Fig 2: Simultaneous high expression of ADAM9 and ANGPT2 correlates with poor outcome in lung adenocarcinoma patients. (A) Kaplan-Meier survival analyses of lung adenocarcinoma patients in each indicated data set, with patients categorized by ADAM9 and ANGPT2 gene expression. Labels are defined as in Fig. 5. (B) Cox proportional hazards regression analyses of overall survival in each dataset of lung adenocarcinoma patients, grouped by ADAM9 and ANGPT2 expression. (C) Ranking the P-value of ADAM9 plus ANGPT2 among all P-values of profile genes by combining a fixed predictor with the other randomly selected genes from the same dataset. Fixed predictor: ADAM9 in the GSE8894 and GSE11969 datasets and ANGPT2 in the Shedden dataset. The P-value of ADAM9high/ANGPT2high versus ADAM9low/ANGPT2low is marked by the red line.

Fig 3: Expression of decorin and collagen type I in ADAM9 deficient fibroblasts. (A) Immunoblot analysis of decorin and collagen type I in cell supernatants and lysates from monolayer cultures of ADAM9 deficient or wild type dermal fibroblasts. Actin was used as a control. Underneath, in the table, is the average densitometric analysis after normalization to control (n = 3; * <0.03–4) (B) Quantitative real-time PCR analysis of decorin and collagen type I was performed using S26 as an internal control for normalization of transcripts. Data are mean values ± SEM (average of 6 different fibroblasts cultures per genotype). (C) Immunoblot analysis of decorin and collagen type I in cell supernatants (sp.) and lysates from monolayer cultures of B16F1, ADAM9-/-, or wild type dermal fibroblasts, without or with stimulation B16F1 conditioned medium (B16F1 c.m.). Underneath, in the table, is the average densitometric analysis of collagen and decorin after normalization to control (n = 4; * <0.01). Ponceau staining of the membrane was used as a control for loading.

Fig 4: Deletion of ADAM9 leads to reduced expression of collagenolytic enzymes and thereby enhanced tumor cell proliferation. (A) MMP-13 and MMP-14 were analyzed by immunoblot in fibroblast supernatants (normalized to amounts of protein lysates) and lysates, respectively. Both ponceau staining of the membrane and actin immunoblot were used as the loading controls. Underneath, the quantification of MMP13 and 14 is shown (n = 5–9 independent fibroblasts isolation each genotype). (B) The proliferation of B16F1 melanoma cells was assessed in the presence of increasing concentrations of purified collagen type I by BrdU incorporation. The graph represents the mean ± SD. p values were calculated for cell proliferation in the presence of collagen type I compared to serum-free untreated control. * p < 0.05.

Fig 5: The functional assays of biomarker knockdown in A549 lung cancer cells using shRNAs against ADAM9, MTHFD2, SLC2A1, and RRM2 individually or simultaneously (4G KD). (A) Western blots of control (shVOID), individual gene knockdown (shADAM9, shMTHFD2, shSLC2A1, and shRRM2), and 4G KD in A549 cells. (B) Colony formation of individual single gene knockdown and 4G KD A549 cells. Colonies were detected with crystal violet staining of the cell culture after 8 days (left) and quantified (right). (C) Cell migration ability of control, individual single gene knockdown, and 4G KD A549 cells. Cell migration was detected by time-lapse video microscopy (left) and quantified (right). (D) Western blots of control, individual gene knockdown, and 4G KD KD CL1-0 lung cancer cells. (E) Colony formation assay of control, individual gene knockdown, and 4G KD CL1-0 cells on day 11. (F) Cell migration ability of control, individual gene knockdown, and 4G KD CL1-0 cells. GAPDH served as the internal control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences were analyzed using the unpaired t test (*P < 0.05, **P < 0.01).