Fig 2: JAK2/STAT3 signaling plays a crucial role in OPN-induced NOX1 upregulation.Western blot was performed to examine the effect of hOPN treatment (10 ng/mL, 100 ng/mL, 1 µg/mL) and OPN knockdown on the levels of pJAK2 and pSTAT3 expression (A, B). SMMC7721 cells were incubated with PBS or 1 µg/mL OPN and fluorescence-stained with STAT3 antibody and DAPI (C). The NOX1 promoter construct pGL3-NOX1 (-200/+1) was transiently co-transfected with pRL-SV40 into SMMC7721 cells treated with OPN or HCCLM3 cells transfected with siOPN. The dual-luciferase activity was determined (D, E). CHIP assays in SMMC7721 cells incubated with hOPN or PBS. Chromatin was immunoprecipitated with anti-STAT3 or anti-IgG, and the amounts of precipitated NOX1 promoter fragments were determined by qPCR using the specific primer sets (F). The levels of NOX1 protein were analyzed in hOPN treated SMMC7721 cells and hOPN treated SMMC7721 cells after treating them with FLLL32 (10 µM, 8 h) (H). Data indicated mean ± SD. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, # Compared with hOPN treatment.

Fig 3: 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 4: Stretch injury causes the activation of ICAM-1 in a time-dependent manner and is regulated by oxidative stress, MMPs, and VEGF signaling. A, Western blot analysis of ICAM-1 and ß-actin 24 h after 3.0 psi stretch injury in the cell lysates of hBMVEC treated with control siRNA, ICAM-1 siRNA, A205804, and IRP. Bar graph represents the quantification of ICAM-1 versus ß-actin (n = 6/group). B, C, ELISA quantification of ICAM-1 in cell lysates (B), and cell culture supernatant (C) of hBMVEC following 3.0 psi stretch injury treated with control siRNA, ICAM-1 siRNA, A205804, and IRP (n = 6/group). D, mRNA expression level of ICAM-1 using qPCR from hBMVEC treated with control siRNA, ICAM-1 siRNA, A205804, and IRP (n = 6/group) 24 h after 3.0 psi stretch injury. E, Western blot analysis of ICAM-1 and ß-actin expression at different time points (1, 6, 12, 24, 48 h) in hBMVEC lysates after 24 h 3.0 psi stretch injury. Bar graph represents the quantification of ICAM-1 versus ß-actin (n = 6/group). F, G, ELISA quantification of ICAM-1 in cell lysate (F), and cell culture supernatant (G) of hBMVEC at different time points (1, 6, 12, 24, 48 h) following 3.0 psi stretch injury. H, Western blot analysis of ICAM-1 and ß-actin 24 h after 3.0 psi stretch injury in the cell lysates of hBMVEC treated with apocynin (NADPH oxidase inhibitor), VEGF-A (recombinant human VEGF), Ki8751 (inhibitor of VEGFR phosphorylation), and TIMP1 (MMPs inhibitor). Bar graph represents the quantification of ICAM-1 versus ß-actin (n = 6/group). I–K, Western blot analysis of NOX1, 4HNE, MMP-2, MMP-9, and ß-actin 24 h after 3.0 psi stretch injury in the cell lysates of hBMVEC treated with apocynin, VEGF-A, Ki8751, and TIMP1. Bar graph represents the quantification of ICAM-1 versus ß-actin (n = 6/group). L–N, Western blot analysis of VEGF-A, VEGFR-2, p-VEGFR-2Y1059, p-VEGFR-2Y1175, and ß-actin 24 h after 3.0 psi stretch injury in the cell lysates of hBMVEC treated with apocynin, VEGF-A, Ki8751, and TIMP1 (n = 6/group). All values are expressed as mean ± SD. Statistically significant, ***p < 0.001 versus uninjured group; ##p < 0.01, ###p < 0.001 versus injury group.

Fig 5: OPN stimulates ROS production and promotes cell proliferation and migration through upregulating NOX1 in HCC cells.A, B DCFH-DA fluorescence was used to evaluate total ROS production in control HCC cell, hOPN treated (1 µg/mL) HCC cells, siNOX1 HCC cells and siNOX1 HCC cells treated with hOPN. Representative images of cell viability (C), and cell migration (E) assays using SMMC7721 control, hOPN treated, siNOX1 treated, and siNOX1+OPN treated SMMC7721 cells. All the experimental conditions in (C, E) were the same as in D, F, except SMMC7721 cells were used instead of HCCLM3 cells. Original magnification, ×100. Data indicated mean ± SD. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, # Compared with hOPN treatment, NS not significant.