Fig 2: PXN and miR-137 expression levels in CRC tissues and their prognostic significance. (A) Immunoreactive intensity of PXN in CRC was translated into a four-tier system as negative (score = 0), weak staining (score = 1), moderate staining (score = 2) and strong staining (score = 3). (B) Relative PXN mRNA and miR-137 expression levels in CRC tissue samples. Relative PXN mRNA and miR-137 expression in N (adjacent non-tumorous tissues), T (tumor tissues), Non-met (non-metastatic tumor tissues) and Met (metastatic tumor tissues) was determined by real-time PCR. The line in the middle indicates the mean value. (C) Kaplan–Meier analysis of overall survival based on PXN protein, PXN mRNA and miR-137 levels.

Fig 3: Knockdown of PXN or ectopic expression of miR-137 inhibit tumor growth and metastasis of CRC cells in vivo. (A) Xenograft tumor growth in nude mice. The graph shows the representative tumor growth 6 weeks after injection. The tumor sizes were measured every week after injection and tumor volume was estimated. At the end, the mice were killed and tumors were removed and weighted; all data are presented as mean ± SD (*P < 0.05) (scale bar, 1cm). (B) Lung and liver metastases in nude mice. The graph shows the representative hematoxylin- and eosin-stained sections of lung metastasis (upper) and liver metastasis (under) (×100); all the data are shown as means ± SD (*P < 0.05).

Fig 4: TMPs from highly metastatic cells or cells exposed to chemotherapy increase the pace of cell biomechanical forces and inhibit focal adhesion signaling. (A–C) MDA-MB-231 cells cultured with TMPs from MDA-MB-231 or LM2-4 cells exposed to PTX or vehicle control were applied on a pillar assay, as described in Materials and Methods. MDA-MB-231 cells cultured with serum-free medium (SF) were used as a control. (A) Left: Example of a cell spreading on an array of 0.5 µm diameter fibronectin-coated pillars (see illustration on the right). (B) Typical pillar displacement curves. (C) Histograms presenting the average number of pillar releases per second binned according to the size of the release. Each of the control conditions was significantly different than the conditions in which the cells were exposed to TMPs from LM2-4 cells or from cells exposed to PTX (a < 0.01; Kolmogorov–Smirnov test). n = 8–10 repeats/group. (D–E) Cells treated as above were seeded on fibronectin-coated plates to evaluate phospho-paxillin (P-Pax) and total-paxillin (Pax) expression by Western blot. GAPDH was used as a loading control (D). The ratio between P-Pax over Pax was calculated based on densitometry, and presented as fold change, for three biological repeats (E). n = 3 repeats/group. * differences compared to MDA-MB-231 control group, * p < 0.05, as assessed by one-way ANOVA followed by Tukey post-hoc test.

Fig 5: Upregulation of PXN induced by miR-137 reduction promotes cell proliferation, migration and invasion. (A) Proliferation rate of HCT116 cells with or without miR-137 mimics and PXN knockdown plasmid treatment and of HT-29 cells with or without miR-137 inhibitor treatment on the basis of MTT assay. The change in PXN protein was confirmed by western blotting (upper), and the absorbance at OD490 is shown at the bottom (*P < 0.05). (B) Representative colony formation assay of HCT116 cells with and without miR-137 mimics and PXN knockdown plasmid treatment and of HT-29 cells with or without miR-137 inhibitor treatment (*P < 0.05). (C) Representative transwell migration and invasion assay of HCT116 cells with and without miR-137 mimics and PXN knockdown plasmid treatment and of HT-29 cells with and without miR-137 inhibitor treatment (*P < 0.05, compared with NC).