N) Kaplan–Meier analysis was performed for our CSCC patients' cohort to evaluate the association between DDX19A protein level and 86 CSCC patients' overall survival. Results represent three independent experiments (A–J). The results were shown as mean ± SD, ***p < 0.001 by two-tailed Student's t-test.

Fig 3: The NADPH oxidase 1 (NOX1)/reactive oxygen species (ROS) axis exerts a pro-metastasis effect downstream of DEAD-box helicase 19A (DDX19A). (A,B) Western blot was employed to evaluate the overexpression efficacy of NOX1 proteins in DDX19A knockdown cell lines (SiHa and MS751) (n = 3). (C,D) Cell migration assay and Matrigel invasion assay were performed to evaluate whether restoring NOX1 expression could increase cellular migration and invasion in DDX19A knockdown cell lines (SiHa and MS751) (n = 3). (E) The 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence assay was performed to investigate whether NOX1 overexpression weakens the increase of ROS production induced by DDX19A knockdown in SiHa and MS751. (F) The DCFH-DA fluorescence assay was used to examine the ROS level in DDX19A-overexpressing cell lines (SiHa and HCC94) treated with or without N-acetylcysteine (NAC) (ROS inhibitor). (G,H) Cell migration assay and Matrigel invasion assay were performed to investigate whether NAC treatment could recover cell migration and invasion ability in DDX19A-overexpressing cell lines (SiHa and HCC94) (scale bar: 200 µm). Results represent three independent experiments. The results were shown as mean ± SD, ***p < 0.001 by two-tailed Student's t-test.

Fig 4: NOX1-induced ROS stimulates JAK2/STAT3 pathway and amplifies the effect of JAK2/STAT3 by OPN in HCCLM3 cells.HCCLM3 cells were transfected with siNOX1 or negative control. A Western blot analysis of NOX1, pJAK2, and pSTAT3 expressions was performed after transfection. The levels of NOX1, pJAK2, and pSTAT3 proteins were analyzed after transfection with NOX1 overexpression plasmid or vector control (B). The levels of pJAK2 and pSTAT3 proteins were analyzed in NOX1 overexpressed HCCLM3 cells and NAC treated NOX1 overexpressed HCCLM3 cells (C). Western blot analysis was carried out for the levels of pJAK2 and pSTAT3 proteins of control HCCLM3 cell, hOPN treated HCCLM3 cells, siNOX1 HCCLM3 cells, and siNOX1 HCCLM3 cells treated with hOPN (D). Data indicated mean ± SD. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, # Compared with NOX1 overexpression or hOPN treatment.

Fig 5: OPN depletion suppresses ROS production, NOX1 expression, and xenograft growth in vivo.A–D Nude mice (ten per group) were injected with negative control or shOPN HCCLM3 cells. The mice were killed after 23 days of injection, and the tumors were excised and weighed (B). The volume of the tumors (C) were measured every other day for an indicated 23-day period. D The effect of OPN knockdown on the body weight of mice. E The level of ROS in the tumor tissue was assayed by DHE. F Western blot analyses of NOX1, pJAK2, and pSTAT3 expressions in the subcutaneous tumor (n = 6). G Immunohistochemistry of tumor tissues stained with OPN, NOX1, and Ki67 antibodies (n = 6). Quantification is on the right side. Original magnification, ×200 (scale bars: 50 µm). Data indicated mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. H Schematic representation of the role and mechanism of OPN-induced ROS in HCC.