Fig 1: ADAM9 abundance in pulmonary arteries. A Representative images of ADAM9 (Red) endothelial cell marker CD31 (green), and DAPI (blue) abundance detected by immunofluorescence performed on human lung tissue from non-smokers and smokers without COPD, GOLD stage 4 COPD, and COPD-PH individuals. White arrows point to pulmonary arteries. Images were captured at 20X; scale bar is 50 µm. B, C Quantification of ADAM9 abundance in (B) small pulmonary arteries and (C) the microvasculature. For all graphs, each data point represents an individual; n = number of individuals in each group; horizontal lines are mean ± SEM; 1-way ANOVA with Dunnett’s multiple comparisons test was used for statistical analysis
Fig 2: Cigarette smoke exposure induces the expression of ADAM9 and IL-17A in human bronchial epithelial (HBE) cells. (A) The cytotoxicity of the cigarette smoker extract (CSE) was measured by the CCK-8 kit (n = 4). HBE cells were cultured with different concentrations of CSE for 24 to 48 h. Gene expression of Adam9 was measured in HBE cells after 24 h (n = 5) (B) or 48 h (n = 5) (C). (D) Gene expression of Adam9 in HBE cells treated with 5% CSE (n = 4). (E) Gene expression of Il17a in HBE cells after 24 h CSE treatment (n = 4). (F–H) Immunoblot and densitometric analysis of ADAM9 and IL-17A in CSE-treated HBE cells (n = 6). (I–K) Immunoblot and densitometric analysis of ADAM9 and IL-17A at 0 to 24 h after CSE treatment (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 3: Knockdown of RUNDC3A-AS1 inhibits thyroid tumor metastasis to lung in vivo. K1 cells transfected with sh-RUNDC3A-AS1 and sh-NC were injected into athymic nude mice via the tail vein. (A) The incidence of lung metastasis in K1 thyroid tumor. (B) The images of lung metastasis of sh-NC and sh-RUNDC3A-AS1 groups by stereo fluorescence microscope and lung metastasis analysis of sh-NC and sh-RUNDC3A-AS1 groups. (C) Hematoxylin eosin (HE) staining assay. (D) Masson staining assay. (E) RT-qPCR detected the expression of RUNDC3A-AS1, miR-182-5p, and ADAM9 in indicated groups. ***P < 0.001 vs. the sh-NC group.
Fig 4: USP39 expression positively correlates with ADAM9 expression in human glioma. (A) The U251 and U87 cells were transfected with lentiviruses encoding USP39‐targeting or negative control shRNA (shUSP39 or shNC), the protein expression levels of USP39 in the cells were analyzed by western blotting. (B) The total RNA from U87‐shUSP39 and U87‐shNC cells was isolated to perform gene expression profiling. Scatterplots display differential gene expression between U87‐shUSP39 and U87‐shNC (fold change > 2, n = 1242) from the data of gene expression microarray. The blue point chart and table on the right exhibit the downregulated genes related to the migration and invasion of cancer cells in the U87‐shUSP39 cells. Significance was calculated using Student’s t‐test. P < 0.05. (C) Expression analysis of the above downregulated genes in human glioma from TCGA samples. (D) Statistical analysis of the correlation analysis between USP39 and ADAM9 expression of TCGA glioma samples from GEPIA databases. The data were examined by calculating Pearson’s correlation coefficients (n = 681, P < 0.001, R = 0.36). (E) IHC images of USP39 (upper) and ADAM9 (lower) in tissue microarray (TMA) slides of paraffin‐fixed human glioma tissues. Scale bar: 200 or 20 μm (as indicated in the picture). (F) Quantification of USP39 and ADAM9 protein levels for the cases shown in (E). (G) Correlation between USP39 and ADAM9 protein expression in human glioma TMA. The data were examined by calculating Pearson’s correlation coefficients (n = 63, P = 0.013, R = 0.31). (H) Protein level of USP39 and ADAM9 in human glioma tissues detected by western blotting, and ratios of gray scale showing the difference in the picture.
Fig 5: Re-expression of ADAM9 reestablished prostate cancer cells (PCa) cell functions and ADAM9 expression is associated with PCa prognosis. (A) Re-expression of ADAM9 was confirmed by western blot analysis following co-transfection of miR-126 mimics and pL-ADAM9. Proliferation effect of DU145 and PC3 cells co-transfected with miR-126 mimics and pL-ADAM9 were analyzed by (B) Cell Counting Kit-8 assays and (C) colony formation assays. Representative results of (D) migration and (E) invasion of aforementioned cells were analyzed by Transwell assays (magnification, ×100). (F) Western blotting was performed to test epithelial-mesenchymal transition associated protein including E-cadherin, N-cadherin and Vimentin. (G) Immunohistochemical images of ADAM9 expression in PCa tissue microarrays. (H) Kaplan-Meier/log-rank analysis for biochemical recurrence-free survival from 93 patients with PCa according to ADAM9 expression in tumor tissues. **P<0.01, Student's t-test or Dunnett's t-test. PCa, prostate cancer cells; EMT, epithelial-mesenchymal transition; ADAM9, ADAM metalloproteinase domain 9; NC, negative control; OD, optical density; pL-ADAM9, pLenti-ADAM9.
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