Fig 2: Down-regulation of IL-1β-induced MMP expression in NP cells by PN-1.Cells were treated with IL-1β (10 ng/mL) alone or combined with PN-1, ERK inhibitor, or NF-kB inhibitor. (A) Equal amounts of protein extract were resolved by SDS-PAGE, and MMP-3, MMP-9, MMP-13, ADAMTS-4, ADAMTS-5, aggrecan, and COL2 proteins were detected by western blot analysis. (B–F) Densitometric analysis shows the suppression of MMP3 (B), ADAMTS-4 (C), MMP-13 (D), MMP-9 (E), and ADAMTS-5 (F) protein levels in NP cells treated with IL-1β, IL-1β + PN-1, IL-1β + ERK inhibitor, and IL-1β + NF-kB inhibitor. Densitometric analysis shows the promotive effect on Aggrecan (G) and COL2 (H) protein levels in NP cells treated with IL-1β, IL-1β + PN-1, IL-1β + ERK inhibitor, and IL-1β + NF-kB inhibitor. Data are representative of three independent experiments, and p-values are shown: *p < 0.05; **p < 0.01 compared to IL-1β stimulation alone.

Fig 3: Autocrine signaling factors FN1, ITGB1, SERPINE2 and LRP1 predict poor survival in early-stage breast cancer. (a) Plots comparing FN1, ITGB1, SERPINE2 and LRP1 levels in RNA-seq data from subtyped breast cancer cell lines. A one-way analysis of variance with Tukey’s test was performed, *P<0.5, ***P<0.001. (b–f) Survival curves from cBioPortal showing survival of patients with stage I, II or III breast cancer with the following genes altered. Log-rank test P-values are shown. (b) Tumors that had a low level (defined by heterozygous loss or homozygous deletion) of MIR200C along with high expression (defined by a gain, amplification or fold expression >2) of FN1 and SERPINE2 (red) compared to those that did not (blue). (c) Tumors that had a low level of MIR183 along with high expression of FN1 and SERPINE2 (red) compared to those that did not (blue). (d) Tumors that had a high level of FN1, ITGB1, SERPINE2 and LRP1 (red) compared to those that did not (blue). (e) Tumors that had a high level of FN1 and ITGB1 (red) compared to those that did not (blue). (f) Tumors that had a high level of SERPINE2 and LRP1 (red) compared to those that did not (blue).

Fig 4: COPII-mediated secretion is essential for VM. (a) Quantitative reverse transcription–PCR (qRT–PCR) for SEC13 in MDA-MB-231 or BT-549 cells transfected with siNT or siSEC13. Error bars represent s.d. of three (MDA-MB-231) or two (BT-549) independent experiments. (b) Representative images of VM in MDA-MB-231 or BT-549 cells transfected with siNT or siSEC13. Scale bars=1000 μm. (c) Quantification of total network length in VM assays in MDA-MB-231 or BT-549 cells. Error bars represent s.d. for five or three independent experiments, respectively. T-tests were performed to compare each to siNT control, ***P<0.001, *P<0.05. (d) qRT–PCR following expression of a NEG or clustered miRNA mimics. Error bars represent s.d. of three independent experiments. T-tests were performed vs NEG control, ***P<0.001. (h) Western blot for FN1 and SERPINE2 in conditioned media from MDA-MB-231 cells transfected as shown. (f) Quantification of three (FN1) or two (SERPINE2) independent replicates of western blots as shown in e. Error bars represent s.d. T-tests were performed for FN1 quantitation compared to NEG control. **P<0.05; ***P<0.001. (g) qRT–PCR following transfection of MDA-MB-231 cells with miRNA mimics. Error bars represent s.d. from three independent experiments. T-tests were performed, *P<0.05, **P<0.01, *** P<0.001. (h) Western blot for LRP1 following transfection of MDA-MB-231 cells with miRNA mimics. Shown is a representative image of two independent experiments. GAPDH is loading control. (i) qRT–PCR following transfection of MDA-MB-231 cells with miRNA mimics. Error bars represent s.d. from three independent experiments. T-tests were performed, *P<0.05, ***P<0.001.

Fig 5: Increased expression of FN1 and SERPINE2 is regulated by serum withdrawal and is essential for VM. (a) Quantitative reverse transcription–PCR (qRT–PCR) in breast cancer cell lines cultured with 10 or 0% serum for 48 h. Error bars represent s.d. of three independent experiments. T-tests were performed for each line comparing 0vs10% conditions, **P<0.01, ***P<0.001. (b, d) Representative images of VM in control and FN1 (b) or SERPINE2 (d) knockdown cells. Scale bars=1000 μm. (c, e) Quantification of total network length for VM assays as shown in b and d. Error bars represent s.d. for at five and four independent experiments, respectively. T-tests were performed to compare each to siNT control, **P<0.01. (f) Representative images of VM in control and FN1 or SERPINE2 knockdown in BT-549 cells. Scale bars=1000 μm. (g) Quantification of total network length. Error bars represent s.d. for three independent experiments. T-tests were performed to compare each to siNT control, *P<0.05. (h) Representative phase images of cell morphology in siNT or siSERPINE2 cells. Scale bars=100 μm. (i) qRT–PCR for FN1 and CDH1 in cells transfected with siNT or siSERPINE2. Error bars represent s.d. of three independent experiments. T-test was performed, ***P<0.001. (j) qRT–PCR for FN1 and SERPINE2 in cells transfected with NEG or clustered miRNA mimics for 48 h, then cultured with 10 or 0.5% serum for 48 h. Error bars represent s.d. of three independent experiments. T-tests were performed comparing 0.5vs10% in the control or comparing cells transfected with miRNAs vs NEG in matching serum conditions, ***P<0.001. (k) Western blot for FN1 for cells as in h. ACTB is loading control. (l, m) Quantification of total network length of VM assays from VM-incompetent cells plated in 0% serum (l) or VM-competent cells plated in 5% serum (m) in the presence or absence of recombinant FN1 and SERPINE2 as shown. Error bars represent s.d. of two independent experiments.